Fundamentals of Pharmacology

VM522P / VM7522 Pharmacology WIMU Fall

WIMU Regional Program in Veterinary Medicine Fundamentals of Pharmacology

VM522P, VM7522

Handouts of Pharmacology Lectures by Washington State University and Utah State University faculty, Drs. Suzy Appleyard, Steve Simasko, Nicolas Villarino, Mirella Meyer‐Ficca, and Ralph Meyer

VM522P / VM7522 Pharmacology WIMU Fall Table of Contents PART1 – Pharmacodynamics (S. Appleyard) ...... 5 L1: PHARMACODYNAMICS – TARGETS ...... 5 I. Specific Drug Targets ...... 5 II. Non‐targeted mechanisms ...... 9 L2: PHARMACODYNAMICS – RECEPTOR ‐ DRUG INTERACTIONS ...... 10 I. Drug‐Receptor Interactions – General Concepts ...... 11 II. Measurement of Binding Affinity ...... 12 III. Measurement of Effective Concentration...... 13 IV. Relationship between binding and effect ...... 14 V. Potency ...... 16 VI. Selectivity: The Structure‐Activity Relationship ...... 17 L3: PHARMACODYNAMICS – AGONISTS AND ANTAGONISTS ...... 19 I. Receptors ...... 19 II. Agonists ...... 19 III. Antagonists ...... 20 IV. Partial agonists ...... 23 L4: POPULATION PHARMACOLOGY ...... 26 I. Quantal Dose‐Response Curves ...... 26 II. Therapeutic Index (TI) ...... 27

III. LD50 ...... 28 IV. Adverse Reactions ...... 29 PART2 – Pharmacokinetics (S. Simasko) ...... 32 L5: Absorption ...... 32 L6: Pharmacokinetics: Distribution ...... 39 L7: Pharmacokinetics: Metabolism ...... 44 L8: Pharmacokinetics: Excretion ...... 53 L9: Pharmacokinetic Models ...... 59 A. One‐Compartment Open Model ...... 59 B. Two‐Compartment Open Model ...... 63 C. Multi‐Compartment Models ...... 66 L10‐12: Pharmacokinetic Variables ...... 68 a) Clearance ...... 69 b) Volume of Distribution ...... 70 c) Bioavailability ...... 71 d) Half‐life ...... 73 2

VM522P / VM7522 Pharmacology WIMU Fall L13: Dosing regimens ‐ Infusions ...... 74 L14: Dosing regimens – Intermittent dosing ...... 82 L15: TOLERANCE AND DRUG INTERACTIONS ...... 94 I. Tolerance ...... 94 II. Drug‐Drug Interactions ...... 96 L16: Pharmacokinetics: Adjustments ...... 99 L16: Clinical Pharmacokinetics ...... 105 Drug label or package ...... 105 Drug label or package: sections ...... 105 PK section of the label ...... 105 Labels are specific for products and formulations ...... 105 How to use/interpret a PK section? ...... 105 Specific pharmacokinetic parameters ...... 107 List of key works for interpreting labels ...... 109 Part 3 – Neuropharmacology (S. Appleyard) ...... 110 L17: Review of Neurotransmission ...... 110 I. Steps in Neurotransmission ...... 111 II. Examples of how drugs modulate Neurotransmission ...... 115 III. Modulation of Neurotransmission ...... 117 L18: Overview of the Autonomic Nervous System (ANS) ...... 120 I. Divisions and Structure of the ANS ...... 121 II. Chemical Mediators and Receptors within the ANS ...... 125 III. Function of the ANS ...... 129 IV. Somatic efferent nervous system (motor neurons) ...... 131 L19, 20: Cholinergic Pharmacology ...... 134 I. OVERVIEW OF CHOLINERGIC NEUROTRANSMISSION ...... 134 II. MUSCARINIC AGONISTS ...... 136 III. MUSCARINIC ANTAGONISTS ...... 138 IV. GANGLIONIC STIMULANTS AND BLOCKERS ...... 140 V. NEUROMUSCULAR BLOCKERS ...... 141 VI. ACETYLCHOLINESTERASE (AChE) INHIBITORS ...... 144 L21, 22: ADRENERGIC PHARMACOLOGY ...... 148 I. REVIEW SNS ACTIONS ...... 149 II. OVERVIEW OF ADRENERGIC NEUROTRANSMISSION ...... 150 III. ADRENERGIC AGONISTS ...... 151 IV. ADRENERGIC ANTAGONISTS ...... 160 3

VM522P / VM7522 Pharmacology WIMU Fall L23: Integrated Overview of Cardiovascular Drugs ...... 164 Part 4 – Autacoids (S. Appleyard, R. Meyer) ...... 167 L24 ‐26: AUTACOIDS ‐ Additional chemical mediators ...... 167 I. TYPES OF AUTACOIDS ‐ OVERVIEW ...... 168 II. HISTAMINE AND ANTIHISTAMINE DRUGS...... 169 III. SEROTONIN (5‐hydroxytryptamine, 5‐HT) ...... 175 IV. PURINES (Adenosine, and Purine Nucleotides, ATP and ADP) ...... 180 V. EICOSANOIDS (PROSTAGLANDINS & LEUKOTRIENS) ...... 181 VI. PEPTIDES (NEUROKININ, ANGIOTENSIN, BRADYKININ) ...... 187 Part 5 – Diuretics (R. Meyer) ...... 196 L27, L28: Diuretics ...... 196 1. General Concepts of Diuretics ...... 196 2. The Diuretics ...... 196 3. Summary ...... 198 4. Overview...... 200 Part 6 – Calculations (M. Meyer‐Ficca) ...... 201 L29‐34: Calculation Issues ...... 201 Calculation Issues for Unit 1 ...... 205 Calculation Issues for Unit 2 ...... 211 Calculation Issues for Unit 3 ...... 215 Calculation Issues for Unit 4 ...... 219 Calculation Issues for Unit 5 ...... 223 Calculation Issues for Unit 6 ...... 227 Calculation Issues for Unit 7 ...... 231

VM522P / VM7522 Pharmacology WIMU Fall

PART1 – Pharmacodynamics (S. Appleyard) L1: PHARMACODYNAMICS – TARGETS

Reading assignment for first 3 Lectures: Riviere and Papich 9th Ed. Chapter 4, pages 75‐85. Alternative reading Rang et al. 6th Ed. Chapters 2, 3 and 6. This script was authored by Drs. Steve Simasko and Suzy Appleyard and edited by Dr. Ralph Meyer

Objectives:

1. Be able to explain the difference between targeted vs non‐targeted drug mechanisms of action. Know the examples of targeted and non‐targeted mechanisms discussed in class and listed in the notes.

2. Be able to list the four classes of endogenous receptor proteins. Know an example of each mechanism and the relative speed by which each mechanism induces an effect.

Outline: I. Specific Drug Targets II. Non‐targeted methods

CONCEPTS OF DRUG ACTION

Major tenet of modern pharmacology – a drug binds to some component of the cell to exert an action. That component is the drug ‘target’.

Targeted vs non‐targeted drug mechanisms

Most drugs act by binding to a specific target. They produce their effect by either activating or inhibiting the action of that target protein. This allows drugs to have specificity in their effects. In general, the more targeted the mechanism of action, the more specific a drug’s effects will be. However, a few drug preparations are non‐ targeted. That is they do not specifically bind to any one protein target. Rather these drugs produce their effects by inducing a more global change, e.g. by altering pH, ion concentration or osmolarity. Precisely because these effects are more global these preparations tend to have more general effects, often including more unwanted or adverse effects.

I. Specific Drug Targets

1. Receptors – the most common drug target. ‐ receptors are proteins that monitor the cell’s external environment for the presence of endogenous chemical mediators e.g: neurotransmitters, neuromodulators, hormones, cytokines, prostaglandins ‐ their normal role is to respond to endogenous mediators. Drugs also bind to them and essentially mimic (or block) their effect. There are multiple types or classes of receptors. These are defined by their signaling mechanism. The class of receptor or how the receptor signals will often predict how fast the onset time is to a drug’s effect. However, remember the onset time of a drug’s effect is also going to depend on pharmacokinetic factors, e.g. an i.v. injection that goes straight to the blood vs. a pill that has to be absorbed. 5

VM522P / VM7522 Pharmacology WIMU Fall

Classes of receptor proteins:

a. Ligand‐gated ion channels (stimulate response in msecs ‐ secs), e.g. nicotinic acetylcholine, glutamate, GABAA, 5‐HT3 Receptors

b. G protein‐coupled receptors (GPCR) (stimulate response in secs ‐ mins), e.g. adrenergic, dopaminergic, muscarinic, histamine receptors

c. Receptor‐regulated enzymes (stimulate response in mins ‐ hours) e.g.: ‐ tyrosine kinase receptors (insulin, growth factors), ‐ serine / threonine kinase Receptors (TGF)

‐ guanylyl cyclase (Atrial Natriuretic Factor, ANF)

d. Intracellular (= nuclear) receptors (stimulate response in hours ‐ days) e.g.: steroids, thyroid hormone, vitamin D, retinoic acid

Why you should care: don’t expect a drug that is a steroid to have an effect within minutes! However, watch out for a quick effect of epinephrine given i.v.!

G-protein-coupled receptors EFFECTOR 7-transmembrane regions receptors adenylyl cyclase ligand-gated  K channel   Ca channel ion channels phospholipase C nicotinic R cGMP phosphodiesterase glutamate R Na+ or Ca2+ or Cl- glycine R adrenergic R GTP GDP muscarinic R GABAA R 5-HT R dopaminergic R 3 serotonin R histamine R peptide R

intracellular receptor receptor-regulated ligand-regulated hormone-like molecule transcription factor steroids enzyme thyroid hormone vitamin D retinoic acid BINDING CATALYTIC DOMAIN ribosome DOMAIN insulin transcription AAAAA EGF mRNA PDGF tyrosine kinase TGF AAAAA translation serine/threonine kinase DNA ANF guanylyl cyclase NUCLEUS out in membrane new protein

VM522P / VM7522 Pharmacology WIMU Fall 2. Ion channels

Some drugs bind directly to ion channels in the cell membrane. Many types of ion channels are drug targets including voltage‐dependent and calcium‐ or cAMP‐activated channels. These drugs will also have fast effects (depending on their route of administration).

Ligand gated vs. non‐ligand gated. Remember one class of receptors described above were also ion channels. Those were ligand‐gated ion channels, meaning that a ligand, e.g. a hormone or neurotransmitter, had to be occupying a receptor binding site on the channel for them to open. The drug targeting that channel binds to the receptor site to either mimic or block the hormone or transmitter’s effect. In contrast, the drugs listed below target channels that are not considered to be ligand‐gated as they do not have a receptor site that requires the presence of a ligand or neurotransmitter to open the channel.

Examples of drugs acting by regulating ion channels:

i) Local anesthetics : block voltage‐dependent Na+ channels

lidocaine (XYLOCAINE®); procaine (NOVOCAIN®)

ii) Dihydropyridine‐type cardiac drugs : block voltage‐dependent Ca2+ channels

nifedipine (PROCARDIA®); Amlodipine (NORVASC®)

3. Enzymes

Some drugs produce their effects by modulating enzyme activity. The effects of such drugs can be mediated in several ways: i) by inhibiting the action of an enzyme that is responsible for the synthesis or ii) breakdown of a transmitter or hormone (this will either increase or decrease the concentration of the transmitter or hormone and so increase or decrease its effects) or iii) by inhibiting intracellular enzymes to change cell signaling pathways. The speed of their effects depends on the enzyme they are inhibiting and how fast the product of that enzyme is metabolized or removed.

Examples:

i) Drugs that alter enzymes that synthesize a chemical mediator

Cyclooxygenase inhibitors – aspirin like NSAID drugs inhibit cyclooxygenase, which is the enzyme responsible for the production of prostoglandins (PGs). Therefore, NSAIDs decrease PG production and as PGs are key mediators of temperature and pain these drugs help reduce fever and pain.

ii) Drugs that alter enzymes that breakdown a chemical mediator

Acetylcholinesterase inhibitors – decrease the breakdown of a key neurotransmitter acetylcholine (ACh), which leads to more ACh and so an increase in its effects, e.g. neostigmine (PROSTIGMIN®, STIGLYN®) increases ACh at the neuromuscular junction and improves muscle function in myasthenia gravis.

VM522P / VM7522 Pharmacology WIMU Fall iii) Drugs that alter enzymes that produce an effect by altering the levels of an intracellular signaling molecule. Phosphodiesterase inhibitors – decrease the breakdown of cAMP, a key intracellular signaling molecule e.g. theophylline and caffeine.

4. Carriers / membrane transport proteins

Another class of specific drug targets are proteins whose normal role is to transport specific proteins or ions across membranes. Blocking their function alters either transmitter‐ or ion‐ concentrations to stimulate effects.

Examples:

a) SSRI – selective serotonin reuptake inhibitors, e.g. Fluoxetine (PROZAC®). These drugs act by blocking the reuptake of the neurotransmitter serotonin. This leads to an increase in serotonin levels in the brain and an increase in its effects, e.g. elevating mood and decreasing anxiety.

b) Loop diuretics ‐ Na+/K+/2Cl‐ co‐transporter in the kidney, e.g. furosemide (LASIX®). These drugs act to block sodium reuptake in the kidney, which then leads to less water being reabsorbed and diuresis.

c) Cardiac Glycosides ‐ Na+/K+ pumps in the heart – e.g. digitalis (DIGOXINX®). This drug blocks sodium transport out of cardiac cells by blocking the Na+/K+ pump and indirectly leads to an increase in intracellular calcium levels by decreasing the driving force for the Na/Ca exchanger. Increased intracellular calcium in cardiomyocytes leads to increased force of contraction making the heart more efficient at pumping blood.

5. DNA /RNA

Some drugs target DNA or RNA molecules. These are often cytotoxic and are currently mainly used as anti‐ viral or chemotherapy drugs. However, as our understanding of the regulation of both transcription and translation increases future drugs may be able to more specifically target regulation of specific genes and therefore proteins.

Examples:

a) Oncology related compounds such as doxorubicin (ADRIAMYCIN®), which binds irreversible to DNA preventing both RNA and DNA synthesis and Vincristine (ONCOVIN®), which binds to microtubular proteins and disrupts spindle formation during mitosis. Both will therefore inhibit tumor formation by blocking cell division. b) Pyrimidine nucleoside analogs, such as Trifluridine, which is an anti‐viral drug. The nucleoside analogs are incorporated into the DNA as thymidine analogues and disrupt transcription and replication of viral DNA.

VM522P / VM7522 Pharmacology WIMU Fall II. Non‐targeted mechanisms Some drugs do not act by binding to specific targets. Instead they produce a therapeutic effect by altering the environment or conditions of a patient. These effects are therefore non‐targeted. Examples are:

Osmotic effects. Some drugs such as mannitol or glycerin change the osmolairty of the plasma. This can be useful for conditions such as glaucoma, where there is too much pressure in the eye due to too much fluid. By increasing the osmolarity of the plasma this will “pull” water out from the eye, decreasing the pressure. However, clearly anywhere glycerin or mannitol is present the osmolarity will be increased and water balance altered. Therefore, these types of drugs can have a large number of side effects unless they remain in a local environment, e.g. the eye.

Fluid or ion (acid / base) replacement (Na+, K+, Ca2+, etc.). Some treatments involve ion replacement. This is usually done to restore normal balance. Again it is not a targeted effect, and all organs will be affected.

Pore forming agents (ionophores – monensin and lasalocid). These drugs are types of antibiotics often included in feed. They produce their effects by forming pores in the membranes of bacteria. These pores disrupt normally ion and fluid balance and kill the bacteria. Again there is not a specific protein they bind to or target.

Chelators (EDTA, BAL). These drugs chelate (bind up) certain ions to decrease their effective concentration, but do not target a specific receptor protein to produce an effect

Protectants (salves).

Antacids.

III. OTHER

It is not always possible to definitively characterize whether some drugs have targeted or non‐targeted mechanisms, as their mechanism(s) of action is(are) not fully understood.

e.g. Volatile general anesthetics, e.g. isoflurane (AERRANE®) – the target(s) are not clear as their mechanism of action is still not fully understood. Volatile anesthetics traditionally have been thought to produce anesthesia via membrane destabilizing properties, which are non‐targeted. However, recent studies suggest that they also affect the activity of specific proteins in the membrane, e.g. GABA and glycine receptors, which are probably important for their therapeutic effects, making their actions targeted.

VM522P / VM7522 Pharmacology WIMU Fall

L2: PHARMACODYNAMICS – RECEPTOR ‐ DRUG INTERACTIONS

Reading assignment:

Riviere and Papich Chapter 4, pages 75‐85. Alternative reading Rang et al. 6th Ed. Chapters 2, 3 and 6.

Objectives:

1. Understand the concept of a dose‐response (concentration‐effect) relationship so that you are able to explain why no effects are observed at extremely low doses and why there is a maximal effect above which no further therapeutic response is observed regardless of dose. You should also be able to extract the EC50 concentration from a dose‐response graph.

2. Know the difference between affinity, efficacy and potency and be able to define each term.

3. Understand why drug receptor interactions are reversible (i.e., describe the forces that mediate binding).

4. Understand how the concept of reversible binding defines plots of binding data (Law of Mass Action) and be able to determine the affinity of the drug for the receptor (Kd) from these plots.

5. Provide mechanistic explanations for disparities between dose‐response observations (EC50) and binding result (Kd).

6. Explain the relationship between binding affinity and potency and why sometimes high affinity drugs may not be the biologically most potent.

7. Explain how the concept of drug binding can account for a high degree of selectivity in drug action.

Outline: I Dug‐Receptor Interactions – general concepts II Measurement of Binding Affinity III. Measurement of Effective Concentration. IV. Relationship between binding and effect V. Potency VI. Selectivity ‐ The Structure‐Activity Relationship

VM522P / VM7522 Pharmacology WIMU Fall I. Drug‐Receptor Interactions – General Concepts

Most receptors are transmembrane proteins – this means the drug binds to the external portion of the receptor, induces a confirmation change that activates the receptor to produce a signal inside the cell.

1. Receptors exist in an inactive (resting) state or an active state (signaling)

2. Only drugs with structures that match (are complimentary to) the binding site will bind to the receptor (highly specific)

AFFINITY: Ability to bind to the receptor

3. Drugs that are agonists bind to the receptor and activate it. Antagonists bind to the receptor, but do not activate it as they do not induce the necessary conformational change.

Efficacy: Ability to produce a response

Therefore agonists produce an effect by stabilizing the active or signaling state of the receptor while antagonists stabilize the inactive state.

Receptor (in active state)

Drug A is an example of a drug that doesn’t bind the receptor. Drug B is an example of an antagonist Drug C is an example of an agonist

There are many degrees of agonists from one that only slightly activates the receptor (low efficacy – partial agonist) to agonists that fully activate the receptor (high efficacy – full agonist).

A fundamental concept in pharmacology is how well a drug produces an effect, e.g. its efficacy. However, efficacy can be defined in many ways. First, how well the drug causes a confirmation change to activate the receptor protein (receptor efficacy); second, how well that activation produces a cellular response (cellular efficacy) and third, how well the drug produces an effect at the level of the whole animal response (clinical efficacy).

VM522P / VM7522 Pharmacology WIMU Fall

At the level of receptor activation antagonists have no efficacy (ability to activate the receptor).

Antagonists do have “clinical efficacy”. That is the ability to produce an effect in the whole animal. However, they only produce an effect clinically by blocking the action of an agonist, normally the endogenous hormone or transmitter for that receptor. Therefore, the size of the effect of an antagonist clinically will depend on the concentration of the endogenous hormone/transmitter whose actions it is blocking and how well that endogenous mediator is activating the receptor being targeted.

II. Measurement of Binding Affinity

How well a drug binds to its receptor is determined by binding assays. A cell line or tissue that is enriched in the receptor being targeted is used. The unknown drug’s binding characteristics are then compared to a known high affinity antagonist for that receptor that is labeled (normally with radioactivity or fluorescence). Drug binding to its receptor can be defined by:

Law of mass action (applies to reversible interactions only)

k 1 D + R DR k-1

D - drug R - receptor DR - drug-receptor complex

k - association rate constant k - dissociation rate constant 1 -1 Define the equilibrium dissociation constant as:

k-1 Kd = k1

at equilibrium: complex formation = complex breakdown

Kd = concentration of a drug that is required to occupy 50% of

the receptors at equilibrium

Proportion of receptors occupied PA = [D]

[D] + Kd

When [D] = Kd then PA= 0.5

VM522P / VM7522 Pharmacology WIMU Fall

The Kd of a drug tells you about the affinity of the drug for a receptor.

It is inversely proportional to the binding affinity of a drug.

A low Kd means a drug has a high affinity for its receptor.

A high Kd means a drug has a low affinity for its receptor.

A Kd tells you nothing about the efficacy of a drug. Both agonists and antagonists can have high affinities or a low Kd .

III. Measurement of Effective Concentration.

During drug development a bioassay is used to compare the relative effects of drugs.

For example, you can measure how well a drug causes contraction of a piece of ileum by dissecting a piece of ileum, placing it in a apparatus that measures force transduction and applying different drugs to the bath to determine how much contraction they produce. The ability of a drug to contract or relax ileum tissue normally reflects how well those drugs will causes contraction or relaxation of gut in vivo.

force transducer add drug

water intestinal bath strip

Initial drug screening can also be performed using cell culture systems, e.g. cells that are derived from GI cells. A given response, such as calcium entry, can be measured and quantified.

VM522P / VM7522 Pharmacology WIMU Fall You can plot a concentration/effect curve or dose response curve:

Concentration - Effect Curve Concentration - Effect Curve (or Dose - Response Curve) (or Dose - Response Curve) (semi-log scale) (linear scale) 12 12 10 10 drug A drug B 8 8

6 6

4 drug A 4

(arb force units) drug B (arb force units) 2 2 0 0 contraction of intestinal strip 0 500 1000 1500 2000 2500 3000 3500 contraction of intestinal strip 0.01 0.1 1 10 100 1000 10000 concentration of drug (mcg/L) concentration of drug (mcg/L)

25 The doses are normally expressed in log units so that it is easier to read the concentration that produces a certain effect, i.e.20 the EC50

10 EC50 = the dose (or “Effective Concentration”) that produces a

half‐maximal (or 50%) effect. (arb force units) 5

contraction of intestinal strip drug A drug B drugs A + B

test compounds (max effect)

IV. Relationship between binding and effect

There is often a good correlation binding affinity, or Kd and the EC50. However, this isn’t always the case as there are sometimes many steps from the activation of the receptor to a clinical response. k1 D + R DR EFFECT k‐1

Remember:

Kd = concentration required to occupy 50% of the receptors at equilibrium

EC50 = concentration that produces a half maximal effect

RT = Total number of receptors RT = R+DR

VM522P / VM7522 Pharmacology WIMU Fall

So how do binding curves compare to

dose‐response curves? DR complex ( )

100 R T

Good correlation: 75 R To get a full effect the drug needs to 50 T occupy all the receptors. 2 25

response ( ) % 0 0.1 1 10 100 1000 K EC50 d Kd > EC50 (spare receptors):

Normally suggests the drug is an agonist. DR complex ( )

100 R T Effect occurs when only a small 75 proportion of receptors are activated. R Therefore you do not need to occupy all 50 T the receptors to produce a full effect. 2 Hence “spare receptors”. 25 response ( ) % 0 0.1 1 10 100 1000 K EC50 d Kd < EC50 (threshold effect): Only see a response when a high DR complex ( ) percentage of sites are occupied. 100 R T Normally suggests an antagonist. If the 75 antagonist is blocking a response R produced with “spare receptors”, you will 50 T need to occupy all the spare receptors 2 25 before you begin to block the response. % response ( ) In rare cases this can also occur if an 0 agonist is inhibiting a physiologically non‐ 0.1 1 10 100 1000 K EC rate limiting step. d 50

drug (nM)

VM522P / VM7522 Pharmacology WIMU Fall V. Potency Potency: the concentration of drug required to produce an effect, i.e. the desired therapeutic effect. This is in contrast to “clinical efficacy”, which refers to the size of the response that is produced. Beware, just because a drug has a low potency doesn’t mean it can’t eventually produce the same effect or an even greater effect than a drug with a high potency. In the graph below the order of potency is A > B > C > D. However they all have the same efficacy!

120

100

60 A B 40 C D

20 responseunits) (arb

0 0.0001 0.001 0.01 0.1 1 10 100

In the graph below drug A has a high potency, but low clinical efficacy. In contrast, drug B has a low potency, but high clinical efficacy.

B 70

60 50

40 A 30

Response (arbitrary units) 0

0.01 0.1 1 10 100 1000

Dose

VM522P / VM7522 Pharmacology WIMU Fall Potency in the clinics applies to agonists, partial agonists, and antagonists (i.e., you can have high or low potency agonists or antagonists).

High affinity drugs tend to be highly potent drugs but not always. Some reasons maybe that the drug:

1. is not good at stabilizing the receptor in its active state, i.e. it does not have high efficacy.

2. does not gain access to its site of action.

3. is rapidly metabolized or excreted.

Generally highly potent drugs are desirable because you do not need so much to exert an effect and they may be useful when the injection volume is limited (darting an animal), however, they are much more dangerous (easy to overdose).

VI. Selectivity: The Structure‐Activity Relationship

1. Think in 3D, a drug will often have many points of attachment within the binding pocket of its receptor. This leads to a high degree of specificity. Therefore, even chemically related drugs, such as catecholamines can have very different binding affinities for receptors within the same family, e.g. adrenergic receptors:

catecholamines receptor preference H HO CH2 CH2 N DA >>  >  dopamine H HO

H HO CH CH2 N  >  >> DA norepinephrine H HO OH

CH3 HO CH CH2 N  >  >> DA epinephrine H HO OH

CH3 HO CH CH2 N  >>  > DA isoproterenol CH HO OH 3

VM522P / VM7522 Pharmacology WIMU Fall 2. Because of the special orientation of binding pockets stereoisomers may have substantially different potencies (requires three or more points of attachment)

N N H H

N S S N

levamisole S (-) R (+)

tetramisole

3. Receptor structure is used by modern pharmaceutical industries to develop new and more selective agents.

Most drugs bind reversibly to their receptors and therefore their effect can be reversed either by an antagonist, if the drug is an agonist, or when the concentration of the drug drops and therefore the drug dissociates from its receptor.

Most drugs bind reversibly with non-covalent interactions

Forces involved (strongest to weakest):

a) ionic interactions

b) hydrogen bonds

c) hydrophobic interactions

d) Van der Waal's forces

However, some interactions are irreversible (covalent interactions). This means the effect of that drug cannot be reversed by giving an antagonist, or in the case or a drug that is an antagonist, an agonist. The effect of the drug will only be lost when the receptor protein is replaced, or the cell compensates for the effect of the drug. One example of this is Phenoxybenzamine, an antagonist that binds irreversibly to alpha1 adrenergic receptors (see page 85).

VM522P / VM7522 Pharmacology WIMU Fall

L3: PHARMACODYNAMICS – AGONISTS AND ANTAGONISTS

Reading assignment:

Riviere and Papich Chapter 4, pages 75‐85. Alternative reading Rang et al. 6th Ed. Chapters 2, 3 and 6.

Objectives:

1. Be able to explain the difference between agonists, partial agonists, and antagonists at the level of the receptor activation.

2. Be able to explain the difference between competitive and non‐competitive antagonism both at a mechanistic level and how you can identify the different types of action in dose‐response studies.

3. Be able to explain the differences between physiological vs. pharmacokinetic vs. pharmacological antagonism.

Outline: I. Receptors II. Agonist III. Antagonists IV. Partial Agonists

I. Receptors

Remember: 1. Receptors are allosteric proteins that can change their confirmation, or shape. 2. An allosteric protein can exist in two states, active and inactive. 3. In most cases, when the receptor protein is unoccupied it exists in the inactive (or resting) state.

II. Agonists 1 Agonists are molecules that bind to the receptor protein and shift it into the active state. 2. This produces a simple saturation curve for the concentration‐effect plot. 3. The magnitude of a drug’s response is related to the 120 absolute number of receptors in their active or max response signaling state, not the percentage of receptors 100 agonist occupied. Remember that agonists have different 80 alone

efficacies. A high efficacy agonist will cause most of 60 the receptors it has bound to be in their active, or 40 signaling state. A low efficacy agonist will cause less effect (arb units) 20 to be active or signaling, so that even if 100% of the EC50 receptors have drug bound, only a few will be in 0 0.001 0.01 0.1 1 10 100 1000 their active or signaling conformation. agonist concentration (nM) 19

VM522P / VM7522 Pharmacology WIMU Fall

III. Antagonists There are several ways to antagonize (or inhibit) the action of a drug

1. Pharmacological Antagonists Pharmacological antagonists are drugs that bind to receptor proteins and push them into their resting or non‐signaling state as they stabilize their inactive confirmation. Their occupation of the receptor prevents agonists from activating it. Note: as antagonists do not produce a signaling response on their own the only way they can have an effect is by blocking the action of an agonist. In most cases clinically used drugs that are antagonists produce an effect because they block the action of an endogenous agonist, e.g. histamine. Another use of antagonists is to reverse an overdose of a drug that is an agonist, e.g.the opioid antagonist naloxone is used to reverse an overdose of opioid agonists, such as morphine or fentanyl. There are 2 types of pharmacological antagonist:

A. Simple competitive antagonists

1. A simple competitive antagonist binds the same site as the agonist and physically blocks the agonist from binding to the receptor. 2. However, the binding of a competitive antagonist is reversible and equilibrium will occur. The higher the concentration of the antagonist the more receptors will be occupied by that antagonist at any given time. However, because the binding is reversible and an equilibrium exists, increasing the concentration of the agonist will outcompete the antagonist, displacing it from the receptor and allowing the agonist to occupy the binding site and activate the receptor. Which drug is bound will depend BOTH on their respective concentrations AND their binding affinity for the receptor. 3. Remember the size of the response to an agonist is determined by the number of 120 receptors in an active of signaling same max response 100 confirmation. If a competitive antagonist agonist alone agonist is present it will take more agonist to 80 alone results in displace the antagonist and produce the presence of same size of response compared to in the 60 competitive absence of antagonist. antagonist 40 4. However, if enough agonist is added, effect (arb units) effect new 20 eventually all the receptors will be EC50 occupied by agonist. Thus the maximal 0 response can still be obtained, but the 0.001 0.01 0.1 1 10 100 1000 concentration‐effect curve will shift to the right. agonist concentration (nM)

VM522P / VM7522 Pharmacology WIMU Fall B. Non‐competitive antagonists

1. A non‐competitive antagonist either binds irreversibly to the agonist binding site or binds at another site that prevents receptor activation. In either case the agonist cannot compete off the antagonist.

2. Since the agonist cannot compete off 120 the non‐competitive antagonist less

receptors will be available for the 100 agonist to bind to. This causes the lower agonist maximal response of the agonist to be 80 max alone decreased. response 60 results in 3. In addition, since the agonist is not presence of 40 competing with the antagonist for its non-competitive same antagonist

binding site the EC50 will not be shifted. effect (arb units) 20 EC 50 0 0.001 0.01 0.1 1 10 100 1000 Mechanistic example of agonist action with a competitive vs. non‐competitive antagonist: agonist concentration (nM)

agonist only agonist + competitive antagonist agonist + noncompetitive antagonist

agonist antagonist agonist non-comp agonist + + + + + + + + + + + + + + + + + + + + + + + +

membrane membrane membrane ion ion ion channel channel channel

+ + + + + + + + + + + + + + + + + + + + + + + + + + agonist + antagonist + agonist non-comp agonist + + membrane ion membrane membrane membrane channel ion ion ion + channel channel channel +

VM522P / VM7522 Pharmacology WIMU Fall

2. Physiological Antagonists

i. Physiological antagonists oppose or antagonize a drug’s action by activating an opposing receptor signaling pathway of the same physiological system: e.g.: ‐ Histamine causes contraction of airways through activating the histamine receptor (it is an agonist at the histamine receptor). ‐ Adrenaline is an agonist at the adrenergic receptor. Activation of the adrenergic receptor dilates the airways and reverses the effect of histamine. ‐ Two drugs that have opposing effects on blood pressure but both act on blood vessels, one to contract one to dilate.

ii. They may also target an entirely different physiological system e.g.: ‐Two drugs that have opposing effects on blood pressure, one through an effect on blood vessels and one through an effect on the heart.

iii. They represent generally a much more 'risky' approach to treating an overdose. However, in the case of an allergic reaction where adrenaline is used to reverse the effects of histamine, the risk is outweighed by the fact that adrenaline acts so fast. Blocking the actions of histamine with an antagonist would take longer and may not work in time. Also an allergic reaction often involves more than just histamine release. Also in some cases you may not have the time or ability to know exactly what is causing the problem, e.g. what the animal overdosed on. Therefore you treat the symptoms, which may mean using a physiological antagonist.

3. Pharmacokinetic Antagonists

This type of drug works by removing the drug from the system. For example by:

i) binding to the drug and eliminating it’s ability to bind to a target and produce a response, e.g. chelators ii) reducing the absorption or enhancing the elimination of the active drug, e.g. charcoal

VM522P / VM7522 Pharmacology WIMU Fall Dose-response curves: IV. Partial agonists Partial agonist A. Some compounds produce some effect but even at high 35 concentrations they do not produce a maximal effect – they are called partial agonists 30

25 A 20 D 1. Partial agonists have a lower efficacy. B 15

C 10 2. Full agonists stabilize the active confirmation of the receptor (compounds A and D in the graph). 5 response (arb units) E 0

3. Antagonists stabilize the resting confirmation of 0.001 0.01 0.1 1 10 100 1000 the receptor (compound E in the graph). drug concentration (nM)

4. Partial agonists have mixed affinity for the two Partial antagonist confirmations (compound B and C in the graph).

35 5. Because partial agonists do not fully activate the 30 receptor, at higher concentrations when they displace the full agonist, less receptors will be in 25 Give enough the signaling confirmation and partial agonists compound A 20 actually act as partial antagonists to reduce the to get maximal response. maximal response. 15

10 Add in other compounds.

units) response (arb Question: which line 5 belongs to which compound? 0 0.001 0.01 0.1 1 10 100 1000

concentration of partial agonist (nM)

VM522P / VM7522 Pharmacology WIMU Fall

overview drug out of signal G-protein adenylyl cyclase in transduction PP receptor ATP cAMP GTP GDP

full agonist agonist agonist receptor G-protein GDP receptor GDP inactive active G-protein GTP

antagonist antagonist antagonist

receptor

receptor GDP inactive inactive G-protein GDP G-protein

partial partial agonist agonist receptor G-protein

GDP

partial active agonist GTP partial agonist

receptor

receptor inactive inactive GDP GDP G-protein G-protein

VM522P / VM7522 Pharmacology WIMU Fall

A FEW THINGS TO KEEP IN MIND:

1. More complexities exist – e.g., inverse agonists. Some receptors have a low amount of basal activity. That is that even in the absence of any agonist a small proportion of the receptors are in the signaling confirmation. Binding of an agonist will shift more of the receptors into a signaling confirmation and produce a response. However, binding of an inverse agonist shifts the confirmation of all the receptors into an inactive or non‐signaling state. Therefore inverse agonists will have the opposite, or inverse, effect to agonists. Furthermore, as inverse agonists silence a receptor that was signaling (in the absence of an agonist) it can have an effect even when no agonist is present.

2. Almost all endogenous chemical mediators are agonists although there are exceptions

3. Drugs that are agonists tend to have more predictable effects because the receptor is activated and induces a change in the signaling state of the cell (leading to a change in the tissue behavior, leading to a physiological response). However, the potency and maximal effect of the agonist will also depend on the number of receptors present as well as pharmacokinetic factors ‐ so there is always some unpredictability when giving a drug.

4. Antagonists can have extremely variable responses – from virtually nothing if the there is no agonist present (i.e., endogenous mediator), to very profound and possibly fatal results if the event blocked is ongoing and vital for life.

For example, a beta receptor antagonist does not have a clinically important effect on the lungs in most animals, but in asthmatics that rely on a basal tone of an endogenous agonist at the beta receptor to keep the airways dilated, blocking this dilation can be fatal. Another example is that an anti‐histamine (histamine antagonist), such as diphenhydramine (BENADRYL®) has little effect normally, but can have profound effects during an allergic reaction when a large amount of histamine is released.

VM522P / VM7522 Pharmacology WIMU Fall L4: POPULATION PHARMACOLOGY

READING ASSIGNMENT:

There is no good general background consolidated in one chapter in Riviere and Papich. Pharmacogenetics is covered in Chapter 50 in Riviere and Papich, page 1313. For a more general background see Rang et al., “Pharmacology” 6th edition Chapters 52 and 53.

Objectives: 1. Be able to explain a quantal dose‐response curve.

2. Be able to extract ED50, LD50, and other such values (e.g., TD90), from quantal dose‐response curves. 3. Recognize that there is no standard definition of a therapeutic index or ‘safety factor’.

4. Understand the usefulness and limitations of an LD50 determination. 5. Be able to list the typical reasons for adverse drug reactions. 6. Understand the differences between dose related and non‐dose related adverse reactions (both the reasons for and the consequences of).

Outline:

I. Quantal Dose‐Response Curves II. Therapeutic Index

III. LD50 IV. Adverse Reactions

I. Quantal Dose‐Response Curves

A quantal dose‐response curve is a graph that allows you to look at the effect of a drug on a whole population of animals. A clinically relevant all‐or‐non criterion is established; for example, whether body temperature is reduced by 3 degrees, or the animal falls asleep of not, or heart rate or blood pressure are reduced by X amount. Then different concentrations of drugs are tested across a population of animals and the number of responders (i.e. animals where the drug produced or exceeded the established criterion) determined. This is in contrast to a dose response curve, where the size of the response produced by different drug concentrations is determined in a few animals. The important points are: i. Quantal dose‐response curves allow for comparisons of population 100 responses. 80 ii. Each patient’s response is all or none, i.e. 60 the drug either had a set effect in that animal or it didn’t. 40 iii. Quantal dose response curves are usually 20 expressed as cumulative frequency curves:

% responding 0 0.1 1 10 100 1000 dose (mg/kg)

VM522P / VM7522 Pharmacology WIMU Fall II. Therapeutic Index (TI)

The TI is the ratio between an effective concentration of a drug compared to its side effects or lethality. It provides some information about the margin between therapeutic effectiveness and toxicity.

ED50 (effective dose for 50% of the population)

TD50 (toxic dose for 50% of the population)

LD50 (lethal dose for 50% of the population)

It is most commonly defined as TI = LD50 / ED50

One problem is that the "Therapeutic Index" can have several definitions or meanings. For example: it can also be defined as

TI = TD50 / ED50 or even as TI= LD3 / ED97 or TI= TD3 / ED97

ED 97 LD 3

100

80 therapeutic response lethal response 60

% responding ED 50 LD 20 50

0 0.1 1 10 100 1000

dose (g/kg)

LD50 TI = = = 20 40 ED50 These 2 values are very different from 2 each other. This is why in order to interpret a therapeutic index. You need to LD3 10 TI = = = 1 } know what the number is referring to! ED97 10

In addition to the multiple ways of defining the Therapeutic Index there is the added complication of knowing how the therapeutic effect and toxic effects are defined; e.g. was it mild pain relief or significant pain relief, were the toxic effects mild constipation or severe cardiac or respiratory failure?

For veterinary medicine an added complication is to know what species the data was generated in. Therapeutic, toxic and lethal doses can vary greatly between species.

VM522P / VM7522 Pharmacology WIMU Fall The therapeutic index is sometimes also referred to as the 'safety factor'. However, again there is no firm definition for this term.

This is not to say that a TI or safety factor does not have any value. If you know how the TI is precisely defined and which species it was determined in it can have value. For example, the TI (as defined as LD50/TD50) for digoxin, a cardiac glycoside used during heart failure is very low in most species (close to 2), which means the dose of digoxin that will cause lethal effects, is only twice the dose that causes therapeutic effects. Therefore, obviously you are going to have to very carefully monitor the concentration of digoxin you are giving.

Other terms you might see: LOAEL = lowest observed adverse effect level (lowest concentration of drug associated with observed adverse effects) NOEL / NOEAL = no observed (adverse) effect level (greatest concentration of drug that causes no detectable adverse effects)

III. LD50

This is the median lethal dose, or the dose that will kill half of the animals of a tested population. This sounds simple as death is all or none. However, again you have to be clear about what lethal refers to. Is it how many animals die within 1 hour, 24 hours, a week or a month of treatment, i.e. does lethality refer to acute or delayed lethality. Normally the dose that will cause issues chronically is lower than that which causes problems acutely. Furthermore, the LD50 is only one number. It is helpful to an extent, but unless you know how steep the response curve is, it can still be hard to know what doses are safe.

100

80 LD 10 60 40 LD 50

% responding 20

0 0.1 1 10 100 1000

100

80 delayed 60 toxicity 40 acute toxicity

% responding 20

0 0.1 1 10 100 1000

dose (g/kg)

The take home message is: if you hear or read about one of these terms for a drug

you are using, be sure you know exactly what it refers to and understand its limitations before you make any interpretations or judgments from it

VM522P / VM7522 Pharmacology WIMU Fall IV. Adverse Reactions

All drugs have the potential to exert an adverse reaction.

Adverse reactions may or may not be related to the principle pharmacological action of the drug, e.g. its target

1. Type 1 or Type A ‐ Dose related toxicities / overdoses.

The characteristics of this type of overdose are that it: i. is generally very predictable ii. will occur in all individuals if the dose of a drug is pushed high enough iii. may occur by dose calculation error iv. may occur because some factor was not considered in determining dose e.g. ‐ age (young or old) ‐ presence of disease state ‐ excessive adiposity or emaciation

The causes of the toxicity are generally due to:

i. Over‐activation (or inactivation) of the normal target. E.g. Therapeutic doses of opioids cause pain relief and potentially sedation. However, an overdose of an opioid leads to coma, respiratory depression and death. All these effects are reversed by the opioid antagonist naloxone and so are due to activation of opioid receptors.

ii. The drug may bind other receptors or targets at higher doses. E.g. dopamine has a high affinity for dopamine receptors and so at lower doses dopamine binds these receptors and stimulates them to cause vasodilation. However, at higher doses dopamine will cause alpha1 adrenergic receptors to be activated as well and can dramatically increase blood pressure due to this alpha 1 activity.

iii. Cytotoxicity or organ toxicity – this is not necessarily directly related to the main therapeutic effect of a drug. However the effect is still dose‐dependent and fairly predictable. It most commonly occurs in organs with high blood flow (and therefore plasma concentrations of a drug), such as the heart, kidney or liver or an organ that metabolizes a drug, such as the liver and kidneys. Examples include: a) Hepatotoxicty: acetaminophen, diazepam (cats), glucocorticoids, phenobarbital, phenytoin and sulfonamides. b) Renal toxicity: Acyclovir, aminoglycocides, ACE inhibitors, NSAIDs, polymixin B, sulfonamides, tetracycline.

VM522P / VM7522 Pharmacology WIMU Fall c) CNS: Opioids, Phenobarbital and antihistamines (older generation) all cause primarily sedation, but potentially excitation; erythromycin, glucocorticoids and lidocaine can all lower seizure thresholds. d) Many drugs also cause toxicity in the eyes, ears and skin.

iv. Pharmacogenetic ‐ some individuals can have unexpected extreme sensitivity to a drug, this is when an adverse reaction is seen within the normal therapeutic range. However, the effects of the drug are still dose‐related, the adverse effects just occur at very low doses. a) often congenital (family / species) b) may involve differences in metabolism (due to altered levels of P450 enzymes) or the expression levels of a target protein (e.g. mutations or alterations in the level of a drug target). c) in many cases the basis is not understood

2. Type 2 or type B – Idiosyncratic, Non‐dose related overdoses are generally unrelated to the principle pharmacological action of the drug. They are: a) generally much more unpredictable and uncommon. They may not be identified until they are reported by prescribing vets after the drug is on the market. b) cause severe effects in sensitive individuals at doses that are easily tolerated by most individuals

The main cause is generally an allergic reaction to the drug

a) As with all allergic reactions a prior exposure necessary ‐ may only require trace exposure ‐ most drugs are small and non‐immunogenic, but immune responses may be triggered by conjugates between drug metabolite and protein component

b) categories of immune reactions

1) Type I ‐ anaphylaxis ‐ release of histamine and other autocoids from mast cells ‐ quick ‐ may be life threatening (anaphylactic shock) ‐ e.g. penicillin is a common culprit

2) Type II ‐ cytolytic effects ‐ antigens form on the surface of cells leading to antibody‐antigen attack on the cell ‐ drug‐induced anemias e.g. sulfonamides, NSAIDs

3) Type III ‐ (serum sickness) ‐ soluble antigens result in activation of immune reaction

VM522P / VM7522 Pharmacology WIMU Fall ‐ released immune mediators then cause damage

4) Type IV ‐ delayed hypersensitivity, cell mediated ‐ may be minor skin rash or may be fatal ‐ contact dermatitis (therapeutic drugs or industrial chemicals)

VM522P / VM7522 Pharmacology WIMU Fall

PART2 – Pharmacokinetics (S. Simasko) L5: Absorption Objectives for this unit: Using drugs with skill and insight is one of the cornerstones of being a successful clinician. Obviously a critical aspect of drug use involves selecting the best drug for a specific clinical case, but another equally important aspect is using a drug in a manner that enables the drug to exert its beneficial action(s) to the maximal extent possible without causing undue harm. As we shall see, the therapeutic benefits of a drug happen when the drug gets to the site that mediates its therapeutic effect at a concentration that is adequate to cause the effect. The same is also true of undesirable effects; they will occur when the concentration of drug exceeds a toxic threshold in the tissues were these effects occur. How are desired concentrations at the desired targets achieved while avoiding the undesirable concentrations at the undesired target? To the extent possible, these goals are achieved by the proper design of drug dosing regimens (a specific preparation used via the best route, at the right amount, and with the right frequency of administration). This is not a trivial problem and much thought goes into the proper design of a drug dosing regimen. In this unit we will examine what those parameters are so that you can better understand how drug dosing regimens can alter the outcome of your therapeutic intervention. Drugs are often given by a prescribed regimen with little thought beyond the prescribed regimen. In routine cases this may be reasonable. However, in some cases drug use is dangerous and involves detailed knowledge beyond following instructions on a label. For you to properly assess these situations, you must know more about drugs than what is found on a package insert. You must understand both how and why the standard therapies were developed. Such knowledge not only helps you to respond to the unexpected in standard usage (failures or toxicities), but will enable you to use drugs in more dangerous or novel situations. Finally, each patient you will see is a unique being. Because of this, the effect of any specific drug has the potential to be unique in each animal. Thus you need to be ready to make the adjustments neces¬sary to optimize individual therapy. But what adjustments are reasonable and prudent? Again, a basic understanding of how drugs are handled by the body will begin to give you the knowledge base to make these adjustments. Thus, there are two broad objectives in this unit: 1. Understand how basic pharmacokinetic processes (absorption, distribution, metabolism, and excretion; or ADME) influence the manner in which drugs are handled by animals. 2. Develop an understanding of pharmacokinetic models so that you can understand the design and rationale behind dosing schedules. Pharmacokinetic Overview:

VM522P / VM7522 Pharmacology WIMU Fall DISTRIBUTION

muscle fat ABSORPTION lung brain kidney heart inhalation placenta mammary EXCRETION etc. expired LUNG air I.V.

free bound KIDNEY urine parenteral PLASMA

feces enteric LIVER GUT (gut) (and other organs)

unabsorbed drug METABOLISM

metabolite PHARMACOKINETICS: ABSORPTION Objectives: 1. Understand the mechanisms by which drug molecules cross biological membranes and be able to explain how these mechanisms result in different absorption characteristics for different drugs depending on the chemical nature of the drug. 2. Be able to explain how the pHs of different fluid compartments in the body affect the transfer of drugs that are organic bases or acids across membranes that separate these compartments. This requires understanding equilibrium of chemical protonation reactions as described by the H‐H equation. 3. Explain why epithelial membranes are difficult for most drugs to cross and why there are exceptions. 4. Know the four general routes by which drugs are administered and be able to explain which factors affect drug absorption via each route. 5. Be able to explain the role of pharmaceutical preparation in the rate of drug absorption and the logic underlying specific examples of different preparation technologies.

Physical Barriers to Absorption: For a drug to exert an action on an internal organ of an animal, it must first get from the outside of the animal to the inside of the animal. To understand how this occurs (i.e., absorption) we must consider how does the drug get across an epithelial membrane of the animal. First, the drug molecule needs to be in a solution that is in contact with an epithelial surface. Then it must cross the epithelial barrier. While there can be paracellular transport, most epithelial membranes are too tight for this to be a significant route for drug absorption, thus we must consider how the drug gets across the cells of the epithelial membrane. A few factors to keep in mind: • Cell membranes are made of a lipid bilayer. This is a very lipophilic environment that hydrophilic molecules in solution do not particularly care to be in. This sets up a quandary – the drug is in solution (and thus must be somewhat hydrophilic), but it must pass through a hydrophobic environment. How does this happen? • There are certain well defined pathways by which normal physiological/nutritive chemicals use to pass membranes, and drug molecules, being chemical agents, are no different. Thus they utilize the same pathways:

VM522P / VM7522 Pharmacology WIMU Fall o Passive or simple diffusion – requires a concentration gradient and a degree of lipid solubility. o Facilitated diffusion – utilizes a transporter but the force for absorption is still the concentration gradient. Because facilitated diffusion uses a specific protein molecule, it also has selectivity (only certain types of molecules that interact with the transporter mechanism will be moved), and it can be saturated. o Active transport – utilizes a protein based transporter (has specificity and is saturable), but can move molecules against a concentration gradient through energy provided by ATP. o Vesicular trafficking – or pinocytosis. Bulk movement of solution across the cell. • If a drug cannot cross a membrane by one of these routes, it cannot get into the animal. If it can’t get in, there is no effect. This is very important to understand – a compound may have huge effects when injected (and thus bypassing a membrane) but when applied to an epithelial surface (gut, skin, etc), it is totally without effect. You must know this property of every drug you give; otherwise you may use the drug in a completely inappropriate manner! • Before we leave this topic it is worth thinking about the difference between epithelial membranes and endothelial membranes (endothelial membranes will become important when we examine distribution within the animal): o In both membranes crossing can occur by diffusion/transporters/vesicles o Epithelia is very tight compared to endothelia. Endothelia has pores between cells of ~65 A in diameter (although this is different in different tissues). This large enough to pass a molecule of ~60,000 MW, or a moderately sized protein. Most drugs are small molecules (<2,000 MW), so they can cross most endothelial membranes.

Factors Affecting the Rate and Extent of Absorption: Given the nature of the barriers described above, with a little thought it becomes apparent that there are three factors that affect the rate and extent of absorption of a specific drug preparation:  The chemical nature of the drug. Since the drug must cross a lipid environment (the lipid bilayer) the lipid solubility (or partition coefficient) is a consideration, as is the ionization state of the drug. Ionization can occur if the drug molecule is an organic acid or base.  The route of administration. Factors that need to be considered are the specific structure of the absorbing epithelial membrane, the cross‐sectional area of the absorbing surface, and the perfusion rate of the absorbing surface.  The availability of the drug. Two factors are important, how fast the drug goes into solution (controlled by the formulation of the drug), and the stability of the drug both in the preparation and once in solution.

Chemical Nature of Drug Molecules: Most drugs are small organic molecules, so our main focus will be on these types of molecules. However, there are significant exceptions, and when using drugs in these other classes you must be aware of their non‐ traditional aspects. For example, some therapeutic agents are peptides or large proteins (insulin, GnRH, growth hormone, monoclonal antibodies). Others may have large extended carbohydrate residues (glycosaminoglycans, hyaluronic acid, heparin). These agents typically are not absorbed when given orally because they cannot readily cross membranes and they are broken down in the gut. Thus they are usually injected, but there can be exceptions as shall be discussed later. Returning to drugs that are small organic compounds, two factors about their chemical properties are important for absorption:  Partition coefficient – determined by the distribution of a molecule between a water environment and a lipid (oil) environment. The higher the number, the more the drug goes into the oil environment. Drugs with high lipid solubility more readily cross membranes, and therefore make good drug candidates, but there is a limit – if the drug is too lipid soluble it won’t easily go into solution, and thus cannot get into solution in the first place. 34

VM522P / VM7522 Pharmacology WIMU Fall  Ionization state – some drugs are organic acids (proton donors) and some are organic bases (proton acceptors). When they are charged they cannot cross the membrane. Whether they are charged or not depends on the pH of their environment according to the Henderson‐Hasselbalch (HH) Equation: [A‐] Organic acids: log ( ) = pH – pKa [HA] [HA] ↔ [A‐] + [H+]

[B] Organic bases: log ( ) = pH ‐ pKa [HB+] [B] + [H+] ↔ [HB+]

A few reminders: How log units work: How pH works: log(X) = Y is the same as 10Y = X pH is the negative log of the H+ concentration. Thus: if Y = 0, then X = 100 = 1, so log(1) = 0 Thus a pH of 7 means the [H+] is 10‐7 M (neutral). 1 if Y = 1, then X = 10 = 10, so log(10) = 1 A pH of 3 means the [H+] is 10‐3 M (a relatively high if Y = 2, then X = 102 = 100, so log(100) = 2 concentration, aka an acidic environment). if Y = ‐1, then X = 10‐1 = 0.1, so log(0.1) = ‐1 A pH of 10 means the [H+] is 10‐10 M (a relatively low concentration, aka an alkaline environment).

The way to think about the pKa: The pKa is a chemical constant for a compound. It defines the pH at which the amount of ionized compound equals the amount un‐ionized: [A‐] log ( ) = pH – pKa [HA]

[A‐] [A‐] if pH = pKa then: log ( ) = 0 and = 1 which means: [A‐] = [HA] [HA] [HA]

Examples of why this is a significant consideration for absorption:

1. Absorption of weak organic base (pKa = 7) versus a weak organic acid (pKa = 7) from stomach (pH = 2; this is a strongly acidic environment).

B B ‐5 Using the HH equation for bases: log ( /HB+) = 2 – 7 = ‐5 so /HB+ = 10 = 0.00001 In other words, the compound is almost all in the HB+ form (less than 0.001% is in the B form) and virtually none of it (99.999%) is in a form that can be absorbed. Thus drugs that are bases (e.g., morphine) are not easily absorbed from the stomach. (Of course the stomach can be avoided by a direct injection.)

A‐ A‐ ‐5 Using the HH equation for acids: log ( /HA) = 2 – 7 = ‐5 so /HA = 10 = 0.00001

VM522P / VM7522 Pharmacology WIMU Fall In other words, the compound is almost all in the HA form (less than 0.001% is in A‐ form) and almost all of it (99.999%) is in a form that can be absorbed. Thus drugs that are weak acids tend to be readily absorbed from the stomach (assuming there is not another issue at hand).

2. Absorption of weak organic acid (pKa = 7) versus a moderate organic acid (pKa = 4) from stomach (pH = 2). The calculation for the weak acid is given above – weak acids are mostly un‐ionized in the stomach and thus relatively easily absorbed from the stomach.

A‐ A‐ ‐2 For the moderately strong acid (pKa of 4): log ( /HA) = 2 – 4 = ‐3 so /HA = 10 = 0.01 Most of the compound (99%) is still un‐ionized, and thus should cross the membrane. Further consideration reveals that only very strong acids (pKa < stomach pH) would be ionized and thus not absorbed from the stomach.

What happens if the drug can withstand the acidic environment of the stomach and make it into the duodenum (pH ~7) without being destroyed? Now things change, the acidic drugs becomes more ionized (lose their H+) whereas basic drugs become less ionized (give up their H+). If a basic drug can withstand the acidic stomach without being destroyed, once it hits the duodenum it will start to be absorbed. Of course this introduces a delay in onset, and the extent of absorption may be variable as the stability of the drug may depend on the acidity of the stomach and the transit time in the stomach. This can be a reason why some drugs are better absorbed with food (promotes acid secretion) whereas others without food.

Final thought on HH eq.: We will come back to compartment pH and drug pKa when we discuss the concept of ion trapping. It helps when thinking about this phenomenon to come up with an intuitive understanding of what is going on rather than relying on calculating things out (which you are unlikely to do much once you pass pharmacology class). Here is how to think about this in an intuitive manner: An acidic environment is one with an excess of H+ (protons). Thus a compound that accepts protons (i.e., a base) will find it easy to find a proton, and become highly ionized (HB+). On the other hand, a compound that donates protons (i.e., an acid) will find it difficult to give up a proton, and is less likely to be charged (HA). The opposite is true for alkaline environments. Acids will be charged in an alkaline environment because protons are scarce and they can easily give one up (A‐), but a base will have difficulty finding a proton, and less likely to be charged (B). Because charged molecules cannot cross a membrane, this can be summarized as:

Acidic environments trap bases, whereas alkaline environments trap acids.

(Actually, the real statement is when the compartment is more acidic than the pKa of the base, the base is trapped; whereas if the compartment is more alkaline than the pKa of the acid, the acid is trapped, but the above statement is simple, easy to remember, and will lead you to the right answer almost all the time.)

Routes of Administration The route of administration can have a large effect on drug absorption. As a clinician, this can be to your advantage or to your disadvantage depending on what you are trying to accomplish. Thus it is important to take into consideration factors that influence absorption when drugs are given by various routes.

 Enteric: which means the drug is absorbed in the small intestines after given orally. The major advantage of this route is it is relatively easy and requires no special equipment (assuming you can get

VM522P / VM7522 Pharmacology WIMU Fall the animal to swallow the pill). However, it is also the route that has the greatest variation and unpredictability. Some factors to consider are: o The presence or absence of food – this can aid or retard absorption. o pH considerations – the stomach is very acidic, the duodenum is neutral (pH 7). Antacids can cause changes in the pH of the stomach compartment and thus the behavior of drug absorption. o Intestinal flora – can alter (metabolize) a drug or can be altered by a drug. o Rate of gastric or intestinal emptying – presence of diarrhea may expel a drug that is normally absorbed in the intestines. o Drug could interact with other compounds present and precipitate from solution. o Relative to other more direct routes of administration, the onset is slow and amount absorbed can be erratic. o Drug may have significant first pass metabolism (this will be examined in more detail later). o Species variation ‐ monogastric animals versus ruminants.

 Inhalation: This route has several significant advantages for rapid uptake. The surface area is large, the blood flow is high, and the epithelial barrier is thin. The difficulty is that placing solutions in the lung is not advisable. Thus it is typically used for gases and aerosols.

 Topical: Although the skin is a very, very tight epithelia, if the drug is lipid soluble enough, it can be absorbed. Thus you need to be careful with some compounds (for example, prostaglandin analogs and pregnant women). Also, agents can be applied to skin (e.g., DMSO) that reduce the barrier function, making absorption of other compounds more likely (e.g., pour‐on anti‐parasitic preparations or fentanyl patches). On the other hand, because it is such a tight barrier, topical agents can usually be applied with little worry they will be absorbed – but watch out if the integrity of the skin has been compromised!

 Mucosal: Numerous mucosal surfaces are available, each with its own set of considerations (blood flow, surface area, epithelial thickness). Some examples: sublingual, nasal septum, ophthalmic, otic, rectum, urethra, uterus, vagina, bladder. A significant factor to keep in mind is that although these routes are often employed to achieve local effects, systemic absorption can be significant, leading to unplanned toxicities.

 Parenteral: (or outside the enteric system). These routes involve an injection across an epithelial barrier and thus avoid all the epithelial worries we have so far considered. A sterile preparation is a must! General routes include: o Intravenous (IV) – the most direct route to the central compartment. 100% delivery, fast, useful for agents that can damage local tissues. o Intraperitoneal (IP) – takes advantage of the large surface area and high blood flow in the peritoneal cavity. Most useful for small animals (rodents). o Intramuscular (IM) – absorption to the central compartment is usually complete and can be relatively rapid unless a depot preparation is used. Watch out for changes in blood flow (i.e., exercise), it can make a normally slow absorption much, much faster. o Subcutaneous (subcut) – usually has slower uptake than IM, but not always. o Other injection sites are used, typically with the goal to confine the drug to a preferred site of action. But remember, systemic absorption will occur and can lead to unplanned toxicities. Some examples: intra‐arterial, spinal cord (epidural and subdural), intrapulmonary, intra‐cardiac, intra‐ articular (intra‐synovial), intra‐medullary (intra‐osseous), intrauterine, intra‐mammary.

Pharmaceutical Factors Affecting Rate of Absorption Whereas the chemical nature of the drug and the physiology of absorbing surfaces are what they are, and cannot be easily manipulated, the formulation of a drug for administration can be manipulated and have a dramatic effect on how a drug will work therapeutically. Formulation will affect the stability of the drug and 37

VM522P / VM7522 Pharmacology WIMU Fall the speed and amount that is absorbed. These factors in turn directly determine the bioavailability of the drug. The FDA takes a keen interest in this topic and requires that formulations for commercial sale be tested and proved capable of delivering the drug before they can be sold (including generics). Unique formulations (not for resale) can be ordered by working with a compounding pharmacist, who has had special training in preparing drugs from bulk chemical form to a preparation suitable for administration.

All formulations must eventually get the drug into solution from which it can be absorbed. Thus we should think about what are the processes involved, and how can pharmaceutical companies manipulate these processes to get the desired release/absorption characteristics.

For all solid drugs the following events must occur in order for the Examples of different formulations in which disaggregation drug to be absorbed (disaggregation and dissolution have been manipulated and the effects this and dissolution can be controlled by has on the rate of release: the formulation): Dosage form to free drug Rate of release

dosage form RATE OF RELEASE DOSAGE FORM

(disaggregation) SLOW sustained release

particulate coated tablet

(dissolution) tablet

solution capsule

(absorption) powder

absorbed aqueous suspension

FAST aqueous solution

Review of some drug delivery systems. Below are some examples of drug delivery systems that have been manipulated to get desired absorption characteristics. As a clinician you will not directly design these systems, but you will select which preparation you want to use. We do not have time to review these in much detail (pharmacists take entire courses on these subjects) but in order for you to use different preparations with the most skill, you should always examine what is the nature of the system used for a particular preparation and whether something associated with the delivery technology may be responsible for some untoward effect you observe – this is the nature of pharmaceuticals – the technology does change and you will need to constantly educate yourself about new and better tools: o Carriers: macromolecules, liposomes, biodegradable beads, resealed erythrocytes o Implants: osmotic pumps, ceramics, tablets o Repository forms: oil, patches, bound (procaine penicillin, benzathine penicillin) o Epinephrine used with local anesthetics to restrict systemic absorption

Bioavailability: We will develop the concept of bioavailability in more depth later, but as absorption of the active molecule has a large influence on bioavailability, it is worth a preliminary mention. When a drug is given IV, 100% of the drug makes it into the central compartment (i.e., the plasma). This is the reference value for 100% bioavailability. When given by other routes (typically oral, hence the term ‘oral bioavailability’ is often used), some drug may not make it into the plasma in active form (for reasons we have discussed above among others). When dosing regimens are designed to achieve particular plasma levels of drug, or when switching from one route of administration to another and plasma levels need to match, one must take into consideration these differences in bioavailability.

VM522P / VM7522 Pharmacology WIMU Fall

L6: Pharmacokinetics: Distribution Objectives 1. Know the relationship between Vd, Cp, and amount of drug given so as to be able to calculate any one of the parameters when given the other two. 2. Explain why different drugs have different Vd, and be able to apply this understanding to make predictions concerning drug distribution. 3. Use the Vd to of a drug to calculate appropriate dose. 4. Be aware of various internal barriers to the distribution of a drug to its target site and be able to explain the relative ease for drug transfer across each of these barriers. 5. Explain the significance of the following issues on drug distribution: entero‐hepatic circulation, tissue perfusion rate, and various clinical states (shock, exercise, etc). 6. Explain the concept of ion trapping and be able to apply this concept to drug absorption and/or distribution.

Volume of Distribution When a drug is given to an animal, and then a plasma sample is taken and a concentration measured, one can calculate a volume that the drug appears to have been put into. Simple logic would tell you that this volume should be somewhat close to the size of the animal, perhaps a little less, as the animal is not all water. However, when this is done for different drugs, the values obtained for this volume range from a small fraction of the size of the animal, to a volume that vastly exceeds the size of the animal. This value is known as the volume of distribution (Vd), and is more properly thought as an apparent volume (calculated) rather than a real volume that exists somewhere in space. However, the Vd has a very significant function as it relates the amount of drug given (dose) to the concentration found in the plasma (Cp), and is one of the fundamental pharmacokinetic factors used for calculating drug dosages.

dose  Vd defined mathematically: Vd = Cp

 Units: dose (mg) divided by concentration (mg/L), will give a final unit of volume (L). However, we are usually interested in this volume as it relates to the size of the animal (kg). Thus Vd is most often given as a normalized value (L/kg) by dividing the volume calculated for a specific animal by the weight of that animal. Alternatively, a normalized dose can be used (mg/kg), and the resulting Vd will automatically be normalized (L/kg).  Examples: a) Drug A (40 mg) is given IV to a 20 kg dog. The plasma concentration (Cp) measured soon after the drug is given is 0.4 mcg/ml. (Note that mcg/ml is a traditional unit for a plasma drug concentration. However, mcg/ml is the same as mg/L since both the top and the bottom terms are different by a factor of 1,000. Thus, so that units will cancel, we can simply write 0.4 mcg/ml as 0.4 mg/L.) 40 mg Then the Vd = / 0.4 mg/L = 1000 L (it appears the drug has been dissolved in 1,000 L) 1000 L For comparisons normalize to the size of the dog: / 20 kg = 50 L/kg

When one considers that a kg of H20 is roughly ~ 1 L how can there be 50 L of volume in a 20 kg dog? b) Drug B (40 mg) is given IV to the same 20 kg dog. The plasma concentration (Cp) soon after administration is 6 mcg/ml (or 6 mg/L). 40 mg Then the Vd = / 6 mg/L = 6.67 L 6.67 L Normalize the value: / 20 kg = 0.33 L/kg How can the volume the drug dissolves in be so different – it is the same dog? 39

VM522P / VM7522 Pharmacology WIMU Fall

Interpretation of a Vd Vd is a low value – drug is trapped in plasma  If the Vd < 0.2 L / kg (Cp is relatively high for the (~5%) amount given and the apparent volume is small) plasma interstitial B-D D+ water (~15%) Possible causes: D o binding to plasma proteins (e.g., warfarin)

o charged molecule ‐ limited penetration into + tissues B-D D D o large protein (e.g., insulin) – limited penetration into

tissues D+ DDD+ Consequences:

o slow/limited access to site of action in tissue B-D B-D (esp. the brain) o drug at site of action may be lower than Cp non-aqueous (~40%) intracellular water (bone, fat – inc brain) (~40%) o if due to binding renal elimination slowed o if due to charge renal elimination enhanced

Vd is a mid value – drug is more evenly  If the Vd is between 0.2 and 0.4 L/kg distributed o drug is distributed into extracellular fluids (curare, gentamicin)

 If the Vd is between 0.4 and 0.8 L/kg o drug is evenly distributed throughout water compartment (ethanol, neostigmine)

 If the Vd > 0.8 L / kg (Cp is relatively low for the amount plasma given and the apparent volume is large; Vd is a high value – (~5%) interstitial e.g., lidocaine, morphine, digoxin, imipramine) drug is concentrated B-D D+ water (~15%) in tissues Possible causes: D o binding sites within tissues + o absorption of lipid soluble molecules into fatty tissues B-D D D (fat, brain)

o ion trapping in non‐plasma compartment + D+ D D D Consequences: o good penetration into tissues (if reason is B-D unrelated to charge, i.e., not ion trapping) B-D o drug reservoir non-aqueous (~40%) intracellular water (bone, fat – inc brain) (~40%) o possible toxic effects in tissue o significant reabsorption in kidney is likely (drug is lipid soluble so likely to cross membranes)

VM522P / VM7522 Pharmacology WIMU Fall Uses of the Vd to calculate doses  The Vd is one of the fundamental pharmacokinetic variables since it informs a clinician how much drug needs to be given to produce a desired plasma concentration (the plasma concentration is used as a handy reference point since for most drugs it is directly related to the magnitude of drug effects, although there are some notable exceptions). Mathematics: dose = Vd x Cp

 We will see in subsequent sections that when giving a constant infusion or with repeated dosing protocols, the calculations are a bit more complex, but for a single dose, this mathematical relationship holds true. One circumstance for which this specific relationship is useful is in calculating a loading dose for an infusion, since the goal of the loading dose in this situation is to load the total volume of the animal to the desired Cp in a single administration.

 An important aspect to consider is that the dose value used in these calculations is the amount of drug that makes it into the plasma, not the amount given. Thus bioavailability must also be taken into consideration: o For IV administration: amount in plasma = amount given o For oral administration: amount in plasma = amount given x oral bioavailability

Barriers to Distribution Once a drug gets into plasma it still needs to cross endothelial membranes to access its site of action. Thus some considerations of these membranes are important to keep in mind:  Crossing may be by diffusion / transporters / vesicle trafficking  Paracellular diffusion via pores between cells is much more prominent than for epithelial membranes. The significance of this route is dependent on the specific structure of the endothelial membrane (listed from tightest to leakiest): o Blood Brain Barrier (BBB) & Blood CSF Barrier – as tight as most epithelial membranes. . Drugs that are not very lipid soluble (i.e., have a low Vd) will not peneratrate into the brain. This is both good and bad, depending on the drug and the therapeutic goal. It is a significant consideration in drug selection and will often be mentioned as a distinguishing feature when drugs within a therapeutic class are discussed. . For many circulating hormones (protein in nature, such as insulin and leptin) specific transporters are present, thus they have access to brain tissues. . There are specific transporters in these membranes that move certain drugs – this is very significant in regard to ivermectin toxicity in collie‐type dogs. . Some parts of the brain (notable ones: arcuate nucleus, area postrema) are outside the tightest elements of the BBB and thus can sample plasma constituents. The vomiting center is located in the area postrema and thus this critical function can detect absorbed toxins without the toxin crossing the BBB.

o Placenta – although somewhat tight, fetuses are exposed to most drugs

o Mammary Gland – significant for milk withdrawal considerations o Kidney

o Liver – very leaky, even relatively large proteins have access to hepatocytes. This is good because drugs have easy access to metabolic processes.

VM522P / VM7522 Pharmacology WIMU Fall Additional Considerations Entero‐Hepatic Circulation  First pass effects: When a drug is absorbed from the gut the first organ it reaches is the liver, which can metabolize the drug. This limits the amount of drug that makes it into the general circulation and in some cases is the major factor in why there is limited oral bioavailability (for some compounds, e.g., natural androgens, there is almost complete first pass metabolism, and virtually none of these compounds make it into the general circulation after oral administration).  Re‐circulation: Many drugs are transformed by enzymatic processes in the liver. When they become conjugated (you will learn more about this in the next section), they sometimes are excreted via the bile. However, once they return to the intestine, the conjugated moiety can be removed by the enzymatic processes of the gut bacteria. This second transformation may return the drug to a form that can be absorbed. This sets up a re‐circulation cycle – essentially the drug is trapped between the intestines and liver. For some therapeutics this is useful, for examples, some benzimidazoles are subjected to this type of re‐circulation which is beneficial when they are used to treat intestinal parasites. On the other hand, sometimes this situation is bad. For example, re‐circulation of several NSAID drugs in dogs (most notably ibuprofen and naproxen), is thought partially responsible for the high incidence of serious GI erosions caused by these compounds in this species. drug breakdown product transformed drug conjugated drug gut liver BILE DUCT

from to heart heart MESENTERIC HEPATIC PORTAL ARTERY VEIN VEIN

Tissue Perfusion Rate  HIGH: CNS, liver, lung, gut, kidney, heart, exercising muscle  LOW: skin, fat, bone, resting muscle  Drugs will enter into areas of high perfusion first, and then redistribute into areas of low perfusion (we will developed this idea further in consideration of two‐compartment models). This is clinically significant in the action of lipophilic short acting injectable anesthetics (thiopental, propofol).

Unique Situations  Shock –circulatory collapse will slow drug elimination Excitement / Exercise – increase blood flow to muscles may cause more rapid absorption of depot preparations of drugs given IM.

VM522P / VM7522 Pharmacology WIMU Fall Ion Trapping In a previous section we discussed how the pH influences the ionization state of a drug. Because different compartments in the body can have different pHs, this also influences the distribution of drugs. The example below uses theoretical compounds with idealized values (to simplify the calculations), but it similar to the distribution of erythromycin (base, pKa = 8.8) versus cloxacillin (acid, pKa = 2.7) in plasma (pH 7.4) versus milk (pH 6.8). The bottom line is the acidic milk compartment will ‘trap’ significantly more erythromycin (a base). If the target for treatment is an infection in the mammary gland, erythromycin will get there much more effectively than cloxacillin.  Example: Distribution of a drug that is an acid with a pKa of 3.5 (similar to cloxacillin) and a drug that is a base with a pKa of 8.5 (similar to erythromycin) between plasma (pH ~7.5) and intra‐ mammary compartment (pH ~6.5):

[B] Basic drug (pKa 8.5): HH Eq for bases: log ( /[HB+]) = pH - pKa

membrane in milk (pH 6.5): in plasma (pH 7.5): [B] [B] log ( /[HB+]) = 6.5 ‐ 8.5 = ‐2 log( /[HB+]) = 7.5 ‐ 8.5 = ‐1

[B] [HB+] [B] [HB+] /[HB+] = 0.01 or /[B] = 100 /[HB+] = 0.01 or / B = 10

+ + + + HB H + B B + H HB 100 1 1 10 Distribution of total B: 101 11 (or 10 : 1 higher in milk than plasma)

[A-] Acidic Drug (pKa = 3.5) HH Eq for acids: log ( /[HA]) = pH - pKa

membrane in milk (pH 6.5): in plasma (pH 7.5): [A‐] [A‐] log ( /[HA]) = 6.5 ‐ 3.5 = 3 log( /[HA]) = 7.5 ‐ 3.5 = 4

[A‐] [A‐] /[HA] = 1,000 /[HA] = 10,000

‐ + + ‐ A + H HA HA H + A 1,000 1 1 10,000 Distribution of total A: 1,001 10,001 (or 10 : 1 higher in plasma than milk)

Note: if a dosing regimen was designed to produce equal plasma concentrations of the two drugs, the relative concentration in milk between the two drugs would differ by 100‐fold!

VM522P / VM7522 Pharmacology WIMU Fall L7: Pharmacokinetics: Metabolism

Objectives: 1. Explain the purpose of biotransformation reactions in organisms. 2. Explain the difference between Phase I and Phase II biotransformation reactions. 3. Be able to list the most important Phase I reactions (microsomal oxidation, non‐microsomal oxidation, esterases). Know the naming convention for the family of genes that code for proteins involved in microsomal oxidation (CYP) and how these enzymes are involved in pharmacokinetic drug interactions. 4. Be able to list the most important Phase II reactions (glucuronidation, acetylation, sulfation, and methylation), and know the chemical nature of the group added for each reaction. Also be aware of some species differences (i.e., cats) regarding glucuronidation. 5. Describe factors that influence biotransformation rates and explain why these effect biotransformation and in which direction. Be able to relate these specific factors to specific clinical situations. 6. Understand the theoretical enzymatic basis for biotransformation reactions and how the maximal velocity (Vmax) and substrate affinity (Km) influence the absolute rate of transformation and whether the transformation process will be 1st order or zero order (i.e., Michaelis‐Menton kinetics). You will also need to appreciate why elimination via a first‐order versus zero‐order process is important.

Biotransformation Every day an animal’s body is bombarded with non‐nutritive compounds absorbed from their diet. As we shall find out in the next lecture (Excretion), if these compounds are extremely lipid soluble, they are extremely difficult to eliminate from the body. To deal with this situation a system has evolved that enables an animal to convert these compounds into more polar chemicals that are easier to excrete. Thus one purpose of biotransformation is to make foreign compounds more polar and thus better substrates for excretion. Therapeutic drugs are obviously subjected to this same process.

However, absorbed compounds sometimes are also not always very reactive. Thus another aspect of biotransformation is to convert compounds into a more reactive chemical, enhancing the ability to make them more polar.

Biotransformation reactions can be divided into two broad categories: synthetic and non‐synthetic. In a synthetic reaction the foreign compound is covalently coupled to another compound, creating a new, larger chemical (hence the reason they are called synthetic). This new chemical almost always looses any biological activity and is an excellent substrate for excretion (this is because the moiety added makes the resulting compound much more polar). Non‐synthetic reactions do not couple chemicals together but rather changes the foreign compound in some manner. The product of these non‐synthetic reactions are also usually more polar, but just as important, they are also more reactive, making them an excellent reactant for a subsequent synthetic reaction.

These reactions are also referred to as Phases. Phase I reaction are non‐synthetic, and Phase II reactions are synthetic. This terminology is used because the products of Phase I reactions are excellent substrates for Phase II reactions. It should be kept in mind that some compounds do not need a Phase I reaction to make them a good substrate for a Phase II reaction, and sometimes the product of a Phase I reaction is polar enough to be excreted without undergoing a Phase II reaction.

One final general principle is that whereas products of Phase II reactions almost always lose all biological activity, products of Phase I reactions may (or may not) retain some biological activity, and in some cases may be more active than the parent compound (the pro‐drug concept).

VM522P / VM7522 Pharmacology WIMU Fall

Types of Biotransformation Reactions

Phase I reactions (or non‐synthetic). These reactions are either oxidations or reductions (a loss or gain of electrons in the parent molecule, respectively), or a hydrolysis, in which the parent molecule is split by the insertion of a water molecule. Some examples are (no need to memorize these examples):  aromatic and aliphatic hydroxylation (R R ‐ OH)  N‐ and O‐dealkylation (R ‐ O ‐ R' R ‐ OH + R' = O) R represents an aryl or alkyl

 deamination (R ‐ CH2 ‐ NH2 R ‐ CHO + NH3) moiety.  desulfuration (R ‐ SH R ‐ OH)  sulfoxide formation (R ‐ S ‐ R' R ‐ SO ‐ R')

Phase II reactions (or synthetic). These reactions are named after the endogenous compound that is coupled to the foreign compound. Important examples are:  glucuronidation  acetylation  methylation  sulfation  conjugation to amino acids (glycine)  conjugation to glutathione (synthesized from cysteine, glutamic acid, and glycine)

VM522P / VM7522 Pharmacology WIMU Fall Phase I Reactions Microsomal oxidation. The most important Phase I reactions are microsomal oxidations (aka: mixed‐ function oxidases). These occur primarily in the liver, but are also found in other tissues. Within the cell the enzymes that mediate these reactions are found primarily on smooth endoplasmic reticulum. When cells are fractionated and various cellular components separated, the smooth endoplasmic reticulum is found in the microsomal fraction, hence the name ‘microsomal oxidation’. These enzymes are now named CYP (for cytochrome P450, cyto‐ for cell, chrome because of their spectrophotometric characteristics, and 450 for the wavelength of light (450 nm) absorbed by the iron molecule in the heme structure that lies at the heart of the reaction mechanism). There are a large number of CYP enzymes (74 gene families based on amino acid sequence). The naming convention is CYP followed by an Arabic numeral indicating gene family, a capital letter designating subfamily, and another Arabic numeral indicating the specific gene. The three main families are CYP1, CYP2, and CYP3. These enzymes not only metabolize foreign compounds, but also are part of endogenous metabolic pathways (of particular importance in steroid and vitamin D synthesis; see example below).

CH3 CH2 - OH C = O C = O OH 21-hydroxylase OH

O O

17-hydroxyprogesterone 11 - deoxycortisol

If you end up going into industry in which new therapeutic compounds are developed, these enzymatic reactions will have a major contribution on how an ideal drug candidate molecule is selected, and thus you will have to develop a much more sophisticated understanding of these processes. Although an in depth understanding is not as important for the average practicing clinician, there are a few important concepts that a practicing clinician should keep in mind in regard to this system:  Any particular subtype of CYP enzyme may be responsible for metabolism of multiple different types of drugs. Some examples are (no need to memorize the details): o CYP1A2 metabolizes caffeine, ondansetron, acetaminophen, theophylline o CYP2C9 metabolizes ibuprofen, phenytoin, tolbutamide, warfarin o CYP3A4/5 metabolizes cyclosporine, erythromycin, lidocaine, nifedipine, losartan Because these drugs share a common metabolic pathway, it is possible that when given together they will interfere with one another’s metabolism. When competing for a degradation pathway, the presence of one drug could prolong the time it takes to eliminate another drug, causing an unexpected drug interaction. Note that the pharmacodynamic action of the drugs may be completely different, so you would never suspect they would have an interaction based how you think the drug acts. This is why it is always helpful to look up potential drug interactions when giving combinations you are not familiar with. Also, it is becoming more common that the specific metabolic enzymes that inactivate a drug are listed on the package insert. This information is for you – it can help you spot a potential interaction before you give a drug.

VM522P / VM7522 Pharmacology WIMU Fall  There can be wide variations in expression level between species and within a species. This is one reason why the pharmacokinetics of a drug can vary considerably between species. Of even greater concern are the significant variations within species (especially in regard to selective isozymes). This is one reason for idiosyncratic reactions to drugs (i.e., a reaction that occurs only in one or a few animals, but not all animals of the same species). This can have serious consequences when the therapeutic window for a drug is narrow. This is also a major driving force behind the idea of pharmacogenetics – or screening individuals for known variants that might involve drug metabolism (variants of pharmacologic targets is the other side of this field).

 The expression of these enzymes can be induced by particular drugs. This causes the metabolism of these drugs to speed up when the drug is given in a prolonged therapy, and can participate in the tolerance to the drug. This is also another basis for a drug interaction, only in this case, because the common enzyme has increased expression, and the second drug has its elimination enhanced, rather than retarded.

 The effectiveness of this system is altered by age – it is slow in both neonates and geriatric populations. Some adjustment to dose may be needed in these situations (note this applies to many drugs but not all, so a blanket rule of decreasing doses for all drugs given to very young or very old animals is not warranted, but is always a consideration).

 The reaction sequence requires co‐factors NADPH (nicotinamide adenine dinucleotide phosphate) and O2. While we all recognize that lack of O2 is incompatible with animal life, nicotinamide is generated from niacin, vitamin B3. Also FAD (flavin adenine dinucleotide) needs riboflavin, or vitamin B2. While memorizing the specific reaction sequence is relatively unimportant, knowing that the reaction sequence may not work effectively in an animal in poor nutrition (and hence could be a basis for an unexpected drug action), is something to keep in mind.

H2O + NADPH FAD SUB-OH P450 P450 NADP+ FADH 2 SUBSTRATE SUB

VM522P / VM7522 Pharmacology WIMU Fall Non‐microsomal oxidation. These reactions are similar to microsomal oxidation but occur in a non‐ microsomal fraction, usually within mitochondria. They occur in many tissues. Of significance to clinical medicine is monoamine oxidase (MAO). From your course in neuroscience in year 1 you should recall that catecholamines (e.g., dopamine, norepinephrine, epinephrine) are monoamines and they are important neurotransmitters and also can function as circulating hormones (and they are used as drugs). Their action is terminated by MAO, and a class of drugs has been developed that target this enzyme (MAO inhibitors). In addition, drug molecules that have similar monoamine structures may also be acted on by this enzyme.

H MAO HO CH C OH HO CH CH2 N H HO OH HO OH O

norepinephrine 3,4-dihydroxymandelic acid

Esterases. These enzymes break ester linkages (double bonded oxygen next to a single bonded oxygen) by a hydrolysis reaction. They are very fast enzymes and they exist in both tissues and in the circulation. Some esterases are very specific in their substrate specificity (e.g., acetylcholine esterase; AChE), and some have a broad substrate specificity. Because these are found everywhere and are fast, drugs with ester linkages do not make very good drugs unless all you want is a very brief duration of action. In addition, because of the importance of AChE in the action of acetylcholine, drugs the inhibit AChE have been developed and are used therapeutically in several situations (you will learn about these drugs later in the semester when you learn about drugs that interact with the autonomic and somatic nervous systems).

CH3 CH CH3 AChE 3 CH + + 3 CH3 - N - CH2 - CH2 - O - C CH3 - N - CH2 - CH2 - OH HO - C O O CH3 CH 3 acetylcholine choline acetic acid

Reductions. These enzymes reduce azo (double bonded N=N) and nitro (N‐O) bonds.

R ‐ N = N ‐ R' R ‐ NH2 + R' ‐ NH2 R ‐ NO2 R ‐ NH2

VM522P / VM7522 Pharmacology WIMU Fall Phase II Reactions One thing to note about all these reactions is the source of the substrate being added (e.g., glucose, amino acids). These are relatively abundant molecules in physiological systems and thus are rarely limiting, except in extreme circumstances (severe malnutrition) which you will undoubted occasionally encounter in practice.

Glucuronidation. This is the most frequent and important Phase II reaction. It involves coupling the drug to glucuronic acid (a glucose derivative). Reactive groups include: O ‐ OH ‐ COOH ‐ NH2 and ‐ SH2 Note these are the types of structures that are found in the products of HO C microsomal oxidation. Glucuronidation also takes place in the O microsomal fraction, so the enzyme is also very conveniently located near where products of microsomal oxidation are produced. OH Glucuronic conjugates are usually rapidly filtered and excreted HO O UDP by the kidneys (note the carboxylic acid moiety (‐COOH) is likely to be OH charged at physiological pH, also making it a substrate for the anion transporter to be discussed later). For compounds with a molecular weight glucuronic acid >500 biliary excretion may be important (this can set‐up entero‐hepatic recirculation). Of particular importance to veterinarians is that cats have an extremely slow rate of glucuronidation. This is of particular importance in the use of NSAIDs that are salicylates (e.g., aspirin).

Acetylation. These reactions involve coupling to an acetyl group (‐COCH3). Dogs are known not to be able to acetylate aromatic amino groups. Another aspect of acetylation is that with sulfonamides it can reduce their water solubility and make them more likely to precipitate (a problem when these metabolites become concentrated in urine).

Methylation. These reactions involve coupling to a methyl group (‐CH3). A well known example is catechol‐ O‐methyl transferase (COMT), an enzyme of critical importance in the metabolism of catecholamines (dopamine, norepinephrine, and epinephrine). An example is given below.

H H COMT CH O CH CH N HO CH CH2 N 3 2 H H HO OH HO OH

norepinephrine normetanephrine

Sulfation. Formation of sulfate esters (‐OSO3). Of passing interest is that some pigs have fast sulfation and others are slow. Amino acids (glycine).

Glutathione. Glutathione is made by joining three amino acids together (note the carboxylic acid residues (‐COOH) that are likely to be charged at physiological pH).

glutamic acid cysteine glycine

VM522P / VM7522 Pharmacology WIMU Fall Rate of Biotransformation Of critical concern to drug therapy is being aware of factors that influence the rate of biotransformation reactions, as differences in rate have the potential to have profound effects on the reaction to a drug, including fatal reactions. Although many of these situations are well known and often accommodations are made for them, often the significance of the effect is difficult to predict. Thus at all times you need to keep these issues in mind and make appropriate adjustments when you think (or know) they may influence the outcome of a drug treatment. Some general considerations are:

Differences between species. We have already discussed glucuronidation in cats, but for almost all drugs, the half‐life for elimination is different from one species to the next. While there may be some general similarities (for example, dopamine has a short half‐life in all species, and DDT has a long half‐life in all species), it is never good to use dosing information from one species for another.

Difference between sexes. Typically males metabolize faster than females, but this is a generalization and does not always hold. Sex steroids (both androgens and estrogens) are known to have influences on the expression of metabolic enzymes in the liver. However, different dosing schedules for males and females are almost never given.

Genetic polymorphisms in the expression and structure of metabolic enzymes. This is one of the most significant current issues in drug metabolism. These polymorphisms are also thought to be important in susceptibility to cancer causing chemicals founds in the environment. In human populations the existence of fast and slow acetylators has been known for decades (e.g., isoniazid, a drug used for tuberculosis). Another well established polymorphism in humans is in CYP2D6, an enzyme responsible for the metabolism of several oft used compounds (e.g., anti‐nausea 5‐HT3 antagonists and tamoxifen among others). How many exist in the world of veterinary medicine is not known, but there is no reason to think they do not exist (polymorphisms in the transporter for ivermectin is well established). This is likely to become an increasingly important aspect of the practice of medicine (including veterinary medicine) so you would be well served by making an effort to understand some basics about drug metabolism so that as these concepts become common, you will be in a position to incorporate them into your practice.

Route of administration. For some drugs first pass metabolism in the liver is so fast as to make oral administration worthless (e.g., testosterone).

VM522P / VM7522 Pharmacology WIMU Fall Factors that change the rate of biotransformation. Approved or suggested drug protocols are almost always determined in animals that are healthy with minimal complications. However, as a clinician, you will be confronted with patients that enter your clinic in various pre‐existing states (if they were healthy, you wouldn’t be prescribing a drug treatment). The wise clinician will always be on the lookout for factors that might change how a drug would behave in the animal, as these factors might account for an unexpected toxicity or therapeutic failure.  Increase rate of biotransformation: o Enzyme induction. As mentioned earlier, CYP enzymes are often induced to higher expression levels by certain substrates. These substrates may be drugs, or could also be compounds the animal is exposed to in their environment. If you give a drug to an animal that has elevated levels of its metabolic pathway, the clearance of the therapeutic agent will be increased and there is a chance for therapeutic failure. Some examples (you should know the bolded ones as they are frequently encountered in veterinary situations): . Drugs: pento‐ and phenobarbital, phenylbutazone . chlorinated hydrocarbon insecticides: DDT, PCB o Cold ambient temperature. A general increase in metabolic rates. This would only affect animals that live outside. o Hormones. . Androgens. These tend to increase rates (recall sex differences above). Another situation to keep in mind is when an animal is on anabolic steroids. . Thyroid hormone. Hyperthyroidism will increase rate as general metabolic rate is increased, but prolonged excessive thyroid hormone will eventually decrease rates as protein is metabolized to cover metabolic demand.

 Decrease rate of biotransformation: o Binding to plasma proteins. When a drug is bound to plasma proteins, it cannot get into cells to be metabolized (this issue only applies to drugs that bind plasma proteins – this information is often given on the package insert). If an animal has elevated levels of plasma albumin, drug metabolism will be slowed (of course the opposite also occurs). In situations, changes in plasma proteins will also alter the ratio of free and bound drug, also causing unexpected effects. o Sequestration in non‐metabolizing tissues. Most importantly, fat. This is a concern in obese patients. For example, it is known that recovery from propofol anesthesia (a very lipid soluble molecule) is prolonged in obese patients because fat serves as a reservoir for drug molecules. o Hepatic disease. It follows that since the liver is the main metabolic site, compromised liver function has the potential to alter drug metabolism. If possible, in such cases it is worth considering an alternative drug that is eliminated by renal excretion. o Nutritional deficits. This was mentioned earlier under microsomal oxidation. o Hormones. Glucocorticoids and progestins are known to decrease expression of some microsomal reactions. o Hypothyroidism / low body temperature. Inverse of effects discussed above. o Enzyme inhibition / competition by another drug or environmental chemical. In these cases metabolism of the drug is retarded. It is also worth noting that some P450 reactions are also involved with metabolism of endogenous compounds (e.g., steroids), and such competition / inhibition could change the rate of these metabolic pathways. o Age. . Neonates: low in both microsomal oxidation and glucuronidation. . Elderly: low in microsomal oxidation.

VM522P / VM7522 Pharmacology WIMU Fall Kinetics of Biotransformation Because metabolism is one of the two mechanisms by which active drug molecules are eliminated, the kinetics of biotransformation reactions have a direct effect on the kinetics of drug elimination, which is an important factor in the design of drug dosing protocols. Thus it is worth taking a brief moment to review some basic ideas surrounding the kinetics of biochemical reactions and thinking about how this might influence drug elimination. Michaelis‐Menton kinetics (MM):  Model

k1 k2 ENZYME + SUBSTRATE ENZYME‐SUBSTRATE ENZYME + PRODUCT

k‐1

 Equation (saturation isotherm). ([Sub] is the 40 substrate concentration, Vmax is the maximum Vmax velocity of the reaction, and Km is the MM binding 30 constant.) (Note: There is no need to memorize this equation.) Vmax 20 rate [Sub] x Vmax 2 Rate = 10 (Km + [Sub] ) Km

0  When enzyme is not saturated the rate is first 0.1 1 10 100 1000 order. [substrate] (nM) o This occurs when [Sub] < Km o In this case (Km + [Sub])  Km (note this is the denominator of the above equation) o Substituting Km for (Km + [Sub]) into the above equation (with slight rearrangements):

Rate = [Sub] x (Vmax / Km)

o Since Vmax and Km are constants, it shows that the rate of the reaction is directly proportional to the substrate concentration. This is what is meant by a first‐order reaction, that is, the rate is dependent on the concentration of one component. (Rem: a second‐order reaction is dependent of the concentration of two components, a third‐order on three, etc.)  When enzyme is saturated the rate is zero order. o This occurs when [Sub] > Km o In this case (Km + [Sub])  [Sub] (note this is the denominator of the above equation)

o Substituting [Sub] for (Km + [Sub]) into the above equation: Rate = Vmax o Since the rate of the reaction is not dependent on any component, the reaction is said to be zero‐ order.

Why is knowing reaction order relevant to anything clinical? In a first‐order process, a constant percentage of substrate is eliminated per unit time, and one can know when half of the compound present is eliminated, and this relationship holds true regardless of the substrate concentration (i.e., there is a constant half‐life for the drug). In a zero order process, a constant amount is eliminated per unit time, and the time to reach half the concentration present depends of the starting concentration. Thus the half‐life is not constant (depends on drug concentration). As we shall see in designing dosing protocols, having a constant half‐life is critical in the design of the protocol, thus drugs eliminated by a first order process are predictable, but drugs eliminated by a zero‐order process are unpredictable. Luckily, most drugs are eliminated by a first‐ order process, but there are notable exceptions (alcohol, high levels of phenylbutazone).

VM522P / VM7522 Pharmacology WIMU Fall L8: Pharmacokinetics: Excretion

Objectives: 1. Know the six routes by which drugs are excreted. 2. Be able to explain the difference between drug removal by filtration vs secretion in the kidney and what kinds of drugs are substrates for each process. 3. Explain why certain drugs are reabsorbed in the kidney, why this is significant to the elimination of the drug, how polarity influences the process, and how and under what circumstances this can be manipulated to enhance drug excretion. Of particular importance is the influence of diuretics on the elimination process via the kidney. 4. Be able to calculate renal clearance and clearance ratio and explain the significance of different ratio values. 5. Explain the extent and limitations of excretion via the GI tract, lung, mammary gland, sweat, and saliva.

Overview of Excretory Mechanisms Once a molecule gets into a body, the body must get it out if it is to maintain a constant internal environ‐ ment. As discussed before, sometimes these chemicals (natural or man‐made) are by themselves a good substrate for excretion, but if they are not, the metabolic machinery in the animal makes them into good substrates (review prior lecture). In this section we shall examine these excretory processes. Major routes for excretion.  Renal  Gastrointestinal (GI)  Lung  Mammary  Sweat  Saliva Renal excretion is the most important, so we will primarily focus on that, but we will examine the other routes briefly.

Renal Excretion In regard to drugs, there are three aspects of kidney function we need to consider, filtration, secretion, and reabsorption:  Filtration. The kidneys filter ~20% of cardiac output. Thus 20% of any component in the blood that is small enough to be filtered in Bowman’s capsule will appear on the luminal side in the forming urine. A few things to keep in mind regarding this process: o Proteins (e.g., albumins) are too large to be filtered. Thus drugs bound to these albumins will not be filtered. If 90% of a drug is bound and 10% is free, just 2% of the total drug coming out of the heart is filtered, as opposed to 20% for an unbound drug. This obviously can have a big impact (10‐ fold) on the rate of excretion. o Filtration is a non‐saturating process. If the concentration of a compound in the plasma doubles, twice as much will be filtered because in both cases the same total fraction of blood flows through the kidney. Thus excretion is always a first‐order process. As discussed above, this is important in the elimination of drugs, because when a drug is eliminated by a first‐order process, the half‐life will be constant.

VM522P / VM7522 Pharmacology WIMU Fall  Secretion. Renal secretion is a process in which compounds in the plasma are taken up by transport proteins in the renal vasculature and then delivered to the luminal (urine) side. A few things to keep in mind about this process:

o Specific transport proteins exist. Of most significance to pharmaco‐ afferent arteriole efferent arteriole kinetics are the organic anion transporter and organic cation transporters. Anion excretion is Bowman’s particularly effective – this is why capsule glucuronide conjugates are such glomerular excellent substrates for excretion. capillary peritubular filtration capillaries o This is an active process. In other words, the energy from ATP is used and substrates can be moved against a reabsorption passive (active transport) concentration gradient. (Rem: as the reabsorption luminal fluid is processed it is shrinking in volume because water is secretion reabsorbed, thus solutes in the luminal (active transport) fluid are becoming more concentrated. renal This causes a force for reabsorption to renal vein develop if the molecule is membrane tubule permeant). to urine o What about drugs bound to albumins? The main concept to keep in mind is everything is in equilibrium, and as free molecules are removed from the plasma to the urine by the transporter proteins, the plasma concentration of drug falls, thereby causing further release of drug molecules bound to carrier proteins, which then are further substrates for secretion. In other words, drug can be ‘stripped’ from the carrier proteins in the blood. How significant this is depends on the affinity of the compound to the various proteins involved (albumins and transporters), the rate that the transporter moves molecules, and the length of time the blood is in contact with the peritubular capillaries.

 Reabsorption. As the luminal contents concentrate it forms a concentration gradient that can drive passive reabsorption, however, a molecule can only be reabsorbed if it can cross a membrane. Thus charged molecules (such as glucuronide conjugates) do not have significant reabsorption, but fat soluble molecules do. This process is very significant to a number of clinical issues discussed below. In addition to passive reabsorption, there are active transport processes (use energy from ATP and are protein based, thus there is selectivity for substrates). These are mostly concerned with nutritive molecules (e.g., glucose) and ions (e.g., Na+ and Ca2+), and thus are usually of minimal concern to pharmacokinetics.

VM522P / VM7522 Pharmacology WIMU Fall Renal Clearance Clearance is an extremely important pharmacokinetic concept. The role of clearance in overall pharmacokinetic function will be more fully developed later, but for now you need to recall a few things about clearance from the physiology course you took last year:

 Clearance is the process by which drugs (and other components) are removed from the plasma. It has units of vol/time (for example, ml/min). One way to think about this is it is the amount of plasma completely cleared of drug per unit time. However, it is important to remember that like the volume of distribution (Vd), this is a theoretical volume and a pool of plasma completely devoid of drug does not exist somewhere in the body.

 Like Vd, reported clearance values are normalized to the size of the animal (ml/min/kg). Clearing 100 ml/min would be astronomical for a Chihuahua, but insignificant in an elephant. Typical values range from 0.2 to 30 ml/min/kg.

 Clearance can be calculated for individual organs and total body clearance is the sum of clearance values from all individual organs. (CLs is the abbreviation used for whole body clearance.)

CLs = CLrenal + CLhepatic + CLlung + ….

 Calculating renal clearance. Renal clearance can be calculating by determining the amount of drug that appears in the urine over a period of time, and dividing by the concentration of drug in the plasma:

drug in urine (mg/min) Renal clearance = = plasma cleared of drug (ml/min) drug in plasma (mg/ml)

o The amount of drug in the urine can be determined by emptying the bladder, allow the bladder to fill over a period of time, then measure the amount of urine that formed and the concentration of drug in the urine: (ml/min x mg/ml = mg/min)

 Clearance ratio (C.R.). The absolute value of renal clearance is not too interesting in and of itself, however, when one compares renal clearance of a specific drug to a compound that one knows how it is handled in the kidneys, one can gain insight into how the kidneys handle the drug. The standard com‐ pound used for these measurements is inulin, a large carbohydrate that is not metabolized and does not cross membranes (it is given IV). Thus the only way it is removed from the body is by renal clearance, and within the kidneys, is only removed by filtration. This makes inulin an excellent marker for the rate of filtration (which is related to the glomerular filtration rate, GFR). Another advantage of inulin is it can have a radioactive tracer added (14C) and a different tracer can be placed on the drug (3H), thus both inulin clearance and drug clearance can be determined at exactly the same time and under exactly the same conditions. From this a simple C.R. is calculated: renal clearance of drug C.R. = renal clearance of inulin

Examination of the C.R. now gives us some insights:

o If C.R. ~ 1 then drug is filtered and not reabsorbed (just like inulin).

o If C.R. > 1 then drug is filtered and secreted (i.e., cleared faster than inulin).

o If C.R. < 1 then drug is filtered and reabsorbed (i.e., cleared slower than inulin). Another possible interpretation of this finding is that the drug may not be filtered; either because it is too big (you would know this if you know your molecule), or is bound to plasma proteins.

VM522P / VM7522 Pharmacology WIMU Fall Effects of Polarity. From the above discussion it should dawn on you that the lipid solubility of a drug (determined by how polar the molecule is) has extremely important consequence on how the drug is handled by the kidney (as it is also important for absorption and distribution). Knowing this chemical aspect of a drug that you use frequently can give you many insights into the behavior of the drug. A first assumption that gives you this type of insight is to know the Vd of your drug. If the Vd is large, the molecule is likely lipid soluble; and if it is small, it is likely the molecule is not very lipid soluble. The following chart also gives us some landmarks in terms of understanding the absolute time involved in eliminating a drug by kidney excretion. Note that this chart only applies to drugs that are not bound to plasma protein.

a drug that is: and it is: its half‐life will be:

polar & a transport substrate filtered and secreted tens of minutes

polar filtered only hours

non‐polar filtered and reabsorbed days

very non‐polar sequestered in fat months

One thing to note is that even under the most optimal conditions, the half‐life of a compound eliminated solely by renal excretion is tens of minutes. One thing this tells you is that if a drug has a much shorter half‐life (<20 min) it must be inactivated by some other process. These can be enzymatic (in the cases of dopamine and acetylcholine) or by redistribution from the brain to the fat (in the case of thiopental).

Ion trapping in the urine. Because drug reabsorption from the lumen of the kidney requires that a drug pass a through a lipid environment, one way to enhance secretion of a compound is to cause it to become charged in the urine. As discussed earlier, the ionization state of organic bases and acids can be manipulated by changing compartment pH. The urine can be acidified by administering compounds that will add acid (H+) equivalents to the system (ammonium chloride: NH4Cl). Urine can be alkalinized by administering compounds that will add base equivalents (sodium bicarbonate: NaHCO3). This type manipulation can also be used to retard absorption (in which a drug also has to cross a membrane) from the stomach – alkalinization will slow absorption of acids and acidification will slow absorption of bases (and vice versa – alkalinization enhances absorption of bases, etc).

The critical elements to consider for an ion trapping manipulation are the acid/base nature of the drug and the desired goal of the manipulation. In other words, which compartment do you want to trap the compound, and how do you trap the compound in that compartment. Remember that earlier we concluded that acidic compartments trap bases, and alkaline compartments trap acids. Thus if you want to trap an acid in the urine, you would need to alkalinize the urine. If you wanted to trap a base, you would need to acidify.

In addition, one must also consider if you can alter the pH of the compartment enough to alter the ionization state significantly (going from 99.99% ionized to 99% ionized is not a big deal, but going from 90% ionized to 10% ionized is), and whether, given other pharmacokinetic considerations, the manipulation is worthwhile. In one of the homework problems we will examine this issue in relation to pentobarbital versus phenobarbital overdose (ion trapping can be used successfully in the latter, but not so much in the former).

Another aspect to consider in regard to ion trapping is the effect of giving antacids (or other situations) in which urine pH (or stomach pH) could change. This could have profound effects on the pharmacokinetics of a drug you are using and may underlie unexpected toxicities or therapeutic failures.

VM522P / VM7522 Pharmacology WIMU Fall Effects of Diuretics. Diuretics are drugs that increase urine formation. Usually, the final common mechanism is by inhibition of ion reabsorption in the Loop of Henle or in the collecting ducts, which results in more ions in the lumen, which in turn pulls water by osmotic effects into the urine. It is often concluded that if one increases urine formation, this must speed up the rate at which drugs are eliminated by renal excretion. This is often tried by individuals who want to avoid detection of violating residues in the urine (such as in horse racing). However, careful consideration of how drugs are handled in the kidney reveals this has little chance of success:

 With the exception of osmotic diuretics, these drugs do not increase GFR and thus they do not increase rate of filtration. Since drug molecules primarily enter the lumen by filtration, these diuretics do not result in more drug molecules entering into the urinary lumen of the kidney.

 However, increased water in the lumen of tubules will decrease the concentration of drug in the lumen of the tubules.

 Since the driving force for passive reabsorption is the concentration gradient, if a drug has significant reabsorption (i.e., is lipid soluble), this decrease in concentration gradient could reduce reabsorption, and could enhance elimination.

 However, for most drugs reabsorption in the kidneys has little impact on their elimination – if renal excretion is the primary means of elimination it is because they are already polar, and if they are inactivated by metabolism, the metabolites are generally very polar with little reabsorption. In these cases diuretic treatment will not accelerate the rate of elimination of either the parent molecule nor detectable metabolites. Drug screens can also measure both components.

 Diuretics will, however, reduce the concentration of drug in urine due to greater urine volume. This is why diuretics are often called for when dealing with drugs that have potential renal toxicity (e.g., cisplatin) – it is not because the manipulation enhances elimination, but rather because it prevents toxic concentrations in the bladder.

 Another exception alluded to above is to use osmotic diuretics (e.g., mannitol). These compounds do increase GFR and thus will cause more drug molecules to be filtered. However, if reabsorption is significant (the drug is lipid soluble), the drug will still be passively reabsorbed.

VM522P / VM7522 Pharmacology WIMU Fall Other Pathways for Excretion. Renal excretion is important for two reasons, it is the primary route that absorbed molecules are eliminated from the body, and, along with metabolism, it is one of the primary mechanisms by which the activity of a drug can be terminated (this is going to be the important element when considering dosing regimens). However, drug molecules are also excreted by other pathways and there are a few considerations that a clinician should keep in mind in regards to these pathways:

 GI Excretion o Significant for poorly absorbed molecules. This can sometimes be an issue for oral insecticides or antibiotics – active drug will show up in the feces in concentrations that maybe toxic to organisms that would normally breakdown the waste product (the poop will not disappear!). o Drugs can enter into GI tract from blood. In this pH can be important, i.e., ion trapping. o Some drugs enter the GI tract via biliary excretion (conjugated molecules) or salivary excretion. Conjugated molecules can undergo bond breakdown in the intestine, regenerating the parent molecule, which could be reabsorbed. This is what causes entero‐hepatic recirculation.

 Excretion via the Lungs o This route is important for gases (i.e., general anesthetics). o It is dependent on the relative tissue / blood/ alveoli equilibrium. o Because this is so important to the use of anesthetic gases, it is a topic that will be covered in much detail in your Anesthesiology course.

 Excretion into the Mammary Gland o Remember that milk has lower pH than plasma (pH ~6.8 vs. ~7.4). Thus bases will enter into milk better than acids. o Mammary excretion is significant in dairy cows for establishing withdrawal times. o Also remember that often drugs are given intra‐mammary to deal with mastitis and reduce systemic absorption, there can be absorption from the mammary gland back into the general circulation. o May also be significant for neonatal exposure if nursing mothers are given drugs. This can be bad, but is also a method to treat suckling animals without having to handle the babies.

 Sweat o This is not a significant elimination pathway but is of more concern if the animal is treated by a chemical of potential toxicity that would appear in the sweat.

 Saliva o Also not a major pathway for elimination (besides, most saliva is swallowed so drugs in saliva are recirculated), but like sweat can be of concern as a route for unintended exposure (e.g., a drooling St. Bernard and a chemotherapeutic agent).

VM522P / VM7522 Pharmacology WIMU Fall L9: Pharmacokinetic Models

Objectives: 1. Be able to describe a one‐compartment open pharmacokinetic model, what assumptions are made in this model, and what the various terms used in the model (Cp and Ke) represent. 2. Explain the difference between zero order kinetics and first order kinetics and be able to describe how these kinetic processes relate to the concept of half‐life. 3. Be able to use the concept of half‐life to make predictions about future plasma concentrations of a drug. 4. Be able to describe a two‐compartment open pharmacokinetic model, what assumptions are made in this model, and what the various terms used in the model (Cp, Ct, k12, k21, and Ke) represent. 5. Be able to explain the significance of the  phase and  phase in a two‐compartment model. 6. Understand the insights gained into plasma and tissue concentrations of a drug given by a two‐compartment model. 7. Be aware of the shortcomings of the two‐compartment model vs. real organisms (multiple half‐lives), but also the usefulness of the simplifying assumptions made in the two compartment model. 8. Understand the concept of bioavailability and be able to use knowledge of oral bioavailability to correct for incomplete drug absorption. 9. Be able to explain how manipulating absorption rate can influence drug exposure (therapeutic and/or toxic) and how different absorption kinetics can greatly influence dosing schedules for a drug.

Models: Before we get into the nitty‐gritty of designing dosing regimens, we will take a brief detour (1 to 1 ½ lectures) to examine some theoretical issues through a discussion of one and two compartment pharmacokinetic models. While you may find this topic a bit abstract in regards to giving a drug to an animal, these models enable us to define and label some basic processes. Once we have developed a good conceptual understanding of these processes, it will be easier to talk about them in a precise manner in the future. Further, examining where reality and models diverge enables us to appreciate in more detail what is likely to be going on in the animal. Thus keep in mind that the goal of the material in the next few pages is not to make you budding pharmacokinetists, but is to define fundamental processes. For these purposes the mathematics are relatively unimportant as long as you understand the starting point and the bottom line.

A. One‐Compartment Open Model

Ke plasma IV dose elimination (Cp)

 It is one compartment because only the plasma compartment is considered.  It assumes that when the drug is given it instantaneously distributes throughout the compartment.  It is an open model because once drug is eliminated it is gone from the system.  Ke is the elimination rate constant. This is the sum of all elimination mechanisms (i.e., all mechanisms that excrete active drug and all metabolism that inactivates the active drug).

VM522P / VM7522 Pharmacology WIMU Fall What is an elimination rate constant (Ke) and how does it relate to the rate of elimination? o An elimination rate constant has a fractional value (< 1) and units of inverse time (min‐1). For example:

Ke = 0.03 min‐1. What this says is the 3/100 (or 3%) of whatever is present is eliminated in 1 min.

o To calculate how much is eliminated (or rate of elimination), the amount present is multiplied by the elimination rate constant:

(if 10 mg is present) x (0.03 eliminated/min) = 0.3 mg eliminated in 1 min.

o However, if less is present (say 5 mg), then less is eliminated:

(if 5 mg is present) x 0.03/min = 0.15 mg is eliminated in 1 min

o Under these conditions the fraction eliminated is constant, and the absolute amount eliminated is dependent on how much is present. Because of this we say the elimination process is first order.

o Graphing a first order elimination is shown to the right. The resulting change in plasma Cp is a curved line that decays toward zero. This decay is said to be ‘exponential’ because it can be fit with an exponential function.

o Note that we can also consider how long it takes to eliminate half of what is 100 present. Because the fraction eliminated is constant, the half‐life will be con‐ stant, regardless of the initial 80 concentration.

o Most drugs are eliminated by a first 60 order process (which is good because it means the half‐life is constant – we shall Cp see how important this is in the near 40 future). It should also be noted that the concept of 1st order kinetics doesn’t just apply to drugs, many biological 20 parameters change according to 1st order kinetics. It is applicable to anything that changes as a percentage of what is present. This could apply to physiology 0 (e.g., turnover of platelets), or to ecology 0 20406080 (growth of populations), or even your retirement account. t1/2 time

VM522P / VM7522 Pharmacology WIMU Fall  What if the amount eliminated is a constant amount, not a constant fraction?

o In this case the rate of elimination is a constant value independent of how much is present. For example, 1.3 mg is eliminated per minute.

o In such cases the rate of decay would be linear (see graph to the right). But the 100 half‐life varies – it is dependent on the starting concentration. In this case we 80 say the decay is zero order.

o This type of elimination occurs when Cp the elimination process is saturated 40 (recall MM kinetics from before). Thankfully, in regard to drugs this only occurs in a few situations (typically low 20 potency drugs given in high concentrations) but they are notable: alcohol (in all species), phenylbutazone 0 (in human, horse, dog), salicylates 0 20406080 (aspirin; in cats and humans). t1/2 time

VM522P / VM7522 Pharmacology WIMU Fall  Relationship between the elimination rate constant and half‐life. o Mathematical models of 1st order elimination:

Cpt = plasma concentration at time t Cpt ln ( ) = -Ke x t Cpo = plasma concentration at time 0 Cpo Ke = elimination rate constant t = time since injection (Note: ln is the natural logarithm, logs are the inverse of an exponential, thus this equation models an exponential decay )

o The definition of a half‐life (t1/2) is when the plasma concentration is ½ the original concentration,

or when Cpt = 0.5 x Cpo

0.5 Cpo substituting into the above equation: ln ( ) = -Ke x t1/2 Cpo

0.5 Cpo solve the left side of equation: ln ( ) = ln (0.5) = -0.693 Cpo

thus -0.693 = -Ke x t1/2 or t1/2 = 0.693 / Ke

o In other words, the half‐life is inversely proportional to the elimination rate constant with a factor of 0.693 (the natural log of 0.5) thrown in. We will see this value (0.693) pop‐up in subsequent equations, and this is where it comes from. Beyond that, there is no need to get concerned about the mathematics involved.

o What is the t1/2 good for? Knowing the half‐life enables one to easily predict future plasma levels of drug: . One could use the equation given above and do a precise, but somewhat awkward calculation.

. On the other hand, by using the t1/2 one can do the calculation in their head. For example, in one t1/2 the Cp is half the original value. In another t1/2 it is half the second value, and so on…

. Using specific numbers: if at time = 0 the Cp is 24 mcg/ml, in one t1/2 it will be 12 mcg/ml, in another t1/2 it will be 6 mcg/ml, in another t1/2 it will be 3 mcg/ml, etc… . We will use this concept in the design of dosing regimens, so make sure you understand the concept – you just need to divide by 2 on each half‐life.

. This does require that the drug have a constant t1/2, and thus only applies to drugs eliminated by a 1st order process.

VM522P / VM7522 Pharmacology WIMU Fall B. Two‐Compartment Open Model Even the briefest of physiological considerations leads one to recognize that a one‐compartment model is not a very complete model of a real animal (although it is useful for giving us the relationship between half‐life and the elimination rate constant). In a real animal there are multiple separate compartments that are in contact with the plasma. The next simplest model that can deal with some of these complexities is a two‐compartment open model:

compartment 1 compartment 2

k12 tissue IV dose plasma (Cp) (Ct)

k 21 Ke

elimination

 Assumptions and definitions in the two‐compartment model:

o k12 and k21 represent the rate constants for drug moving from the central compartment (Cp, or plasma) to the peripheral compartment or tissue (Ct), and vice versa, respectively. Other parameters are as defined in the one‐compartment model. o The IV dose is given and assumed to instantaneously distribute throughout the central compartment, but redistribution to the peripheral compartment takes time. o Sampling is from the central compartment.

 Mathematic model of two‐compartment kinetics:

dCp = -(Cp x k12) - (Cp x Ke) + (Ct x k21) dt

What this equation says is the change in Cp with time (dCp / dt) is equal to the loss of drug moving from the central compartment to the peripheral compartment (note the negative sign: ‐(Cp x k12)); plus the loss of drug via elimination (note negative sign: ‐(Cp x Ke)); plus the movement of drug from the peripheral compartment to the central compartment: + (Ct x k21).

 This equation can be solved precisely by use of calculus. The mathematics are complicated, but not important for our purposes. The important facet is the resulting equation is comprised of two exponential components: Note: there is no need (- x t) (- x t) where: A + B = Cp k = A x  + B x  to know these relation‐ Cpt = A x e + B x e o 21 ships; they are given just A + B  x  to show you how the Ke = k12 =  +  - k21 - Ke starting and ending k 21 equations are related.

VM522P / VM7522 Pharmacology WIMU Fall  Graphs of changes in Cp with time have two components showing exponential decays:

logarithmic scale linear scale A + B = Cpo

64 dog 60 dog -phase 32

40 -phase 16 -phase  = 0.00407 min-1 8 20 -phase 4  = 0.0559 min-1 [erythromycin] (ug/ml)[erythromycin] [erythromycin] (ug/ml) 0 2 Cp Cp Cp 0246 A B 0246 time (hr) time (hr)

These graphs are from a study in which erythromycin was given IV to a dog. The points are actual data points collected, and the lines are fitted to the data with the rate constants indicated. The fact that the experimental results fit so well to the theoretical results suggest that a two‐compartment model is a reasonable approximation of the natural system.

 The important part: Interpretation of the two‐compartment model o The ‐phase is the "distribution" phase: immediately after introducing the drug to the central compartment there is a period of time in which the movement of the drug is dominated by movement from the plasma into the tissue. o The ‐phase is the "elimination" phase: once the tissues (slowly perfused compartments) are filled with drug, the movement of drug is dominated by eliminating the drug through the processes represented by the elimination rate constant Ke (this can be either renal excretion of active drug or metabolism of active drug to an inactive metabolite).

 Clinical significance of two‐compartment kinetics. The most significant point is that when the drug is rapidly introduced into the plasma, distribution does not happen instantaneously, but takes time. This has several clinical consequences that you should keep in mind: o When a drug is introduced by an IV injection, it should not be given rapidly unless it is a very safe drug. The levels observed in the ‐phase are the levels that are targeted by drug dosing regimens, as the ‐phase is a short temporary phase. However, also note in the example above that during the ‐phase, plasma levels can be greater than 4 times the peak of the ‐phase (64 mcg/ml vs 15 mcg/ml). Is the drug you are giving safe at these higher levels? If not, the injection should be given slowly to allow time for the drug to distribute and thus avoid these high ‐phase levels. Some clinical examples: . An excellent example is cancer chemotherapeutic agents. These agents are dosed very close to maximal tolerated levels. Thus these drugs are often given by a slow IV infusion over a period of a few hours to ensure that unsafe ‐phase levels are not achieved. . Another less drastic example is tetracyclines. Tetracyclines can chelate calcium and if given as an IV bolus can cause cardiac abnormalities because the high ‐phase levels produced by an IV bolus can bind up enough calcium to cause ionic disturbances in the heart. Thus if given via an IV route, the administration should be by a slow IV push.

VM522P / VM7522 Pharmacology WIMU Fall  Clinical significance of two‐compartment kinetics (continued).

o How long do ‐phases usually last? Although this will depend on the chemical nature of a specific drug and the circulatory status of the specific animal, but as a general rule the ‐phase lasts 20‐60 minutes.

o What about non‐IV administration? One of the advantages with non‐IV administration (such as oral or IM) is that the absorption of the drug is delayed. This delayed absorption can often closely match the kinetics of the ‐phase. Thus high plasma levels of drug are avoided because the drug distributes into tissue on approximately the same time scale as it is absorbed. In these cases you do not need to worry about the distribution phase. We will address other aspects of the kinetics of absorption in more detail below.

o Special consideration with short‐acting lipid soluble anesthetics. Short acting injectable anesthetics such as thiopental are very lipid soluble. The reason they are short acting (10‐20 min) is not because they are rapidly metabolized (also note this is too fast for renal excretion), but because they redistribute from the brain (very lipid rich with a high perfusion rate) into fat depots (very lipid rich with a slow redistribution). Thus the ‘therapeutic’ effect of the drug (anesthesia) actually occurs during the ‐phase, rather than the ‐phase, and therapeutic action is terminated by redistribution.

o What happens if you give repeated doses of thiopental? With repeated dosing the plasma levels achieved during the ‐phase approach anesthetic levels. Now action is prolonged because the drug has nowhere to redistribute to. Because the drug now needs to be metabolized to be inactivated, the anesthetic action can last hours rather than 10‐20 min.

VM522P / VM7522 Pharmacology WIMU Fall

C. Multi‐Compartment Models

tissue 1

k 12 k oral Kabs 21 dose gut plasma k13 k 31 k tissue 2 4 Kebile Kefeces bile

Ke Keurine metabolism

Further physiological considerations (illustrated above) lead one to conclude that even a two‐compartment model is likely to be an over simplification of the real system. However, often the two compartment model is a good approximation (see the erythromycin example above). Although a simplification, the two‐compartment model helps us to understand the plasma profile and makes us aware of the distribution phase that must be taken into account in certain clinical situations. However, one place the two‐compartment model really fails is with very lipid soluble drugs that become concentrated in fat (or other situations in which the drug may form a depot that slowly releases drug into the system). In these cases ‘therapeutic’ levels may fall with a reasonable kinetic rate (hours to days), but there is another component in the kinetics, a very slow terminal half‐ life, that produces plasma levels below that needed to produce significant pharmacological effects, but not below the levels that can be detected. These slow terminal half‐lives can be a significant complication when it comes to establishing withdrawal times and avoiding drug residues in tissues, a very important veterinary issue if you are either involved in regulatory affairs, or on the other side, trying to avoid violating residues.

VM522P / VM7522 Pharmacology WIMU Fall Manipulating the Rate of Absorption ‐ Effects on Cp and Duration of Action

A brief reflection on absorption reveals two (In this graph the very interesting aspects: elimination rate  Changing the rate of absorption is likely constant was dose Ka (rate of absorption) to have profound effects on the plasma profile held constant so A 100 0.1 min-1 the half-life is of a drug. B 200 0.1 min-1 the same in all -1 C 200 0.02 min cases.)  Absorption is one of the processes that -1 can be manipulated by the pharma‐ D 400 0.005 min ceutical preparation. 120 B

toxic effects 80

Shown to the right is a graph of a situa‐ Cp tion in which the rate of absorption was manipulated: 40 D C M.E.C. A

0 02468 time (hr)

 A is the standard situation. The drug remains below toxic levels but only remains therapeutic (above minimum effect concentration (M.E.C)) for a fairly short period of time (~1.7 hr). If the MEC needed to be maintained for 24 hours, one would have to come in and re‐administer the drug ~every 2 hours.

 B shows what happens when twice as much drug is given. Note that plasma levels are twice as high at every point compared to A. Also note that toxic levels are encountered, but duration was not doubled; now it is ~2.1 hr) – this is what happens in a 1st order process (higher Cp causes the absolute amount of drug to be eliminated faster. Thus if MEC is needed for 24 hours, not only does the drug need to be injected ~every 2 hours, but repeated toxic exposures occur. Thus doubling a dose is NOT an effective way to double the duration of drug action!

 C shows what happens when twice as much drug is given and absorption is slowed. Therapeutic levels are maintained for a longer period of time and toxic levels are avoided. Now the drug might need to be given ~every 3 hours, a slight improvement over situation A.

 D shows a more extreme case, even more drug is given and absorption is slowed even more. We have developed a sustained release formulation that enables therapeutic levels to be maintained for a reasonable period of time (~6 hr; 4x a day dosing) with no toxicity.

 Thus the rate of absorption is the parameter that pharmaceutical manufacturers manipulate to optimize their preparations. Remember to think about this when you use preparations from a compounding pharmacist (this requires a special license that with additional training a pharmacist can get) or other source (natural preparations or home‐made concoctions) in which the specific preparation has not been tested – you really have very little idea of what the absorption characteristics are. Does the active compound get absorbed at a rate fast enough to produce therapeutic levels? Does the active compound get absorbed so fast as to produce toxic level? How long does the preparation continue to release drug and thus maintain therapeutic levels? This is the reason why the FDA is very persnickety about ‘compounding’ (or the preparation of drug in its final form for use), especially if you compound a preparation and then sell it. Even mixing of two preparations from commercial sources so you can inject them in one shot is considered compounding, and is illegal in the eyes of the FDA.

VM522P / VM7522 Pharmacology WIMU Fall L10‐12: Pharmacokinetic Variables

Unit objectives: We will now turn our attention from the theoretical to the practical – actual design of dosing regimens. This will require that we first review and develop a slightly more sophisticated understanding of fundamental pharmacokinetic parameters. After this we can delve into dosing regimens in detail.

Prior topics have taught you most of what you need to consider when giving a single dose of drug in safe and efficient manner. But often drug therapy is a continuous process (drug given by a constant infusion or by periodic administration of a set dose, aka, intermittent dosing), and additional considerations are needed. Although in practice you will find that the mechanics of these regimens are almost always provided to you, by examining how these protocols are developed from the ground up, you will develop a better appreciation for how the elements of the regimen are translated into changes in plasma levels. From this understanding you will be in a better position to make judgments and changes when you are using a drug in a clinical situation. In addition, some of you may eventually find yourself in the position of having to design and test novel regimens. In these situations you will need a fundamental knowledge of pharmacokinetics to help design optimal regimens. Our overall objectives for the next series of lectures are to:

1. Develop an understanding of the three core pharmacokinetic variables: volume of distribution (Vd), clearance (CLs), and bioavailability (F). This includes what these variables represent, how they are determined, and what impact they have on dosing regimens. You will also need to understand how t1/2 relates to these variables.

2. Know the relationship between CLs and half‐life and Vd so that you can calculate any one of the terms given the other two, and why changes in CLs and/or Vd can alter the half‐life of a drug (or toxicant).

3. Use the knowledge we have developed about pharmacokinetics to understand how dosing regimens are designed. From this you should be able to understand what parameters can be manipulated (amount and frequency) to achieve desired outcomes.

Overview

Clearance (CLs), along with volume of distribution (Vd) and bioavailability (F) comprise the three pri‐ mary pharmacokinetic parameters used in designing drug dosing regimens. CLs indicates how fast drug is removed from the body, Vd indicates the volume that needs to be filled if a drug is to reach therapeutic concentrations, and bioavailability indicates how much of the administered drug gets into the central compartment (plasma). Together, these parameters dictate how much (Vd and F) and how often (CLs) a drug should be given to achieve a desired plasma concentration profile. This assumes, of course, that the plasma profile is relevant to the therapeutic effect of the drug. This is often the case, especially in drugs that need to be dosed carefully because the margin for error is small. However, there are some cases in which the plasma concentration is not particularly useful; for example, if a drug has a delayed effect (if it influences gene regulation) or active metabolites are generated. It is important that you recognize these situations by always knowing how the drugs you use work, and thus you can apply your knowledge in an appropriate manner.

VM522P / VM7522 Pharmacology WIMU Fall a) Clearance Compounds (drugs) are removed from the plasma by a process known as clearance. In this process the plasma in contact with an organ loses drug through some process ongoing in the organ (metabolism or excretion). The amount removed will be a product of the amount extracted from the blood as it courses through the organ multiplied by the blood flow through the organ. An example that readily illustrates this process is hepatic clearance:  Calculated by examining the concentration of to vena cava drug in arterial blood entering the liver (CA) and the concentration of drug in the venous blood Schematic leaving the liver (CV) and taking into of liver central consideration blood flow (Q): lobule hepatic vein C L hepatic = Q ( (CA ‐ CV) / CA) liver space of Disse  cells (CA ‐CV) / CA is known as the extraction ratio Kupffer cells o Example: If CA is 20 mcg/ml and CV is 15 mcg/ml then the extraction ratio = sinusoids (20 – 15) / 20 = 0.25 (or 25% of what is lymphatic present is removed). vessel

portal vein  Further considerations reveal that hepatic (from gut) clearance (or clearance from any organ) can hepatic artery be altered by one of two means: bile duct

o Either the organ becomes less (or more) effective at extracting the drug from the blood (disease and enzyme inhibition cause decreases whereas enzyme induction causes increases). o Or blood flow to the organ increases or decreases.

More about Clearance:

 The above discussion can apply to clearance by any organ (CLorgan = Qorgan x extraction ratio)

 The kidney is a special case, in which CLrenal can be calculated by determining the amount of drug that shows up in the urine (since this is the drug extracted from the plasma ‐ see earlier lecture for details).

 Total body clearance (CLs) is the sum of the clearance from all organs:

CLs = CLhepatic + CLrenal + CLlung + ….

 Alternatively, if you can estimate the total absolute rate of elimination (in mg/min), you can calculate total body clearance directly:

CLS = rate of elimination (mg/min) / concentration in plasma (mg/ml)

or CLs = [(amt of drug present) x Ke (elimination rate constant)] / Cp)

 From the above equation it can be seen that clearance has units of vol/time or ml/min. This value represents the volume of plasma that would be completely cleared of drug in the unit of time.

Note: Reference values are normalized to the size of the animal: ml/min/kg.

VM522P / VM7522 Pharmacology WIMU Fall b) Volume of Distribution  The concentration of a drug is a critical factor for its action. The concentration (mg/ml) will depend on how amount of drug placed into a particular volume. While it seems reasonable that an animal of a certain size would have a specific volume, observations have shown this is not the case. In an earlier lecture we developed the concept of Vd and discussed why different drugs could appear to be dissolved into different volumes even within the same animal. Nevertheless, if the goal of a dosing regimen is to achieve and maintain a particular concentration of drug, we need to know that volume in order to calculate an appropriate amount of drug. This is determined by using a reference values for Vd (given in L/kg; found in compendia of drugs) and multiplying the value by the size of the animal (kg). From this an amount of drug can be determined. For example: dose = Cp x Vd

 Alternatively, the Cp produced by giving a particular amount of drug can be calculated by rearranging the above equation: Cp = dose / Vd

 An aspect of Vd that frequently confuses students when they first start to use Vd in calcula‐ tions is that sometimes the value is multiplied by the weight of the animal, and sometimes it is not. This is because the reference value is normalized, but when you use Vd in a calculation for a specific animal, you need to know the total absolute volume: o For example, if you give 20 mg of a drug that has a volume of distribution of 0.5 L/kg to a 10 kg dog, what concentration of drug would be produced: Cp = dose / Vd = (20 mg) / (0.5 L/kg x 10 kg) = 4 mg/L Note: by multiplying the reference (or normalized) value by the weight of the animal, the specific volume (5 L) that the drug (20 mg) was placed was determined. o At other times the dose, instead of an absolute amount, may also be normalized (in the case above 20 mg/10 kg = 2 mg/kg). If this normalized dose (2 mg/kg) is used with a normalized Vd (0.5 L/kg), then the weight of the animal is not used: Cp = dose / Vd = 2 mg/kg / 0.5 L/kg = 4 mg/L Note: this is because the weight of the animal was considered in normalizing the dose.

o How do you keep this straight? . One approach is to always multiple normalized values by the size of the animal. This will always get you the right answer, but you will do a lot of extra multiplications (not that this is wrong). From the above example: Cp = dose / Vd = (2 mg/kg x 10 kg) / (0.5 L/kg x 10 kg) = 20 mg / 5 L = 4 mg/L . A second approach is to recognize from the get go that you are dealing with two normalized values, so animal weight is not needed, or to recognize that only one normalized value is used, so you do need to correct for the size of the animal. This is not always obvious when you start a calculation, especially when you are new to these calculations. . A final approach is to be very careful when you write out your units, and always make sure your units cancel correctly in the final answer. If you have a final answer with a kg as a unit and you don’t want it that way, you need to multiply (or divide) by the weight of the animal to remove the unwanted kg. For example, from the original calculation above: Cp = dose / Vd = 20 mg / (0.5 L/kg) = 40 mg ∙ kg / L ÷ 10 kg = 4 mg/L Note the first answer (mg ∙ kg) / L is not a concentration because of the kg term.

VM522P / VM7522 Pharmacology WIMU Fall c) Bioavailability Bioavailability is the fraction (F) of the drug given that is absorbed into the systemic circulation. By definition, when the drug is given IV, all of it makes it into the systemic circulation. But how does one know how much of an oral drug actually gets into the systemic circulation, especially since we know that the absorption characteristics are different and the resulting plasma profile will be different? Since the removal process is 1st order and proportional to the concentration of drug in the system, the integrated area under the curve (AUC) is the same regardless of the speed at which the drug was absorbed. This area can be determined by giving a drug and then frequently sampling the Cp over time, and plotting the resulting data. The AUC could be determined by integral calculus, brute numeric calculations, or, in the old days, cutting out the area and weighing the scrap of graph paper on a balance! One does this for the IV dose and then the oral dose, and then calculates the fraction absorbed into the general circulation (which becomes the bioavailability):

 The left hand graph shows the shape of the curves when 100% of the drug is absorbed orally (F = 1). Note at the beginning Cp from the IV dose > Cp for the oral dose, but later the Cp for the oral dose > Cp for the IV dose. These two areas exactly balance, thus all the drug made it into the systemic circulation.

 The right hand graph shows the shape of the drug plasma concentration curve when only 50% of the oral dose was absorbed (F = 0.5). Now the areas under the curves are substantially different – not all the drug given orally was absorbed and the bioavailability is <1 (in this example the value was set to 0.5).

AUC oral Bioavailability = F = AUC I.V.

100 100 F = 1 F = 0.5 80 80 I.V. 60 60 I.V.

Cp 40 40 oral 20 20 oral 0 0 0123 0123 time (hr) time (hr)

VM522P / VM7522 Pharmacology WIMU Fall  Most students do not have difficulty grasping the essence of the concept of bioavailability, but what frequently confuses students is when to use bioavailability to correct a dose, and when to not used it. o When you are told to give some amount of drug orally (say 5 mg/kg), either by a supervisor, or from a package insert, or from a compendia of drug doses, or in a problem set, you give the recommended amount. This value was determined in previous studies and found to be effective. No one would give you a dose and then expect you to somewhere find the oral bioavailability and make the correction! That is a recipe for disaster. o If you are asked to calculate a resulting plasma concentration from a dose of drug given orally, then you must correct for the fact that not all the drug made it into the systemic circulation. If you gave 10 mg by an oral route, but if oral bioavailability is 80% (i.e., F = 0.8), then only 8 mg made it into the systemic circulation system. The value used to calculate Cp is the amount that made it into the systemic circulation (i.e., dose x F), not the amount you gave! Thus in our equations we must start to replace ‘dose’ with ‘dose x F’ when oral dosing is under consideration, but do not use ‘dose x F’ if the drug is not given orally. o If you need to match an oral dose to a dose given IV, you must also consider oral bioavailability. If 10 mg is working fine when given IV, and oral bioavailability is 50%, then you will need to give twice as much drug orally to get the same effect. Alternatively, if 10 mg is working orally, and oral bioavailability is 50%, then to match the oral dose when the drug is now given by an IV route, the dose needs to be cut in half. The following equations apply (as they ensure that absorbed amounts are equal): IV dose = oral dose x F Rearranging: oral dose = IV dose / F

 Another consideration is AUC. AUC is a measure of the total exposure of an animal to a drug over time. Where this can be a critical consideration in the clinics is in chemotherapeutics. Chemotherapeutic considerations are important with antibiotics, but most critically, in cancer chemotherapy. In these situations the effectiveness of the drug is often related to the total exposure, not just the peak concentration or total duration of treatment. Thus you will often read about AUC in situations in which chemotherapeutic regimens are developed, and careful consideration of absorption, distribution, and elimination need to be thought about to design protocols that have maximum therapeu‐ tic efficacy (which is thought to be related to total exposure as represented by AUC) but that produce minimal toxicities. While toxicities can be related to AUC (for example, doxorubicin has a limited life time exposure before significant cardiotoxicities arise), toxicities may also be related to peak plasma concentrations, and can be minimized by protocols that maintain an adequate total exposure (AUC) but avoid excessive peak levels. If you plan to enter into oncology, AUC will be a concept that you will frequently need to consider.  A final consideration is bioavailability when the drug is given parenterally but not IV (e.g., IM, or subcut, or IP). As a general rule these parenteral routes are associated with 100% bioavailability as they circumvent the major processes that are responsible for limiting oral bioavailability (acid stomach environment, intestinal flora, ion trapping in the gut, precipitation in the gut, incomplete release of drug from the preparation, significant first pass metabolism), but not always, such as a depot injection that contains a compound that is labile. But as a general rule, unless you have reason to believe otherwise, it is probably safe to assume you are achieving 100% bioavailability with these other routes – but the absorption characteristics will be different from IV administration (no  phase is one consideration).

VM522P / VM7522 Pharmacology WIMU Fall d) Half‐life Earlier we developed the idea of a half‐life (t1/2) and one often hears discussion of a drug’s t1/2 – but t1/2 is not considered a fundamental pharmacokinetic variable! Why is this, and what is the relationship of t1/2 to the variables we have considered?

 Half‐life is a construct of theoretical 1st order models, whereas drugs are actually eliminated by clearance. That is, drug is removed by drug laden plasma being in contact with an organ extracting drug from the plasma. Thus the removal of drug is influenced by items such as changes in blood flow to the organ, or changes in the extraction ratios of the organ. These events change the clearance of the drug and thus they alter the dynamics of the profile of Cp.

 While it is intuitively obvious that changes in clearance would alter a drug’s t1/2, it is less obvious that t1/2 is also influenced by the Vd for the drug. The way I like to think about this is that if the Vd is expanded (i.e., and obese patient with a lipid soluble drug), a much smaller amount of drug is in contact with the organ that is actually clearing the drug, thus increases in the Vd can prolong (slow) the t1/2.

 The mathematical relationship between t1/2 and CLs and Vd is:

t1/2 = (Vd x 0.693) / CLs

(Note this equation can be rearranged to solve for any one of the three variables.)

 Thus:

o If the Vd increases the t1/2 slows (if Vd gets larger/ the t1/2 gets longer)

o If clearance gets faster (larger number) then the t1/2 gets faster (smaller number).

o t1/2 is a derived variable, that is changes in t1/2 do not cause changes in Vd or CLs, but rather, changes in CLs and Vd cause changes in t1/2.

 If the t1/2 is a derived value that is altered by many clinical situations why do we even bother learning about it?

o The power of the t1/2 is that it can easily help one predict future plasma concentrations. We will use this understanding in a variety of situations that enable us to understand how dosing regimens produce particular plasma profiles. There are a few items you should keep in mind as be start the next unit:

. These are models that are only good to a first approximation. Thus while they help organize your thinking, they are not absolute laws of behavior.

. The t1/2 is a value determined by both the drug and the animal. While literature values are given, they are averages (approximations) and variations exist in any specific animal you work with (also, book values are determined in healthy animals).

. The most frequent encountered source of variation is obesity. This is especially significant for lipid soluble drugs (those with a large Vd). Thus you should be aware that when giving a drug with a large Vd of distribution to an obese animal, compared to the standard animal the circulating levels (Cp) will be lower and the drug will last longer (longer t1/2).

. It is also worth keeping in mind that the t1/2 is not an invariant value in any particular animal, but can change depending on the clinical situation.

o Even with these caveats, we shall see how thinking in terms of t1/2 can greatly improve your understanding of the relationship between dosing regimen and plasma profiles.

VM522P / VM7522 Pharmacology WIMU Fall L13: Dosing regimens ‐ Infusions

Objectives: 1. Be able to calculate the time to reach the plateau and the plateau concentration of a drug during an IV infusion. 2. Understand the consequences of changes in infusion rate on the plateau concentration and the time course to achieve the new plateau concentration so that you can use these insights in a clinical environment.

Overview In this section we will focus on dosing regimens in which a continuous exposure to drug is needed for a desired therapeutic outcome (chronic drug therapy). Continuous exposures can be achieved by one of two methods: an infusion, in which a constant amount of drug is given at a set rate, and intermittent dosing, in which the drug is given in discrete doses at regular intervals. Intermittent dosing has much to recommend, most importantly is that it usually does not require any fancy equipment or trained personnel. However, intermittent dosing has significant draw backs; most important is that the plasma level of the drug can vary considerably over the course of therapy, which can have undesirable consequences. We shall come back to intermittent dosing in the next lecture, but first we will consider an infusion, which although technically more difficult to administer, is conceptually more straight forward.

Infusions Infusions are used primarily in two situations, when the half‐life of the drug is very short and thus maintaining an adequate plasma level of a drug is problematic, or when the drug is rather dangerous and you want to avoid the peaks that are associated with intermittent dosing. An infusion produces a nice smooth drug plasma profile (see graph, next page). However, brief reflection should lead you to several questions:  What controls the level of the plateau (or steady‐state) plasma concentration?

 What controls the manner in which drug levels increase or decrease?

 What happens if you change the infusion rate?

 How can one more quickly establish the plateau?

Before we answer these questions, we need to define a few terms:  Infusion rate is the rate of drug delivery (in amount per time, such as mcg/min).  Flow rate is the rate that an infusion solution is given (in amount per time such as ml/min).  Thus the infusion rate is set by the flow rate times the concentration of drug in the infusate: ml/min x mcg/ml = mcg/min

VM522P / VM7522 Pharmacology WIMU Fall

1.0 stop infusion

0.8

0.6

0.4 Cp (mcg/ml)

0.2

0.0

0123456

start infusion time (hr)

In this example:The drug has an infusion rate of 1 mg/min. It is given to a 30 kg dog.

The drug has a t1/2 of 15 min and a Vd of 0.8 L/kg.

What controls the level of the plateau (or steady‐state) plasma concentration? In the above graph Cp plateaus at 0.9 mcg/ml. This presumably is a target set by a therapeutic consideration. What aspects of the infusion procedure resulted in this specific plateau Cp?

Calculating the Plateau Concentration (Cplat): At the plateau rate of input = rate of elimination (thus a steady‐state)

By definition rate of input = infusion rate (I) (mg/min)

Recall CLS = (rate of elimination) / Cplat (ml/min)

Thus rate of elimination = CLs * Cplat (mg/min)

Substituting I = CLS * Cplat (Eq. 1)

Rearrange to find Cplat Cplat = I / CLS (Eq. 2)

Calculating using t1/2:

Recall CLS = (Vd x 0.693) / t1/2 (Eq. A)

C * 0.693 * Substitute into Eqs. 1 and 2 I = plat (Eq. 3) Vd

I * t1/2 Cplat = (Eq. 4) 0.693 * Vd

VM522P / VM7522 Pharmacology WIMU Fall Questions:

Use the formulas derived above and the values given under the graph to calculate Cplat:

(I * t ) (1 mg/min * 15 min) Use Eq 4: Cplat = 1/2 / (0.693 * Vd) = / (0.693 * 0.8 L/kg * 30 kg) = 0.90 mg/L Note that mg/L is the same as mcg/ml so 0.90 mg/kg = 0.9 mcg/ml

From the pharmacokinetic parameters given, calculate the whole body clearance for this drug (in ml/min/kg): (0.8 L/kg x 0.693) Use Eq. A: CLs = (Vd x 0.693) / t1/2 = / 15 min = 0.03696 L/min/kg Convert to ml/min/kg: 0.3696 L/min/kg x 1,000 ml / L = 37 ml/min/kg

What controls the manner in which drug levels increase or decrease?

 In a first order process, when one stops an infusion, the plasma level decreases according to the t1/2.

That is, in one t1/2 plasma levels would be 50% of the starting value, in a second t1/2 it falls 50% again, or to 25% of the starting value, in a third t1/2 it falls another 50%, or to 12.5% of the starting value, etc (see below at point marked stop infusion).

 It is also true that if a process is first order the kinetics of the approach to a plateau level is identical to the kinetics of falling to zero. In other words, in each t1/2 the plasma level closes the gap by 50%. Thus in the first t1/2 Cp achieves 50% of the eventual plateau value, in a second t1/2 it climbs another 50% of

the way to the plateau level, or to 75% of the eventual plateau value, in another t1/2 it climbs another 50% of the way to the plateau level, or to 87.5% of the eventual plateau level, etc (see below at point marked start infusion).

 Thus if you know the t1/2 of a drug, and you know the ultimate plateau value, you can predict both the time course of the rising phase and falling phases. Such information can be critical in making an appropriate clinical decision during an infusion.

1.0 stop infusion Cplat For this course we will

0.8 Col 1 vs Col 2 assume it takes 5 t1/2 to Col 1 vs Col 2 reach the plateau (actually t1/2 0.6 96.875% of the plateau) but some clinicians also t 1/2 like to think in terms of 3 0.4 t1/2 because at that time Cp (mcg/ml) t 1/2 the level is almost 90% of 0.2 the plateau (actually 87.5%). 0.0 0123456t 1/2 t1/2 start infusion time (hr)

VM522P / VM7522 Pharmacology WIMU Fall What happens if you change the infusion rate? We will first consider what happens when we start at a different infusion rate from the beginning.  Note the equation (Eq. 1) that relates the infusion rate to the plateau concentration:

I = CLs * Cplat  This equation shows that the plateau concentration is directly proportional to the infusion rate. Thus if you double the infusion rate, the targeted plateau will double. If you half the infusion rate, the targeted plateau will be one half.

Questions:

Use the same patient and pharmacokinetic parameters from above (the drug has a t1/2 of 15 min and a Vd of 0.8 L/kg and it is given to a 30 kg dog). What will be the targeted plateau concentration if the infusion rate is 0.33 mg/min rather than 1 mg/min?

(I * t ) (0.33 mg/min * 15 min) Use Eq 4: Cplat = 1/2 / (0.693 * Vd) = / (0.693 * 0.8 L/kg * 30 kg) = 0.30 mg/L Note that mg/L is the same as mcg/ml so 0.30 mg/kg = 0.3 mcg/ml

How long will it take to achieve this level (0.3 mcg/ml) with the lower infusion rate (0.33 mg/min)?

The 5 half-lives rule applies: Thus it will take 5 x t1/2 = 5 x 15 min = 75 min

Rather than asking how long it takes to plateau, what if the question is the time to achieve a particular level? For example, approximately how long will it take to achieve a plasma concentration (Cp) of ~0.3 mcg/ml (the plateau level with the slower infusion) with the original infusion rate (1 mg/min)? In this situation (1 mg/min) the eventual steady state level is 0.9 mcg/ml. Recall from above that the Cp gets half-way (0.45 mcg/ml) to the final level (0.9 mcg/ml) in one half-life (15 min). Thus to achieve 0.3 mcg/ml would take <15 min. A reasonable value would be ~9 min (look at graph below). (Thus although this isn’t calculated precisely, we are capable of making a reasonably educated guess without a lot of fancy calculations.)

Graphical Examples of Changes in Infusion Rate:

C 1.0 infuse 1 mg/min stop infusion plat 0.8

change 0.6 infusion to 1 mg/min 0.4 t1/2

(mcg/ml) Cp C plat 0.2 infuse 0.33 mg/min 0.0 0123456t 1/2 t1/2 start infusion time (hr) 77

VM522P / VM7522 Pharmacology WIMU Fall We will now consider what happens if we change the infusion rate after the infusion has started.  Continue with the above example (start infusion at 0.33 mg/min). After 1.5 hours you now change the infusion rate from 0.33 mg/min to 1 mg/min (the original infusion rate). (See point marked change infusion rate to 1 mg/min on the graph.) Questions: Is 1.5 h after you started the infusion at 0.33 mcg/min long enough for a plateau to be established? Why or why not?

This is long enough because 1.5 h (90 min) is greater than 5 x t1/2 = 75 min = 1.25 h

Once you change the infusion rate to 1 mg/min what is the new eventual steady state or plateau level? At an infusion rate of 1 mg/min the eventual plateau level is 0.9 mcg/ml (from the first calculation above).

What is the starting Cp when the infusion rate was changed? At an infusion rate of 0.33 mg/min the plateau level is 0.3 mcg/ml.

Here is the really important insight: Recall in the discussion above concerning the approach to the plateau – in each half‐life the gap between the current Cp, and the eventual Cp (or Cplat) closes by 50%. On the climb towards the plateau this held true whether the current Cp was 0, or 0.45, or 0.6, etc. Thus in a first order process the exact starting value is not important, what holds true is in one half‐life the gap between where the Cp is at and where it is going (Cplat) decreases by 50%. We can use this insight to estimate plasma levels when you change the infusion rate. We will call this the ‘Gap Rule’.

Questions: Consider the above change in infusion rate. At what time after the change would Cp be ~0.6 mcg/ml? The current Cp is 0.3 mcg/ml. The eventual plateau Cp is 0.9 mcg/ml. The difference between these is 0.6

mcg/ml. In one t1/2 (15 min) half of this gap will be achieved [(0.6 mcg/ml) / 2 = 0.3 mcg/ml]. Thus in 15 min the Cp will be: 0.3 mcg/ml [starting level] + 0.3 mg/ml [amount added in one half-life] = 0.6 mcg/ml. Thus 15 min after the change the Cp will be 0.6 mcg/ml.

At what time after the change would the Cp be ~0.75 mcg/ml? We know the Cp is 0.6 mcg/ml at 15 min after the change. At that point in time the remaining gap to the

eventual plateau level is: 0.9 mcg/ml – 0.6 mcg/ml = 0.3 mcg/ml. We know in the next t1/2 that the gap will close by 50%, or [(0.3 mcg/ml) / 2 = 0.15 mcg/ml]. Thus in another t1/2 (or an additional 15 min) the Cp will be: 0.6 mcg/ml [starting level] + 0.15 mcg/ml [amount added in one half-life] = 0.75 mcg/ml. Thus in another 15 min (or two 15 min blocks) the Cp will climb from 0.3 mcg/ml to 0.75 mcg/ml. Thus it will take 30 min after the change to achieve a Cp of 0.75 mcg/ml.

This process can be repeated for each half‐life (the gap decreases by 50% per half‐life). With a few landmark points (i.e., calculate Cp value for each additional half‐life), one can draw a reasonable smooth curve and basically look at the graph and know exactly what Cp should be at any time.

VM522P / VM7522 Pharmacology WIMU Fall Further insight: In addition, the Gap Rule is independent of whether the Cp is climbing or falling. Thus as long as you know approximately what the current Cp is and what it will eventually plateau at, you can estimate exactly where you are on the curve. This only requires that you divide the gap by 2 and note the half‐life.

Questions: After the plateau level of 0.9 mcg/ml is achieved, you now half the infusion rate (to 0.5 mg/min). What is the eventual Cplat with this new infusion rate?

(I * t1/2) (0.5 mg/min * 15 min) Use Eq 4.: Cplat = / (0.693 * Vd) = / (0.693 * 0.8 L/kg * 30 kg) = 0.45 mg/L Note that mg/L is the same as mcg/ml so 0.45 mg/kg = 0.45 mcg/ml

How long after you make the change to 0.5 mg/min would it take for the Cp to fall to ~0.7 mcg/ml? The gap between the current Cp and eventual Cp is 0.9 mcg/ml – 0.45 mcg/ml = 0.45 mcg/ml. So half of

this gap (0.45 / 2 = 0.23) is achieved in one t1/2. In one half-life (15 min the Cp will fall from 0.9 – 0.23 = 0.67 mcg/ml. Thus 0.7 mcg/ml will be achieved in just under 15 min.

A final consideration: The ‘Gap Rule’ may seem kind of odd that it should work so well. However, consider the situation we first started with, that an infusion was first started at a rate of 1 mg/min, and we estimated Cp at various times making steps at one half‐life at each step. In that calculation we had been applying the Gap Rule even at the first step; it just was that the starting Cp was 0 so the size of the gap was the entire change in Cp (from 0 to 0.9 mcg/ml). The same thing applied to the falling phase when the infusion was terminated; only in this case the eventual Cplat was 0. Again the Cp changes in half‐steps from where the current Cp was to where Cp was going. In both cases we still applied the gap rule only we had the special circumstance that one of the values (starting Cp or ending Cp) was 0.

VM522P / VM7522 Pharmacology WIMU Fall Some clinical thoughts:

 Often in the clinic, when you start an infusion, you may not know exactly what the targeted Cplat is. Does this make these pharmacokinetic insights into plasma concentrations worthless? The answer is NO ‐ you can still think about the changes in a relative manner. For examples: o You still know it will take ~5x the half‐life to achieve the plateau, regardless of what it is. This is when you can start to make judgments about the effectiveness of your therapy (or if you are comfortable with 87.5% of the final Cp, you can make judgments after 3 half‐lives).

o If the animal exhibits a desired or undesired reaction before the final level is achieved – how long was it since you started the infusion? One half‐life? Two‐half‐lives? A fraction of the half‐life? In each case understanding first order kinetics give you some insights:

. If the reaction occurred at a time equal to one t1/2, then the Cp at the time was half the targeted Cplat. Since you know the infusion rate is directly proportional to the Cp, you can adjust your infusion rate accordingly. For a desired reaction you might scale it back to just 50% of the original value and still maintain the desired reaction, for an undesired reaction you might scale it back to below 50% of the original rate to stay below those levels.

. If the reaction occurred ~2 t1/2 after the start of the infusion. Again your insight is this happened at a level that was close to 75% of your targeted Cplat. The infusion rate could then be adjusted accordingly (>75% or <75%) to stay above or below the level determined to be effective or problematic (respectively).

. If the time of the reaction was a fraction of a half‐life, then the reaction occurred at below 50% of Cplat. Again appropriate actions can now be taken (an adjustment that is greater than 50%).

o Once you have made the changes described, you also know that the new equilibrium between existing and target will take ~5 x t1/2. On the other hand, since the Cp may already be close to the new targeted Cplat, the new level may effectively be achieved in much less time. Again this insight can be determined by thinking about the time since you started the infusion and the relative level of the new Cplat.

o It is important to remember these insights come at little calculation effort. Serially dividing by 2 and then adding or subtracting – in other words, the ‘gap rule’, gives you tremendous under‐ standing that does not require fancy calculations or the memorization of equations. What it does require is the understanding of first order processes (those processes that change as a constant percent of what is present).

VM522P / VM7522 Pharmacology WIMU Fall How can one more quickly establish the plateau?

Perhaps you have reason to want to establish a plateau level much faster than the 5 t1/2 life rule. Are there mechanisms to get around this? We will examine two ways. One is to rapidly give a loading dose, only you need to calculate an appropriate dose. The second is to manipulate the infusion to achieve the desired goal.

 Precise calculation of a loading dose for an IV infusion: o The goal of a loading dose is to rapidly achieve a desired Cp. Since we know the relationship between Cp and dose is given by the Vd, we can use this to calculate an appropriate dose: Definition of a volume of distribution: Cp = dose / Vd Rearrange to solve for dose: dose = Cp x Vd o Thus you could calculate this dose and give it, then immediately start the infusion which would then maintain the desired Cp. o However, while this is a precise calculation, there are practical issues that need to be considered: . Giving such a dose all at once does not give you time to evaluate whether the Cp achieved causes any undesirable reactions. You are stuck at the level you achieve until the drug is eliminated. . Even more relevant is application of concepts we developed in the two compartment model. We know for a fact that distribution does not take place instantaneously. Thus by giving a sudden injection, the high levels associated with the ‐phase will occur, and perhaps cause an undesired reaction, even if the final level is appropriate.

 Manipulating infusion to achieve Cplat faster: o One can also apply the ‘gap rule’ to accomplish much of the same goal without the dangers associated with a sudden injection.

 Recall that Cplat is directly proportional to the infusion rate. Thus, if the infusion rate is doubled, then Cplat is doubled.  If at the beginning of an infusion if you double the infusion rate and left this alone, you would eventually produce a Cplat that is twice as high as that produced by the original infusion rate. .  However, application of the gap rule tells us that one half‐life after starting the infusion at the doubled rate, you would achieve 50% of the higher Cplat, which happens to be exactly the value targeted by the original infusion!

 Thus, you can double the initial infusion rate and then wait one t1/2 before you cut it back to the original infusion rate. This would produce a smooth increase of Cp over a single t1/2 (no ugly ‐ phase) until the desired Cp is achieved, and then when the infusion rate is returned to the original rate, the original desired Cp would be maintained.

Questions: Calculate a precise loading dose using the formula above and the parameters from the original infusion at the start of this section: Use the Eq: dose = Cp x Vd = 0.9 mcg/ml (or mg/L) x 0.8 L/kg x 30 kg = 21.6 or 22 mg

Using the second method described above, what is an appropriate infusion rate to start and how long would you allow this rate to go before you cut back to the original rate? Start the infusion at 2 mg/min and 15 min later turn it down to 1 mg/min.

VM522P / VM7522 Pharmacology WIMU Fall L14: Dosing regimens – Intermittent dosing

Objectives:

1. Understand the similarity between Cpavg obtained with an intermittent dosing protocol and Cpplat obtained in an infusion. Also understand the relationship between Cpavg and Cpmax and Cpmin.

2. Explain what is meant by the range of plasma concentrations (Cpmax/Cpmin) for an intermittent dosing protocol, and how selection of a particular protocol influences the variation.

3. Know the kinetics of accumulation and magnitude of accumulation that occur with intermittent dosing protocols.

4. Know what a priming (or loading) dose is and how to calculate one for either an infusion or intermittent dosing.

5. Understand how manipulation of the absorption rate affects the overall Cp profile during an intermittent dosing regimen.

Overview: In the previous session we examined parameters involved when a drug is given by a constant infusion. We will now focus on intermittent dosing protocols. With intermittent dosing a drug is administered in discreet doses at a regular interval for an extended period of time (it could be for the life of the animal, so getting the parameters set right is an extremely important consideration). As previously mentioned, the mechanics of intermittent dosing usually are quite easy, especially with an orally administered drug, but the nature of the protocol introduces two significant clinical issues. The most important is that the plasma level of drug varies between doses, and thus there is a chance on the high side that some threshold will be exceeded that either produces an unwanted side‐effect or toxicity, and on the low side there is a failure to maintain therapeutic efficacy. Thus it is critical that first, you are aware that this variation exists; second, what aspect of the dosing regimen is responsible for the variation; and third, how adjustments or changes in the protocol will change the profile. Another issue that you need to become aware of is that with these protocols drug will accumulate to a plateau level. It is important that you understand the nature of this accumulation process and what manipula‐ tions can be undertaken if you want to circumvent the issue.

VM522P / VM7522 Pharmacology WIMU Fall

Cpmax

200

steady-state 150 or plateau

Cp 100

Cpmin 50 DI 0 012345 time (days) Above is a graph that illustrates the profile of the concentration of drug given via an intermittent protocol. The open arrows indicate when the drug is given (the plot assumes virtually instantaneous absorption, but no ‐ phase). It is assumed all doses are equal. Illustrated on the graph is the dosing interval (or DI), which is simply the time between dosing. As with dose, it is usually desirable to keep this constant during the protocol so as to maintain predictability in the Cp. We also need to identify two important points on the graph Cpmax and Cpmin. These values are determined after a plateau (or steady‐state) has been achieved.

Reflection on the plasma profile should lead you to several questions:

 How do different parameters in the regimen control the level of drug at the plateau (or steady‐state)?

 How does one control the peaks (Cpmax) and valleys (Cpmin) in the protocol to obtain a safe and effective therapy?

 How can you know the degree of drug accumulation before the plateau is reached?

 What causes the delay in reaching a plateau (or steady‐state), and how can this be circumvented?

VM522P / VM7522 Pharmacology WIMU Fall How is the level of drug at the plateau (or steady‐state) established?

 There are two approaches by which this issue can be approached:

o One is to establish what average level of drug is desired (Cpavg), and then calculate the amount of drug necessary to accomplish this level. After this is established we can then investigate how often to give the drug in order to control the variation. (Approach A)

o A second approach is to determine how much variation in Cp we can tolerate. Once the range of tolerated levels is established, then a protocol can be designed to obtain the desired absolute values of Cpmax and Cpmin. (Approach B)

o Either way can work (and they are somewhat related), so it doesn’t really matter which is used. There are certain aspects that are nicely illustrated by each approach so we will examine both.

Approach A (using Cpavg):

 First establish the average amount at the plateau (Cpavg):

o Recall that for an infusion the plateau is reached when the rate of elimination is equal to the rate of administration (which for an infusion is the infusion rate). The rate of elimination was given by Cp * CLs.

o The plateau in an intermittent protocol will also be reached when the rate of elimination is equal to the rate of administration. The average rate of elimination is given by Cpavg * CLs, and the rate of administration is the dose/dosing interval (or D/DI). Thus:

Cpavg * CLs = D / DI solving for Cpavg: Cpavg = D / (CLs * DI) (Eq. 6)

o Eq. 6 can also be rewritten using Vd and t1/2 by making the proper substitutions:

( recall that CLs = (Vd x 0.693) / t1/2 ) thus:

Cpavg = (D x t1/2) / (0.693 x Vd x DI) (Eq. 7)

o Note: essentially all we have done compared to the equations generated for an infusion is substitute Cpavg for Cpplat, and D / DI for the infusion rate (note both D / DI and the infusion rate are in units of amt/time).

o Also note: If there is limited bioavailability then the term (D * F) should be substituted for the term D to correct for the fact that not all drug given is absorbed.

 Then establish the range of plasma concentrations at the plateau:

o The absolute range of Cp is: Cpmax to Cpmin For example, we might find that Cp ranged from 200 mcg/ml to 100 mcg/ml o However, as we will see below, for our purposes the fold‐range is of more interest: Using the above example and expressed as a ratio (or fold‐range):

range = Cpmax / Cpmin = 200 / 100 = 2 fold

In other words, the Cp varied over a 2‐fold range (or Cpmax was 2 times as great as Cpmin)

VM522P / VM7522 Pharmacology WIMU Fall o Really important concept in estimating the range: Simple reflection will reveal the following critical insight (make sure you understand each point before you move on):

. The difference between Cpmax and Cpmin will depend on how much time passes between doses. If you only wait a short time they will not be very different, but if you wait a long time they will be quite different. But what is a ‘short’ time and what is a ‘long’ time?

. One time at which we know the exact difference between the starting value and the ending value is the t1/2 for the drug. If the time between doses is exactly equal to the t1/2 for the drug, then Cpmin will be ½ of Cpmax, and the range will be 2‐fold.

. We can repeat the above logic for another t1/2, since we also know the difference between the starting value and ending value if you wait over two half‐lives – after the first t1/2 the level will be ½ the starting value, and after the second t1/2 it will be cut in half again, or be ¼ the starting value. Thus if the dosing interval is equal to 2 times the t1/2, the range will be 4‐fold. In other words, as a unit of time equal to the drug’s half‐life passes, the remaining Cp will be cut in half.

. With these thoughts in mind let us return to the question we posed above – what is a ‘short’ time and what is a ‘long’ time? What matters is not the absolute time, but the time relative to the t1/2 of the drug. If the dosing interval is a fraction of the drug’s t1/2 it is a short time (and the variation in Cp will be small), and if the dosing interval is a multiple of the drug’s t1/2 it is a long time (and the variation in Cp will be large).

. Note that because every drug has a different t1/2, what is a short time and what is a long time is unique to the specific drug. Because of the importance of this relationship, we will give it a name: relative dosing interval (RDI) and it is defined by the DI / t1/2 for the drug:

RDI = DI / t1/2

o Now let us return to the issue of variation in plasma levels: the RDI is useful because it allows us to estimate the variation in Cp in an intermittent dosing protocol. We can also use it to develop a calculation formula:

RDI Range = Cpmax / Cpmin = 2

o Although we will use this formula and refer to the RDI in developing some charts below, it is not important that you learn this formula unless you want to. This is because you can figure out the major landmarks in the chart (see next page) by thinking about t1/2 – all it involves is dividing the plasma concentration in half each time a t1/2 passes.

VM522P / VM7522 Pharmacology WIMU Fall Examples of variation in plasma levels for particular dosing intervals relative to the t1/2:

If dosing interval (DI) = 1 * t1/2 Cpmin is ½ of Cpmax (Cpmin is 50% of Cpmax) the range will be 2‐fold

If DI = 2 * t1/2 Cpmin is ¼ of Cpmax (Cpmin is 25% of Cpmax) the range will be 4‐fold

1 If DI = 3 * t1/2 Cpmin is /8 of Cpmax (Cpmin is 12.5% of Cpmax) the range will be 8‐fold

1 If DI = 4 * t1/2 Cpmin is /16 of Cpmax (Cpmin is 6.25% of Cpmax) the range is 16‐fold

Some examples that require the formula:

1 If DI = 0.5 * t1/2 CPmin is /1.41 of Cpmax (Cpmin is 71% of Cpmax) the range is 1.41‐fold

1 If DI = 0.25 * t1/2 CPmin is /1.19 of Cpmax (Comin is 84% of Cpmax) the range is 1.19‐fold

o Note in the chart that when the dosing interval is a large multiple of the t1/2, very little drug is left at the end of the dosing interval. In fact, if the dosing interval exceeds 5 times the t1/2, one can essentially assume that therapeutically the drug has completely been eliminated between doses (although residues might be a different issue). To use a protocol in which the DI is a large multiple of the t1/2 assumes you do not need constant drug coverage between doses.

o On the other hand, if the dosing interval is a fraction of the t1/2, then a lot of drug is left at the end of the dosing interval, and Cp has minimal variation. You would use a protocol in which the DI is a fraction of the t1/2 when constant coverage is needed and needs to be kept within a tight range (for example, anti‐seizure therapy).

Example of variation in plasma level of drug for different protocols. To better appreciate how this works, let us examine a specific example: o A drug has the following pharmacokinetic parameters: volume of distribution (Vd): 1 L/kg

half‐life (t1/2): 6 hr

clearance (CLS): 115 ml/hr/kg

o You target an average plasma concentration of 15 mcg/ml (Cpavg). o The drug comes in 50 mg and 100 mg tablets.

How much drug do you give and how often to a 10 kg dog? From Eq. 6 you can calculate how much drug per day is needed:

Eq. 6: Cpavg = D / (DI x CLS)

Solve for D: D = Cpavg x DI x CLS

Now put in numbers and figure how much drug is needed for a day:

Per day (i.e., DI = 24 hrs): D = 15 mcg/ml x 24 h x 115 ml/h/kg = 41,400 mcg/kg or 41 mg/kg/day

Solve for 10 kg dog: D = 41 mg/kg x 10 kg = 410 mg/day (because of limited pill sizes, we will round to 400 mg/day) A second consideration is that you want to keep your dosing schedule simple and reproducible from day to day. Thus you should only consider a whole number multiple per day (once a day, twice a day, three

VM522P / VM7522 Pharmacology WIMU Fall times a day, four times a day). Given the tablet sizes you have to work with and keeping your dosing interval to a convenient schedule your choices are:

400 mg once a day (4 x 100‐mg tablets): (DI = 24 hr; RDI = 4) total dose/day = 400 mg

200 mg twice a day (2 x 100‐mg tablets): (DI = 12 hr; RDI = 2) total dose/day = 400 mg

100 mg four times a day (1 x 100‐mg tablet): (DI = 6 hr; RDI = 1) total dose/day = 400 mg

50 mg eight times a day (1 x 50‐mg tablet): (DI = 3 hr; RDI = 0.5) total dose/day = 400 mg

Note that in every case the total amount given (total daily dose) is always the same. Because of this Cpavg will be the same for all protocols (14.5 mcg/ml). What will be different is the amount of variation. As described above, the variation will depend on the RDI, which is listed for each protocol above. (REM: RDI = DI / t1/2 and the t1/2 for our example was 6 h)

The schedule you pick depends on choosing a tolerable Cpmax and range:

o (Note: Cpmax and Cpmin for the first case (upper left, RDI = 1) are indicated on each of the subsequent graphs for comparison purposes – the vertical axis are different on the lower graphs.) o If you can tolerate a wide variability, then once a day dosing is reasonable (lower left), but be aware that the peak levels will be almost 3x the average level and 16x Cpmin. o If you cannot tolerate much variation, then 8 times a day is a good choice (lower right). But this is a demanding schedule for the owner. o Further note that even twice a day dosing (upper right) produces levels that vary 4‐fold over a day.

100 mg every 6 hrs (RDI = 1) 200 mg every 12 hrs (RDI = 2) 30 30

Cp (RDI =1) 20 max 20 Cpmax (RDI =1) g/ml) Cp g/ml) Cp  avg avg 

Cp (RDI =1) 10 min 10 Cpmin (RDI =1) Cp ( Cp (

0 0 01234 01234 days days

400 mg every 24 hrs (RDI = 4) 50 mg every 3 hrs (RDI = 0.5)

Cp (RDI =1) 40 20 max

30 Cpavg g/ml) g/ml)

 DI  Cp (RDI =1) 20 Cpmax (R =1) 10 min

Cpavg Cp ( Cp (

10 Cpmin (RDI =1)

0 0 01234 01234 note change note change in axis scale days in axis scale days

VM522P / VM7522 Pharmacology WIMU Fall Take home messages: In a standard practice you are unlikely to set your own dosing schedule, but rather you will be following an already established schedule. However, there are three important aspects of this analysis that are useful even when using a standard protocol that you should appreciate: o First, you should appreciate that when you put a patient on an intermittent drug therapy the Cp of the drug is varying, and that when you evaluate the patient, you should be aware of where you are on the curve (e.g., near the peak or near the valley). o Second, although you are likely to follow a standard protocol, there might occasionally be a situation in which you change the schedule. You should be aware of what that change is doing to the variation in plasma level.

o Third, for any drug you use frequently you should learn the drug’s t1/2, and how often you typically give it (in other words, the RDI). This can tell you in an instant how much Cp varies during the therapy. At some point this may be a critical consideration in making an evaluation or adjustment. Note this insight does not require you know the specific concen‐ tration of the drug, but rather you simply understand the magnitude of the variation in Cp. o In the problem sets we will examine some established protocols just to give you some insights into these therapies.

Approach B (designing a protocol to obtain exact values for Cpmax and Cpmin): In the above approach we targeted a general level of drug and then estimated how much variation the patient could tolerate. But what if the drug is a relatively dangerous drug (for example, an anti‐arrhythmic compound) that needs to be kept within precise levels to avoid toxicity but maintain therapeutic levels? For these drugs the toxic level and minimal therapeutic level are often known. Thus how can we design a protocol that meets these demanding limits?

To do this we have to develop a more precise understanding of how Cpmax is achieved (this will also be impor‐ tant when we think about drug accumulation, so this exercise has two purposes). As discussed above, in a standard practice it is unlikely you will be designing exacting protocols, however, as with understanding the variation in Cp developed above, understanding what aspect of the protocol controls how much accumula‐ tion occurs will be useful in any clinical situation. Fortunately this can also be appreciated with a simple insight; and as you might guess – it is related to the RDI!

 First determine a tolerable therapeutic range (often times maximum and minimum plasma levels are of more concern than average plasma levels).

Cpmax < toxic dose (one needs to stay as far below the toxic dose as possible)

Cpmin > M.E.C. (one should stay above the minimum effective concentration)

 From this calculate a convenient value for the range:

range = Cpmax / Cpmin

VM522P / VM7522 Pharmacology WIMU Fall  From this determine a relative dosing interval (RDI): mathematical procedure: range = 2RDI (take log of both sides and solve for RDI) RDI = log (range) / log (2) or RDI = log (range) / 0.301

practical procedure: if range is ~2 then RDI is 1 (i.e., dose on the half‐life) if range is ~4 then RDI is 2 (i.e., wait two half‐lifes between doses) if range is ~8 then RDI is 3 (i.e., wait three half‐lifes between doses) (Note: for any problem in this class the practical procedure is adequate.)

 Then calculate how much each dose should be to achieve the desired Cpmax:

o We know how to determine peak Cp after a single dose: Cp1 = dose1 / Vd

o We need to develop an understanding between Cp1 and Cpmax if we are to exactly calculate Cpmax. This requires a little insight: . Review the graphs given two pages earlier – note that if very little drug is eliminated between doses (RDI = 0.5; bottom right graph), then Cpmax>>>Cp1 (i.e., there is a lot of accumulation) . On the other hand, if a high percentage of the drug is eliminated between doses (RDI = 4; bottom left graph), then Cpmax is just a tad higher than Cp1 (i.e., very little accumulation) . Another way to look at the issue is to see that if only a small fraction of the existing amount is eliminated between doses (small RDI) then a lot of drug will need to accumulate before the amount given at each dose is matched by the amount eliminated between doses (which defines the plateau). On the other hand, if a high fraction of the existing amount is eliminated between doses (large RDI), then only a small amount needs to accumulate before the amount given at each dose is match by the amount eliminated. . This can be formulized by an accumulation factor (FAC), which is simply the inverse of the fraction eliminated between doses: FAC = 1 / Fraction eliminated

. This accumulation factor (FAC) gives the relationship between Cp1 and Cpmax:

Some relatively straight forward examples: RDI Fe FAC Comment

1 0.5 2.0 Cpmax will be 2x Cp1

2 0.75 1.3 Cpmax will be 1.3x Cp1

3 0.875 1.1 Cpmax will be 1.1x Cp1 (i.e., virtually no accumulation) Some less straight forward examples: RDI Fe FAC Comment

1.5 0.65 1.5 Cpmax will be 1.5x Cp1

0.5 0.29 3.4 Cpmax will be 3.4x Cp1

0.25 0.16 6.3 Cpmax will be 6.3x Cp1 (i.e., a massive accumulation)

VM522P / VM7522 Pharmacology WIMU Fall . Understanding the above relationships (in a qualitative manner) is the critical take home message from this particular sub‐section: o If the dosing protocol calls for multiple half‐lives between doses (3 or more), there is virtually no accumulation, and you can more reasonably assume that if you get the desired therapeutic response after the first dose with little signs of adverse reaction, this is likely to be the case on subsequent administrations. o If the dosing protocol involves giving the drug dose on a time that is a fraction of the half‐life for the drug, then there will significant accumulation, and you should be aware that you should not make a judgment about the effectiveness of your therapy until a plateau is achieved. Again, this insight does not require that you know the exact levels, only that you know the relative degree of accumulation, and thus when it is proper to make an evaluation.

. Returning to the specific problem we started this section on ‐ Approach B: calculating how much to give at each dose:

Cpmax = Cp1 x FAC substitute using Cp1 = dose / Vd

Cpmax = (dose / Vd) x FAC now solve for dose

dose = Cpmax x Vd / FAC

. To better understand this approach we will use a specific example. In the example we will:

 Establish a Cpmax and Cpmin, and thus a range for Cp that will keep us within the bounds of toxic levels and therapeutic efficacy (i.e., the therapeutic window). Note that this process is somewhat arbitrary, any Cpmax and Cpmin that maintains Cp in the therapeutic window will work. We will target one that gives us an easy RDI to work with.  From the range we will determine the RDI and FAC.  We will then calculate a specific dose.

Question: An anti‐arrhythmic drug begins to show signs of toxicity when plasma concentrations exceed 20 mcg/ml, but at least 3 mcg/ml are required to see a therapeutic effect. The drug has a t1/2 of 6 hours and a Vd of 0.6 L/kg. What is an appropriate dosing regimen that should be easy for a client to maintain?  Establish how much variation can be tolerated – this establishes the dosing interval: o It is useful to make this as large as permissible so one can dose the animal as few times as possible, but at the same time it needs to be a convenient number (once a day, two times a day, three times a day, etc). o The maximum amount of variation that is tolerable for this drug is 6.7 fold

(Cpmax/Cpmin = 20/3 = 6.7)

o Once a day dosing (DI = 24 h) results in a RDI (DI/t1/2) of 4 (24 h/6 h), but this produces a range of 16 fold ‐ way too much variation. o Twice a day dosing (DI = 12) results in a RDI of 2 (12 h/6 h), and with an RDI of 2 the Cp will vary 4 fold – this works (it is less than 6.7). So we will plan to dose every 12 h.

 Establish targeted amounts for Cpmax and Cpmin: o We need to pick numbers between 20 and 3 and the max should be 4 times the min. o Several sets of numbers will work (20 and 5, 16 and 4, 12 and 3). Let’s pick the middle set of 16 and 4. (Not too much, not too little ‐ always a safe assumption.)

 Calculate a dose that when given 2 times a day produces a Cpmax of 16 mcg/ml: 90

VM522P / VM7522 Pharmacology WIMU Fall o Recall that: dose = Cpmax x Vd / FAC o Cpmax is 16 mcg/ml; Vd for this drug is 0.6 L/kg; and FAC is dependent on the RDI . For an RDI of 2 (12 h / 6 h) the accumulation factor FAC is 1.3 (see chart two pages earlier) o Thus: dose = 16 mcg/ml (or mg/L) x 0.6 L/kg / 1.3 = 7.4 mg/kg

 What students often find odd about this calculation is that the frequency of dosing is established first, and then how much to give is determined (rather than figuring how much to give, and then how often ‐ which is essentially what we did in Approach A). But when you realize one of the most important aspects of intermittent dosing is the amount of variation in the plasma level of drug, the logic of the approach makes more sense. Because this is so important I repeat a statement highlighted above – you do not need to know the absolute Cp to know the variation – simply count the number of half‐lives between doses – this tells you the amount of variation in Cp for any protocol you would encounter! o Dosing equal to a half‐life – the variation is 2 fold o Dosing two times the half‐life, the variation is 4 fold o Dosing three times the half‐life, the variation is 8 fold o Dosing less than a half‐life – the variation is less than 2 fold o Thus you do not need to memorize the half‐lives for every drug you use, but for any drug you use frequently with a chronic intermittent protocol – you should learn the drug’s half‐life relative to how often it is given. How long does it take to reach a plateau (or steady‐state)?  From the above discussion it should be obvious that it is important to understand how long one should wait before assuming your drug therapy is both safe and effective (i.e., you need to wait until the steady‐ state levels of Cpmax and Cpmin are established). What is the critical consideration? Perhaps you should develop a rule such as after the third dose, if nothing adverse has occurred and the therapy seems to be working, you are safe. This is a very bad policy!!! You should all recognize by now that the number of doses by itself does not establish when the plateau will be reached. The critical parameter is the

half‐life of the drug. This is another reason to know the t1/2 of any drug you use frequently.

 Just like we substituted dose/DI for the infusion rate in calculating Cpavg, we can use the analogy to the infusion we developed earlier to understand the approach to the plateau – that is a consideration of first order processes reveals that the approach to the plateau is independent of the manner in which the drug is given (either constant infusion of in episodic boluses).  And, as with infusions, after 5 half‐lives Cp is 97% of the final plateau (50%, 75%, 87.5%, 93.8%, 97%).

 Thus the number of doses needed to achieve the plateau (using the five t1/2 rule) would be:

Number of doses = (5 x t1/2) / DI

Question: A drug has a 48 hour t1/2. It is given every 12 hrs. How many doses are needed to achieve the plateau? Number of doses = (5 x 48 h) / 12 h = 20 doses (these are the numbers for the use of phenobarbital as an anti‐seizure compound in dogs)

VM522P / VM7522 Pharmacology WIMU Fall One can avoid waiting 5 x t1/2 by giving a loading dose: Illustrations of Cp's with and without loading dose  The purpose of a loading dose is to rapidly achieve the plateau values. 400  This can be accomplished by giving enough drug in a D' = 3.4 x D single dose to reach Cpmax. 300  Because the accumulation factor informs us what Cpmax will be relative to the first dose, it can be used

Cp 200 for this calculation: t1/2 = 24 hrs D' = D x FAC 100 RDI = 0.5 FAC = 3.4 (Note D’ is the loading dose and D is the maintenance 0 dose.) 012345 days  Some questions to ask yourself about a loading dose: D' = 2 x D o Is it necessary? For example, if the RDI is large 200 (bottom graph to the right) a loading dose is superfluous. Cp o Do you have to use the precise calculated loading 100 t1/2 = 12 hrs dose? Obviously you don’t. In fact, while you may RDI = 1.0 want to give more than the maintenance dose as a FAC = 2.0 loading dose, you might want to give less than the 0 012345 calculated loading dose. This still speeds up the days time to the plateau, but also allows you to ‘sneak‐ RDI = 3.0 up’ on the plateau level – this might be advisable – D' = 1.1 x D t = 4 hrs 120 1/2 all patients have the potential for unique FAC = 1.1 responses – you never can tell just by looking at 80

the patient if he/she will be more sensitive to the Cp drug than expected. 40

0 012345 days

Question: A drug used to treat seizures is given once every 12 hours at a dose of 4‐6 mg/kg. The drug has a t1/2 of 24 h. It comes in pill sizes of 50, 100, and 200 mg. What is an appropriate maintenance dose (in number and size of pills) and what would be a calculated loading dose for a 66 lb dog?  Dog’s wt in kg is: 66 lb / (1 kg/ 2.2 lb) = 30 kg  High maintenance dose = 6 mg/kg x 30 kg = 180 mg  Low maintenance dose = 4 mg/kg x 30 kg = 120 mg  Given the size of pills the use of 1 medium (100 mg) and 1 small (50 mg) would be appropriate (or 3 small pills would work just as well).  With the given protocol the RDI = 12 / 24 = 0.5  For an RDI of 0.5, the FAC will be 3.4 (see chart 3 pages earlier)  Thus the calculated loading dose would be 3.4 x 150 mg = 510 mg o (note 150 mg this is what you plan to give the dog as a maintenance dose so it is appropriate to use this in the calculation) o You do not need to give the entire 510 mg, but you should not give more than the 510 mg!

VM522P / VM7522 Pharmacology WIMU Fall One final practical issue – effect of absorption rate:  Up to now we have been assuming in both the problems and in the illustrated graphs that absorption is very fast, and thus Cp rapidly shoots up to a peak level determined by the drug. Some practical considerations related to this assumption:

o In most cases, if the drug is given orally, there is a delay in the absorption. As previously discussed, the magnitude of the delay is mostly dependent on pharmaceutical factors.

o In most cases we don’t know the magnitude of the delay, but we can assume it occurs. Although we don’t know the exact timing, delays are usually our friend for several reasons:

. First, in our above discussion we have also avoided consideration of the ‐phase of distribution. However, this is not unreasonable. As previously discussed, with oral absorption, the delay in absorption is likely to limit the magnitude of the ‐phase. (But watch out if you are ever giving the drug IV under such circumstances.)

. Further, any delay beyond that necessary to avoid the ‐phase also is likely to further limit the peak concentration. This is especially useful when dealing with dosing protocols that put Cp near toxic levels, as it is likely the peak Cp will be ‘rounded out’ and remain well below the upper limit you estimate (see graph below).

. Finally, if the delay is made significant enough, it might make a drug that normally has a t1/2 that is too fast to be useful in an intermittent protocol into one that now works well.

Effect of altering absorption on plasma profile of drug given via an intermittent dosing regimen:

200 for this graph 150 A dose = 100

DI = tCp 100 1/2 = 12 hrs ‐1 KB abs for A = 0.5 hr ‐1 K50 abs for B = 0.1 hr DI

0 012345 time (days)

Note how slowing the absorption ‘rounds out’ the peak values and reduces the variation in Cp.

VM522P / VM7522 Pharmacology WIMU Fall L15: TOLERANCE AND DRUG INTERACTIONS

READING ASSIGNMENT:

Riviere and Papich has some related materials in Chapter 4, page 87. Also Rang et al., pp. 19‐20 and 791‐795.

Objectives: 1. Be able to explain the differences between pharmacokinetic, pharmacologic, and physiological tolerances and give mechanistic examples of each and the potential relative magnitude of the tolerance observed with each type. 2. Be able to define the six terms listed that describe various aspects of tolerance and be able to explain the context that each is typically used. 3. Know the three general types of drug interactions (pharmacologic, physiologic, and pharmacokinetic) and be able to give a mechanistic example of each type. (Please note: when the term ‘interaction’ is used it implies two different drugs and is not to be confused with drug ‘action’.) 4. With regard to pharmacokinetic interactions, be able to give a mechanistic example of interactions involving absorption, distribution, metabolism, and excretion.

Outline: I. Tolerance II. Drug‐Drug Interactions

I. Tolerance

Definition ‐ When repeated administration of the same dose of drug fails to produce the same magnitude of response.

Or ‐ To achieve the same magnitude of response, increasing amounts of drug must be given.

Mechanisms:

1. Pharmacokinetic (ADME)

Pharmacokinetic tolerance is normally due to increased metabolism of a drug. This is most often due to a drug inducing CYP P450 enzymes that increase its own metabolism (microsomal oxidation). ‐ effects due to this mechanism are usually modest but are potentially dangerous, e.g. phenobarbitone can induce the expression of the CYP P450 enzymes that metabolize it and this can cause a drop in its plasma level. If phenobarbitone is being given for seizures this drop in plasma levels could cause the levels to be sub‐therapeutic and the seizures could resume. 94

VM522P / VM7522 Pharmacology WIMU Fall 2. Pharmacodynamic

i) Changes in receptor number. Most receptor systems will:

a) down‐regulate receptor number in response to an agonist b) up‐regulate receptor number in response to an antagonist

The best clinical example is beta adrenergic agonists and antagonists. Chronic treatment with beta antagonists will up regulate the number of beta receptors on cell membranes. (Think of it as the cell being under‐stimulated and so it is increasing the number of receptors to try to increase the signal it receives.) If the beta antagonist is withdrawn too fast the receptor number will still higher than normal, but now the endogenous beta agonists (sympathetic nervous system tone) acts unopposed by any antagonist leading to hyper‐activation of beta adrenergic targets, including the heart, causing a dramatic increase heart rate and force of contraction that can be lethal.

Recovery from this type of tolerance is usually slow (days).

ii) Desensitization: In the presence of an agonist some receptors desensitize, i.e. lose their capacity to respond. This is when the receptor protein goes into a confirmation from which it cannot be activated by an agonist. This is another way that a cell or system compensates for what it perceives as over‐stimulation. Desensitization can be fast e.g. nicotinic receptor desensitization at the neuromuscular junction (seconds to minutes) or slow e.g. opioid receptors following chronic treatment (days to weeks) Recovery from the fast type of desensitization is usually fast (min to hr). Recovery from slower desensitization usually takes longer (days).

3. Physiologic

i) Loss of endogenous mediator. If a drug works by releasing an endogenous transmitter, when that transmitter is depleted the drug can no longer produce an effect. E xamples: amphetamines, phenylpropanolamine ‐ phenylpropanolamine and amphetamine work by releasing biogenic amines (e.g., norepinephrine and dopamine); when these amines are exhausted these drugs no longer work.

ii) Physiological adaptations. These could include a compensatory change in a system that works against the effect of a drug. For example, the effects of diet drugs that inhibit food intake can be transitory as other mechanisms that work to increase food intake are increased.

VM522P / VM7522 Pharmacology WIMU Fall Terms used in describing tolerance:

1. Tolerance: General term used for loss of response after repeated exposures to a drug.

2. Cross‐tolerance: When tolerance to one drug leads to tolerance to another drug even though the animal was never exposed to the second drug. This usually occurs when mechanisms of action are identical or very similar, such as both drugs acting on the same receptor.

3. Tachyphylaxis (or desensitization): A term that is usually reserved for very rapid tolerance (onset and recovery).

4. Refractoriness: Mainly used to describe the loss of therapeutic effectiveness.

5. Drug resistance: A term usually used in the context of antimicrobial resistance.

6. Sensitization: When repeated administration produces increased effects (the opposite of tolerance). Thought to be an important action for some drugs of abuse.

II. Drug‐Drug Interactions

1. Pharmacodynamic (receptor mediated)

Most pharmacological interactions can be predicted as the two drugs both interact with the same receptor or target. For example:

i. Two agonists– e.g. two opioids that both act through the mu opioid receptor. If a patient is already receiving a maximal dose of one opioid, giving another will not produce more pain. Furthermore, if a patient has become desensitized or tolerant to the effects of one mu opioid, they will not respond to another opioid that also acts through the same receptor.

ii. Agonist and antagonist‐ e.g. two drugs that both act through beta adrenergic receptors. If a patient is on a beta adrenergic antagonist for blood pressure, they will not be able to respond to a beta agonist given to dilate the airways for asthma.

iii. Two antagonists – e.g. two drugs that block beta adrenergic receptors. If a patient is already being treated with a beta blocker for hypertension, giving another beta blocker to the eye, such as Timolol, will not work to treat glaucoma as the receptors are already blocked and adding another drug that blocks the same receptor cannot produce any more effect. 96

VM522P / VM7522 Pharmacology WIMU Fall

2. Physiologic

1. Induction/suppression of the expression of target receptors. Examples include: i. Thyroid hormone treatment increases the expression of adrenergic receptors and will therefore increase a patient’s sensitivity to adrenergic drugs. ii. Excess glucocorticoids can also both induce and suppress receptor expression. iii. Sex steroids can also produce long‐term changes that alter the responsiveness to drugs (by altering receptor proteins and carrier proteins).

2. Induction of drug binding proteins. Some drugs induce the expression of binding proteins or displace drugs from their binding to these proteins, thereby altering the circulating levels of the bound and free drugs, e.g.: i. Estrogen increases binding globulin and therefore the circulating levels of cortisol, thyroxine and thyronine. ii. Anabolic steroids decrease binding globulins. iii. Penicilin competes with other drugs and hormones associated with globulin, such as cortisol, thyroxine and thyronine, and will displace them and increase their free or unbound levels. iv. Aspirin displaces T4 from its binding to prealbumin.

3. Alteration in physiological parameters, e.g. ion concentrations. Examples include: i. Diuretics that promote the loss of K+ will potentiate actions of cardiac glycosides, which act by blocking the Na+ /K+ pump. ii. Aminoglycoside antibiotics block calcium channels to decrease acetylcholine release at the neuromuscular junction (NMJ). This can potentiate the effect of non‐depolarizing NMJ blocking drugs. NMJ blocking drugs are competitive antagonists at nicotinic ACh receptors on the NMJ. If there is less endogenous agonist around, you need a lower concentration of antagonist to block its effects.

3. Pharmacokinetic (ADME) Any drug that changes the available concentration of another drug will have an interaction, as decreasing or increasing the concentration of a drug can shift it either below the therapeutic range or, if increasing, make it more likely to cause adverse or toxic effects. Therefore, interactions can occur by changing absorption, distribution or metabolism of drugs.

1. Absorption Some examples are: i. Chelation in the GI tract (tetracyclines and Ca2+ or milk) ii. Antacids (change pH thus altering ionization state of drugs) 97

VM522P / VM7522 Pharmacology WIMU Fall iii. Antibiotics (change intestinal flora ‐ vitamin K antagonists may be potentiated)

2. Distribution Some examples are: i. Displacement from carrier proteins (esp. acidic drugs). Examples include: ‐ Tolbutamide or warfarin vs. NSAID (salicylates) ‐ Penicilin competes with the binding of other drugs, such as cortisol, thyroxine and thyronine, to globulin and so will displace them and increase their free levels. ‐ Aspirin displaces thyroid hormone T4 from prealbumin. ii. Sequestration in specific tissues. Examples include: ‐ Ototoxicity from aminoglycoside antibiotics (neomycin etc.) and high‐ceiling diuretics like furosemide. Both these drugs can cause ototoxicity and nephrotoxicity alone and should not be given together as they potentiate each others toxicity. However, the mechanism is not well understood.

3. Metabolism Some drugs can change the levels of metabolizing enzymes, either by inducing or inhibiting their expression. If another drug is metabolized by the enzyme whose levels are altered, especially if it is the main way a drug is inactivated, this can lead to drug interactions. Examples include: i. Enzyme induction: The classic example of enzyme induction is with barbituates, such as phenobarbital and pentobarbital. These drugs induce the expression of hepatic metabolizing enzymes. This can dramatically increase their own metabolism as well as other drugs that are metabolized by the same enzymes, such as cortisone or warfarin. Increased metabolism can reduce the concentration of available drug and drop it out of the therapeutic range. ii. Enzyme inhibition: Some drugs can also inhibit the expression of metabolizing enzymes to have the opposite effect. So that the drug is not metabolized as fast as normal and you have higher than expected concentrations, which can lead to more adverse or toxic effects. For example chloramphenicol suppresses the expression of the CYP P450 enzyme that also metabolizes cisapride.

4. Excretion Several drugs can change the can change the rate at which other drugs are excreted by the kidneys. If excretion is the main way a drug is eliminated this can lead to a drug interaction. Examples include: i. Agents that change urine pH (sodium bicarbonate and ammonium chloride) ii. Probenecid (inhibits anion transport in kidney) iii. Diuretics (limited to sensitive agents ‐ see “Pharmacokinetics” notes)

VM522P / VM7522 Pharmacology WIMU Fall

L16: Pharmacokinetics: Adjustments

Objectives: 1. Be able to describe how various physiological, pharmacological, and pathological conditions can affect the pharmacokinetic parameters of a drug. 2. Be able to describe what will happen to the plasma concentration profile of a drug if drug elimination is reduced but nothing is done to compensate for the reduced elimination. 3. Be able to calculate an impact factor to make corrections for suspected changes in drug elimination. 4. Be able to combine changes in both dosing frequency and amount to develop convenient dosing protocols when making corrections for reduced elimination.

Factors Affecting Drug Dosage The purpose of the entire section on pharmacokinetics is to get you to appreciate how dosing regimen are designed and what controls aspects of the resulting plasma profiles, from elementary properties of ADME, to models of integrated drug kinetics. The importance of you developing a deep insight into these issues is that while you can blindly follow dosing regimens, you must consider the circumstances under which those regimens were developed – they are for an ‘average’ animal in good health. These are unlikely to be your standard patient. Thus, if needed, you must be ready to make judgments and adjustments to tailor your use of a drug in a specific animal. The above materials give you the physiological and pharmacokinetic insights to begin to think about these adjustments. While we have addressed some issues that might alter some particular parameters, below we will make some additional considerations that should enter into your thinking.

Factors Affecting Correlation Between Cp and Effect We have been assuming that the plasma level of a drug is worth knowing because this value is related to the magnitude of the drug’s effect. While this is often a good assumption, there are conditions in which this is not the case. Some of these include:  Pro‐drug or metabolites with activity – in this case you would need to account for all active moieties. This is beyond the capabilities of most models to deal with, but is something you should be aware of.

 A drug with a large Vd ‐ in this case the drug may be at its target site in tissue (e.g., a lipid soluble brain active drug) at a concentration significantly greater than the plasma levels. Thus plasma levels may not accurately reflect drug action.

 A drug with a prolonged receptor effect – thus even though circulating drug may be gone, a biological effect persists. A class of drugs that you will use quite commonly that fall into this category is glucocorticoids (adrenal steroids). Steroids act by changes in gene expression that can take hours to days to fully manifest, long after the circulating steroid is gone.

VM522P / VM7522 Pharmacology WIMU Fall

Some Pathologies that Can Alter Standard Pharmacokinetics:  Absorption o GI administration ‐ GI diseases or bowel resections can reduce oral bioavailability

 Distribution o Compromised perfusion of a specific tissue, or general circulatory collapse, i.e., shock o These can reduce the Vd because the drug is not distributing into tissues. This could cause lack of effect if the drug does not distribute the compartment containing its target, or, if the drug has an action on a highly perfused tissue such as the heart, it may cause an unexpectedly large effect (i.e., a high concentration remains in the central compartment). Note – because the t1/2 is also dependent on Vd, these events can also alter the t1/2.

 Elimination o Liver

. Decrease clearance (and hence prolong t1/2)  Disease, old age, enzyme inhibition

. Increase clearance (and hence shorten t1/2)  Enzyme induction

o Renal . Acid‐base balance and ion trapping – could increase or decrease clearance depending on the nature of the drug and pH change (see renal materials in drug excretion). . Leaky kidneys (loss of plasma proteins) – this could have a profound effect to increase clearance of a drug with significant binding to plasma proteins. . Reduced clearance (age and/or disease)

 Example for adjustments for renal failure: While in most cases you cannot know precisely to what extent a pharmacokinetic variable has been compromised, one exception is renal clearance. The following analysis is particularly relevant to treatments with aminoglycoside antibiotics (e.g., gentamicin). These antibiotics are mostly eliminated by renal clearance and they can become toxic if steps aren’t taken to reduce dosing in animals with compromised kidney function (they have a narrow therapeutic window). Kidney function can be estimated from plasma creatinine or blood urea nitrogen (B.U.N.). Creatinine and B.U.N. are produced at a relatively constant rate and are cleared by the kidneys via filtration just like inulin (a first order process). Since production is constant, changes in plasma levels must be due to changes in elimination, or compromised filtration function in the kidneys. For example, if plasma creatinine (Ccreatinine) is double normal, then kidney filtration must be half of normal (i.e., double the concentration at half the normal rate results in the same amount of creatinine removed). Thus kidney clearance is inversely proportional to the plasma creatinine concentration. We can use this relationship to determine how much kidney function remains:

clearance(failure) Ccreatinine (normal) kidney function remaining = KF = = clearance(normal) Ccreatinine (failure)

100

VM522P / VM7522 Pharmacology WIMU Fall A second factor we need to know is how important is renal clearance to the elimination process for a given drug. If the drug is not eliminated by the kidneys, then kidney function is likely to be irrelevant for terminating drug action. On the other hand, if the kidney is responsible for the entire termination of the action of the drug, then kidney function will be highly significant. This insight is gained by reviewing material about the drug and finding the value given for the fraction excreted unchanged in the urine (FUR). Often this can be found on the package insert, but if not, it can be found in drug compendia. There are two reasons this is a value commonly reported for drugs:  It tells you how important kidney function is to terminating the action of the drug (the current reason we want to know it).  It also informs the physician that active drug is likely to be found in the urine. This can be significant for two reasons: o If the drug is an antibiotic, it might be very useful for treating urinary tract infections. o If the drug is dangerous (e.g., cisplatin), it can tell you to avoid excessive contact with urine from a treated animal.

From these two values (KF and FUR) we can determine how big an impact changes in kidney function will be on the clearance (and hence t1/2) of the drug. The following equation determines the amount of KF that is compromised (1 – KF) and multiplies this by the importance of the kidneys to elimination (Fur), and then subtracts this value from 1: impact factor = If = 1 – [ FUR x (1 ‐ KF) ]

There are several things to note about this equation:

o If FUR = 0 (i.e., no drug is eliminated by this route) then If = 1 (in other words, if the drug is not eliminated by kidney clearance, there is no impact).

o If KF = 1 (i.e., the kidneys have normal function) then If = 1 (in other words, if the kidneys are not compromised, there is no impact). o Note: One of the most frequent reason students make errors using this equation is they forget order of operation: Evaluate variables within parenthesis first. Then do multiplications and division before additions and subtracts. Thus, evaluate (1 – KF) first. Multiply this value by FUR. Then subtract value from 1. If will always a fractional value (<1).

There are two ways that If can be used to make adjustments:

either adjust dose: dose(failure) = dose(normal) x If (Remember the new dose will always be smaller than the original dose.)

interval(normal) or adjust interval: interval(failure) = If

(Remember the new interval will always be longer than the original interval.)

101

VM522P / VM7522 Pharmacology WIMU Fall To appreciate how this equation works in practice, and what happens when different adjustments are made (interval or dose) or not made, we will examine a hypothetical situation: Example of adjusting (or not adjusting) dosing regimen for kidney failure in a dog: Drug is recommended to be given once every 8 hours at a dose of 0.1 mg / kg

Pharmacokinetic parameters of drug in dog: t1/2 = 4 hrs Vd = 0.65 L / kg 90% excreted unchanged in urine In this dog plasma creatinine level was found to be 2.2 mg/dl (normal 0.6 to 1.4 mg/dl)

Use the values for plasma creatinine to estimate kidney function and calculate an impact factor:

KF = Ccreatinine (normal) / Ccreatinine (failure) = ~1.0 / 2.2 = 0.45

FUR = 0.9

If = 1 ‐ (0.9 x (1 ‐ 0.45)) = 1 ‐ (0.9 x 0.55) = 1 ‐ 0.495 = ~0.5

Because the clearance of the drug is essentially cut in half, and the t1/2 is inversely proportional to the clearance, and we have no reason to believe the Vd changed (e.g., dog is not obese), then we can assume the reduced clearance caused the t1/2 to change from 4 hr to 8 hr.

We can now generate values based on these parameters. To see the effects of these changes on the profile of plasma concentrations, see the graphs on the next page.  Note the standard regimen (below, top left):

RDI = DI / t1/2 = 8 h / 4 h = 2; For an RDI of 2 the FAC = 1.3

 In the diseased dog when no adjustments are made (below, top right) the RDI changes:

RDI = DI / t1/2 = 8 h / 8 h = 1; For an RDI of 1 the FAC is 2

 If we adjust only the dose (below, lower left), the change in t1/2 will cause the RDI to change:

RDI = DI / t1/2 = 8 h / 8 h = 1; For an RDI of 1 the FAC is 2

 If we only adjust the interval (below, lower right), the RDI will remain the same as the standard regimen:

RDI = DI / t1/2 = 16 h / 8 h = 2; For an RDI of 2 the FAC is 1.3

Standard regimen in normal dog: Standard regimen in diseased dog: dose: 0.1 mg / kg dose: 0.1 mg / kg dosing interval: 8 hr dosing interval: 8 hr (RDI = 2; FAC = 1.3) (RDI = 1; FAC = 2.0)

Adjust dose in diseased dog: Adjust interval in diseased dog: new dose: 0.05 mg / kg dose: 0.1 mg / kg dosing interval: 8 hr new dosing interval: 16 hr (RDI = 1; FAC = 2.0) (RDI = 2; FAC = 1.3)

102

VM522P / VM7522 Pharmacology WIMU Fall The graphs below illustrate the various plasma profiles at the plateau:

standard regimen standard regimen in normal dog in diseased dog

300 300

expected Cpmax expected Cpmax 200 in normal dog 200 in normal dog expected range expected range in normal dog in normal dog

Cp (ng/ml) 100 Cp (ng/ml) 100 expected Cp min expected Cpmin in normal dog in normal dog 0 0 23 23 days days

adjust dose adjust interval 300 in diseased dog 300 in diseased dog

expected Cp expected Cpmax max 200 in normal dog 200 in normal dog expected range expected range in normal dog in normal dog

Cp (ng/ml) 100 Cp (ng/ml) 100

expected Cpmin expected Cpmin in normal dog in normal dog 0 0 23 23 days days Issues to note:

 In the standard regimen (upper left) Cp varies 4‐fold (Cpmax = 200, Cpmin = 50), as expected for a dosing interval that is 2 times the half‐life for a drug.  If no adjustments are made (upper right): Cp now varies only 2‐fold (300 to 150), as expected for a protocol in which the RDI is 1. However, peak levels are much greater now than the standard regimen (300 vs 200, respectively). This happened because with the smaller RDI, there is greater accumulation. If this was a safe drug, nothing to worry about. But if levels over 250 happened to be toxic, what was a safe regimen is now toxic.  The adjustment was made to dose (lower left): Note that the variation in plasma levels decreased from the standard protocol (it is now only 2‐fold), but because dose was decreased, overall plasma levels were decreased. A decrease in variation will always happen if you make adjustment only to dose because the RDI gets smaller in the diseased animal.  The adjustment was made to the interval (lower right): Note that plasma variation remains identical to the standard therapy. This is because the RDI is the same. The drawback is that now the dosing is every 16 h, which means the timing of the dose during the day will alter from day to day – not a good plan if you want high compliance.  Which adjustment is preferred? There is not a standard answer to this question – it depends on what the goal of the therapy is and how the drug works. As a general rule variations in Cp are usually undesirable, and thus adjusting dose seems to be preferred (lower left), but if for whatever reason peak levels are important then adjusting interval might be preferred (e.g., with an antibiotic or cancer chemotherapy sometimes the peak is important for maximal kill – although you should note that this is not always the case – you need to know your drug and how it works to make this judgment).

103

VM522P / VM7522 Pharmacology WIMU Fall Can one adjust dose and interval to keep drug administration a convenient daily multiple? Consideration of a few ideas will enable us to make even more adroit changes to the dosing scheme:  Under the standard therapy the total daily dose of the drug was 0.3 mg/kg (i.e., 0.1 mg/kg given three times a day).  Take note that the impact factor can be applied to the total daily dose, or to the single dose. o Single dose reduced from 0.1 to 0.05 mg/kg, given three times a day means total daily dose is 0.15 mg/kg. o Alternatively, just reduce the total daily dose (0.3 mg/mg) by 50% = 0.15 mg/kg.

 Recall from our earlier example that when the total daily dose of the drug was kept equal, the Cpavg was a constant. What changed with dosing frequency was the variation in plasma level.  Thus we can take the new total daily dose (0.15 mg/kg), and split it into two doses (0.075 mg/kg) given 12 h apart (RDI would = 1.5).

 Cpavg is the same because the total daily dose is the same (0.15 mg/kg). The variation in plasma level would be somewhere between that in the lower left and lower right graphs shown above. (More variation than when RDI =1 but less than when RDI = 2).

This clever manipulation is also useful even if you are not making changes to the total daily dose! The same principle can be applied when changing dosing frequency for convenience:  Figure out total daily dose.  Divide total daily dose by the number of times the drug is to be taken per day.

 This will produce the same Cpavg, but the Cpmax and Cpmin will change. Questions: A drug is normally given 3 times a day at 10 mg per dose. Your client complains that he cannot maintain such a schedule because he is gone for more than 8 h at a time.

How would you change the schedule to 2x a day dosing and yet keep total drug exposure (Cpavg) constant? Total daily dose = 3 x 10 mg = 30 mg Divide 30 mg number of doses per day: 30 mg / 2 times a day = 15 mg/dose

Would Cpmax be higher or lower in the new regimen versus the old regimen?

Cpmax would be higher – Although Cpavg remains constant because total daily dose is constant, because there is more time between doses, the amount of variation between Cpmax and Cpmin increases. Thus Cpmax would be higher.

Would Cpmin be higher or lower in the new regimen versus the old regimen?

Cpmin would be lower – Even though Cpmax is higher, the greater variation causes Cpmin to fall to a level below the prior Cpmin. Is this a safe alteration? It would depend on the drug and the goals of the therapy!

104

VM522P / VM7522 Pharmacology WIMU Fall L16: Clinical Pharmacokinetics

Objectives:

By the end of the class, you should be able to do the following:

1. Interpret the clinical relevance of pharmacokinetic data reported on a drug label or package.

2. Identify and use critical pharmacokinetics (PK) information on drug label or packages to make therapeutic decisions.

Drug label or package It is a legal document that is given with a prescription or over‐the counter medication to provide additional information about that drug.

The U.S. Federal Food, Drug and Cosmetic Act (FFDCA) defines "labeling" as all labels and other written, printed, or graphic matter upon any article or any of its containers or wrappers, or accompanying such article.

Drug label or package: sections The package insert is divided into several sections and contains information required by the Food and Drug administration (FDA). One of the sections corresponds to CLINICAL PHARMACOLOGY. This section may include subsections, such as mechanism of action, pharmacodynamics, pharmacokinetics, microbiology and pharmacogenomics.

PK section of the label Information in the PHARMACOKINETICS subsection of the CLINICAL PHARMACOLOGY section is generally organized under descriptive subheadings (e.g., absorption, distribution, metabolism, excretion, pharmacokinetics in specific populations, and drug interactions).

Labels are specific for products and formulations Labels are not only drug specific but also product specific; hence, information on a drug label is not representative of a different formulation of the same drug. All brands are not equal. Keep in mind that the information on the labels may have changed. Look for any updates. Always read product labels. How to use/interpret a PK section? The first step is to read the label and identify any critical PK process.

You will need to evaluate the PK section when you change a drug dosage regimen or when you have to add drugs to your treatment plan.

Most of the time you will need to change the dosage regimen when the patient does not respond well to the treatment, based on the therapeutic objectives of your treatment plan. In general, those changes include the following: 105

VM522P / VM7522 Pharmacology WIMU Fall  Increasing the exposure to the drugs by increasing the dose as well as decreasing the dose interval

 Changing the route of drug administration/formulation

 Adding drugs to the treatment plan

When making a decision about treatment change, you must take into consideration the factors associated with drug absorption, distribution, metabolism and excretion of drugs.

 Absorption o Dose proportionality vs non‐proportionality . Dose proportionality is established for a range of doses for certain drugs. For those medications, you can predict the changes in drug exposure when you modify the dose within that particular dose range. If there is no dose proportional absorption, it would be safer to modify the dose intervals, by increasing/decreasing the frequency of administrations.

. Pay attention in situations where there is non‐linearity—especially in cases where the lack of proportionality is associated with greater or lower than proportional changes. If higher doses will lead to greater‐than‐proportional absorption, be wary. This change could trigger toxic effects, particularly if the margin of safety is narrow. If higher doses will lead to less‐than‐proportional absorption, you may have an issue with drug efficacy. o Consider the impact of changing the drug administration route on the bioavailability of the drug. o Evaluate factors that may affect the process of drug absorption: . Transporters: transporters are potential sites for drug interactions, which is important if you are planning to add a medication to the treatment plan. BEFORE treating your patient, rule out the possibility of any interaction between the drugs you are planning to use. . Effect of pH on drug solubility  Some drugs are more sensible to pH changes. A common situation is the co‐administration of gastric antacids. . Effect of food on drug absorption  The PK of some drugs is affected by the presence or lack of food in the gastrointestinal system.

 Distribution o Dose proportionality vs non‐proportionality  Metabolism o Dose proportionality vs non‐proportionality o Site of drug metabolism 106

VM522P / VM7522 Pharmacology WIMU Fall o Auto‐induction o Mechanism of drug biotransformation

. Metabolism by P450 isoenzymes  Excretion o Dose proportionality vs non‐proportionality o Site of drug excretion . Transporters: transporters are potential sites for drug interactions. General observations  Breed: Look for particularities associated with breeds (for examples P‐glycoprotein in collies).  Age: watch for age‐related dose issues, especially with young or geriatric patients.  Drug‐drug interactions: Check the label for any potential drug‐drug interactions.

Specific pharmacokinetic parameters

Half‐life (t1/2) is the PK parameter you will use the most, particularly when you change dosage regimens. As covered in previous lectures, one of the applications of the t1/2 is the estimation of the time to steady state plasma drug concentrations.

You will need to estimate the time necessary to reach steady state because it represents the best time to assess the pharmacological response to a treatment. Remember that steady state is considered to occur around 5 * t1/2.

You will also use the t1/2 of a drug to estimate the time necessary for the plasma drug concentration to decrease by X fold. This will be important to establish washout periods between treatments. For example, if you need to switch NSAIDs, you must wait 10 t1/2 after the last administration of NSAID‐A before treating your patient with NSAID‐B. Check the label for the t1/2 of the drug and use the t1/2 value to estimate an optimal washout period.

In the pharmaceutical inserts, PK is reported as mean +/‐ standard error of the mean (SEM) or standard deviation (SD) or range. You need to decide which value to use. If the variability of the PK parameters is reported as SD or SEM, use the mean value. If the variability of the PK parameters is reported as range, use the upper or lower values of the range, depending on the margin of safety of the drug. If the margin of safety of the drug is low, use the most conservative value. For example, if I need to adjust the loading dose of a drug with a very narrow margin of safety, it would be prudent to use the lower Cave of the range reported on the label.

107

VM522P / VM7522 Pharmacology WIMU Fall Example:

108

VM522P / VM7522 Pharmacology WIMU Fall

List of key works for interpreting labels

Common red light key words Common green light key words

Lack of dose proportional PK /ADME Dose Proportional PK /ADME (or non‐proportional or non‐linear or zero order kinetics) (linear or first order kinetics) or dose proportional PK Transporter mediated ADE, P‐glycoprotein Large, intermediate or low VD Cytochrome P450 Large, intermediate or low protein binding Induction/inhibition and auto‐induction of metabolism Slow or fast clearance Narrow margin of safety / narrow therapeutic window / Short or long HL narrow therapeutic index Drug to drug interactions Small or large bioavailability Age / breed / gender related PK Large inter‐individual variability In ADME Excretion by an affected organ (e.g., renal elimination in a patient with kidney disease)

109

VM522P / VM7522 Pharmacology WIMU Fall

Part 3 – Neuropharmacology (S. Appleyard) L17: Review of Neurotransmission

Reading assignment: NOTE: Riviere and Papich has no section dedicated to this topic An alternative would be:  Rang et al., 6th Ed. Chapter 9, pages 136‐143.  Adams, H.R. (2001) Introduction to neurohumoral transmission and the autonomic nervous system. In Veterinary Pharmacology and Therapeutics. 8th Edition; Iowa State University Press: Ames, IA; pp. 69‐90.

Objectives:

1. Know the major steps in neurotransmission and recognize these as important targets for therapeutic intervention.

2. Know the importance of co‐transmitters and how synaptic transmission can be modulated

Outline: I. Steps in Neurotransmission II. Examples of how drugs modulate Neurotransmission III. Modulation of Neurotransmission IV. Co‐transmitters

Brief Review of Neurotransmission

Nerve impulses are sent to and from the CNS to coordinate smooth, skeletal and cardiac muscle contractions and glandular secretions.

Signals travel down nerves as electrical activity. That signal then has to get from the nerve to the downstream target. This is achieved through neurotransmission, also called synaptic transmission.

A Synapse is the junction of a nerve’s axonal ending with another nerve cell (neuron), a muscle cell or glandular cell.

Neurotransmitters are chemicals which are released to relay signals between neurons or a neuron and another cell.

WHY SHOULD YOU CARE?

Many drugs act by either mimicking or modulating synaptic transmission.

110

VM522P / VM7522 Pharmacology WIMU Fall I. Steps in Neurotransmission

Neurotransmitters are Synthesized and Stored

1. Many precursors are taken up into nerve terminals by specific transporters or uptake molecules:

eg: Choline

2. Transmitters are synthesized by specific enzymes

eg: choline acetyltransferase

3. Transmitters are then transported into vesicles*

Again this is normally by specific uptake molecules

* Some transmitters are synthesized in the vesicle e.g. dopamine is synthesized in the cytoplasm by tyrosine hydroxylase and then transported into vesicles, but norepinephrine is synthesized in the vesicle from dopamine by dopamine beta hydroxylase

Postsynatptic Nerve Cell Terminal

3 NT NT

NT NT NT 2 Syn Enz precursor 1 pre-cursor

111

VM522P / VM7522 Pharmacology WIMU Fall Neurotransmitters are Released Following Stimulation

4. The nerve terminal is depolarized by the arrival of an action potential

Depolarization is due to voltage dependent sodium channels opening, allowing positive sodium ions to enter the terminal, which causes a depolarization

5. Calcium enters the terminal through voltage gated ion channels

The depolarization causes voltage dependent calcium channels to open, allowing calcium to enter the presynaptic terminal.

6. Neurotransmitter is released by calcium‐dependent exocytosis

The entry of calcium activates specific proteins that cause the synaptic vesicles to fuse with the presynaptic membrane releasing the neurotransmitter into the synaptic cleft.

2+ Ca Postsynatptic Nerve 5 Cell Terminal 6 Na+ Ca2+ NT NT 4 Ca2+ Ca2+ Ca2+ NT NT NT Ca2+ (depolarization) NT NT Ca2+ NT Ca2+ NT NT NT Syn Enz precursor pre-cursor

112

VM522P / VM7522 Pharmacology WIMU Fall Neurotransmitter Stimulates Post‐Synaptic Cell

7. Binds post‐synaptic receptor

The neurotransmitter diffuses across the synaptic cleft and binds to specific receptors on the postsynaptic membrane.

8. Causes a conformational change that activates a signal transduction cascade

Neurotransmitters are generally agonists. An agonist binding to its receptor causes a conformational change to stabilize the receptor in its active or signaling state.

The signal transduction mechanism activated will depend on the receptor type:

Many receptors are ligand gated ion channels, e.g. nicotinic acetylcholine receptors (nAChR), glutamate and GABAA receptors.

Many receptors are G protein coupled receptors, e.g. muscarinic acetylcholine receptors, adrenergic receptors.

9. Receptor activation causes the post‐synaptic cell to be either activated or inhibited:

i. Activated examples: Glutamate, nAChR

ii. Inhibited examples: GABA, opioid

Ca2+ Postsynatptic Nerve Cell Terminal

Na+ Ca2+ NT 7a Ca2+ Ion channel

Ca2+ Na+ Ca2+ NT NT Ca2+ Ca2+ NT (depolarization) Ca2+ 9 NT Ca2+ NT 8 NT Syn Enz precursor pre-cursor cAMP 7b Gprotein

coupled

113

VM522P / VM7522 Pharmacology WIMU Fall Termination of Response

10. Cessation of neurotransmitter release: i. Repolarization of the terminal Following depolarization of the presynaptic membrane the voltage dependent sodium channels inactivate (shut) and potassium channels open allowing positive potassium ions to leave the terminal. Positive ions leaving the terminal means that it becomes more negative and the membrane potential returns to its normal hyperpolarized state. Therefore calcium channels close and no more calcium enters the terminal. ii. Sequestering of intracellular calcium There are many mechanisms for removing free calcium from inside cells (e.g. by specific uptake or sequestering into internal stores and by binding to proteins that are present to buffer the concentration of free calcium). Once all the free calcium is either bound or sequestered, vesicle release stops.

11. Removal of neurotransmitter from synaptic cleft:

There are 3 main ways the neurotransmitter is removed from the cleft. Which method dominates depends on what the neurotransmitter is.

i. Diffusion This tends to be slower and therefore for most neurotransmitters there are other active processes that remove the neurotransmitter from the cleft

ii. Degradation Many neurotransmitters are inactivated by metabolism or degradation. e.g. acetylcholine (ACh) is removed from the synaptic cleft or neuromuscular junction within 250‐ 500 usecs by acetylcholine esterases (AChE) rapidly metabolizing or breaking down the active transmitter.

iii. Specific Reuptake Many neurotransmitters are actively removed by specific uptake proteins. e.g. norepinephrine is removed from the synaptic cleft by specific uptake proteins that rapidly take up the active transmitter (back into the pre‐synaptic terminal to be recycled or into other surrounding cells).

12. Inactivation or desensitization of receptors If neurotransmitters are not removed rapidly from the synaptic cleft then the post‐synaptic receptors often will desensitize or inactivate. Normally neurotransmitters, such as ACh, are rapidly removed (see above) and therefore desensitization does not occur. However if you give an drug that is an agonist, but which is not rapidly removed from the cleft, or if you inhibit the enzyme that would normally metabolize the transmitter (such as AChE) then the receptors will be stimulated longer and could desensitize. Desensitization of the receptors could block further synaptic transmission and in the case of nAChRs at the neuromuscular junction this would cause paralysis.

114

VM522P / VM7522 Pharmacology WIMU Fall

Ca2+ Postsynatptic Nerve Cell NT Terminal X + 10 NT Na NT Ion channel NT NT

NT NT NT X

NT 12 NT NT NT NT Syn Enz NT precursor pre-cursor 11c X NT 11a NT Gprotein 11b NT metabolite coupled Deg Enz

Why you should care!

Synaptic Transmission is required for life!

Many drugs interfere with some point of synaptic transmission.

II. Examples of how drugs modulate Neurotransmission

All These Steps are Potential Targets for drugs or toxins:

1. Modulating production of transmitter:

There are currently no good therapeutic examples in vet med. However, L‐DOPA the precursor for dopamine is used in human Parkinson patients to increase dopamine transmission.

2. Modulating release of transmitter:

i. Depolarization

Example: Local anesthetics block sodium channels and are used to reduce pain sensation. Blocking sodium channels means that the nerve terminal will not depolarize and synaptic transmission cannot occur. Interestingly they preferentially affect sensory nerves (that are unmyelinated and have small diameter axons). This is why they are useful as analgesics and don’t just stop all nerve transmission.

115

VM522P / VM7522 Pharmacology WIMU Fall Many toxins also block sodium channels. Tetrodotoxin, from the puffer fish, is a very potent sodium channel blocker. As it is so potent it affects all nerves and causes paralysis and death.

ii. Calcium entry

Example: Calcium channel blockers reduce calcium entry, both in the nerve terminal and in the postsynaptic cell, e.g. blood vessels. For example, less calcium entry decreases the contraction of blood vessels and causes vasodilation, which is why calcium channel blockers are useful as anti‐hypertension drugs. However, at high enough concentrations calcium channel blockers will also reduce neurotransmitter release.

Omega conotoxins from the venom of the marine cone snail and some snake toxins also block calcium channels.

iii. Vesicle release

Examples: Botulinum and tetanus toxins both cleave calcium‐sensitive proteins that are required for vesicle release. Therefore, both toxins prevent transmitter release.

However the symptoms are completely different. Why?

Botulinum toxin causes a “flaccid paralysis”, where there is no muscle contraction. Symptoms include muscle weakness and decreased muscle tone (often seen in the tail, eyelids and tongues first). The horses or cows are often lying down and have difficulty swallowing.

This is because botulinum toxin preferentially affects the neuromuscular junction and acetylcholine release. Therefore, the symptoms are related to a loss of muscle function.

Therefore, the symptoms are related to a loss of muscle function.

Tetanus causes a “rigid paralysis”. Where there is unopposed muscle contraction and spasms. The ears will often be held pricked, the gait will be stiff and the tail held out and stiff.

This is because tetanus preferentially blocks inhibitory neurotransmission (e.g. glycine and GABA release). With no inhibitory transmission the excitatory pathways act unopposed and so you see spasms and rigidity.

3. Modulating the effect of the neurotransmitter:

This is one of the most common mechanisms by which drugs produce their effects, either at a synapse or on the target organ for a nerve or hormone

i. Binding to post‐synaptic receptor

An agonist mimics an endogenous neurotransmitter or hormone

 examples include: epinephrine, morphine (mimics endogenous opioids)

An antagonist mimics an endogenous neurotransmitter or hormone

 examples include: beta receptor antagonists, such as sotalol; muscarinic antagonists, such as atropine

Another snake venom alpha‐bungarotoxin is an irreversible antagonist at the nicotinic acetylcholine receptor at the neuromuscular junction. Blocking this receptor causes paralysis and death.

116

VM522P / VM7522 Pharmacology WIMU Fall

ii. Altering the post‐synaptic response

A few drugs act by increasing or decreasing the size of a drug effect through modulating the size of the cellular response initiated.

 Example: dandrolene is a muscle relaxant that reduces the amount of calcium released in muscles following motor nerve stimulation, reducing the size of the muscle contraction.

4. Modulating termination of the effect:

i. Diffusion can’t be modulated!

ii. Removal of neurotransmitter from cleft

a) Metabolism

The classic example we will be discussing is acetylcholine esterase (AChE) inhibitors. These drugs are used clinically to prolong/increase the actions of ACh. For example, myasthenia gravis (MG) is an autoimmune disease that attacks the receptors for ACh in the muscle. Increasing the concentration of ACh in the neuromuscular junction improves ACh signaling and muscle function. Remember if you increase either agonist concentration or receptor concentration you increase bound/activated receptor. Therefore, when the receptor concentration decreases, like in MG, increasing the agonist concentration will compensate.

b) Re‐uptake

An example we will be discussing is fluoxetine (prozac), which blocks serotonin reuptake, increasing serotonin levels and prolonging its action. As serotonin is a key neurotransmitter for mood and to relieve anxiety, fluoxetine can help with some obsessive‐compulsive disorders, especially in dogs.

iii. Inactivation or desensitization of receptors:

There are few widely used examples, but succinylcholine, an agonist at the nAChR, causes muscle paralysis by prolonged activation of the nAChR leading to desensitization of the muscle.

III. Modulation of Neurotransmission

1. Pre‐synaptic Modulation

As well as binding to and activating receptors in the post‐synaptic terminal some neurotransmitters will bind and activate receptors in the pre‐synaptic terminal. Depending on whether those receptors are excitatory or inhibitory this can either ↑ or ↓ probability the neurotransmier (NT) is released

i. Auto‐regulation: NT regulates its own release

ii. Cross‐regulation: Another NT or hormone regulates release of the NT

117

VM522P / VM7522 Pharmacology WIMU Fall This is important as some drugs act at these presynaptic receptors to alter (generally inhibit) another transmitter’s release and action. For example: xylazine, an alpha2 adrenergic receptor agonist used as a sedative. It inhibits synaptic transmission in part by decreasing transmitter release. cisapride, metoclopramide are pro‐kinetics in the gut that work by activating pre‐ synaptic 5HT4 receptors to increase the release of acetylcholine, which then acts at muscarinic receptor to increase GI movement.

2. Post‐Synaptic Modulation

The size of a response to a neurotransmitter and any drug that is mimicking its effect by activating the same receptor can also be altered at the level of the post‐synaptic cell. Some examples that could affect the size of a drug response are:

i. Desensitization: As discussed before, strong and prolonged stimulation of a postsynaptic response often results in desensitization. This can happen quickly (in seconds to minutes), but more often occurs following longer exposure to a drug (days to weeks)

a. nAChR in muscle desensitize to prolonged activation (conformational change). This occurs quickly (seconds). You can think about it in terms of the muscle not being able to tolerate constant activation as this would cause pain and rigid paralysis.

b. ‐adrenergic receptors are internalized following prolonged (days to weeks) activation (these receptors are phosphorylated and internalized). In this case the desensitization occurs more slowly and is thought to be a way that the body compensates for excessive prolonged beta‐ adrenergic receptor activation.

ii. Hypersensitivity: Chronic under stimulation by denervation (especially of muscle) or drug antagonism (e.g. beta blockers) can cause postsynaptic hypersensitivity (generally due to increased expression of post‐synaptic receptors). This is the reverse of desensitization. The cells are “starved” of the beta adrenergic or nerve signal and so up regulate the receptors as a way to try to “find” their normal input.

iii. Synergism. Synergy is when two compounds given together have a much larger effect than when either is given alone. This can occur physiologically when two neurotransmitters have synergistic effects. It can also occur between a neurotransmitter and drug as well as between two drugs

For example benzodiazepine, barbituates and alcohol all produce effects by increasing the efficacy of the inhibitory transmitter GABA at its receptor.

This is also important as if these two drugs were given at the same time you cause dangerously high levels of sedation due to excess CNS inhibition.

e.g. Opioids and barbituates or other CNS depressants. Opioids decrease excitatory transmission and barbituates increase inhibitory transmission. If both occur at the same time you can again cause excess CNS depression (inhibition).

118

VM522P / VM7522 Pharmacology WIMU Fall IV. CO‐TRANSMITTERS

1. Other transmitters can be co‐released (probably the rule rather than the exception).

i. Some are co‐packaged in the same vesicle, which means they will be released at the same time.

ii. Some are packaged in separate vesicles, which mean they may be differentially released, e.g. peptides often require a greater (larger or more prolonged) stimulation than fast neurotransmitters like glutamate.

2. In the case of the ANS, these co‐transmitters are also autonomic transmitters and are frequently referred to as non‐adrenergic, non‐cholinergic or NANC neurotransmitters

 Examples include: ATP, Vasoactive intestinal peptide (VIP), substance P and calcitonin gene related peptide (CGRP)

Why should you care? This means that even if you block the effect of one transmitter, e.g. ACh, if there is a co‐ transmitter you are not blocking all the effects of that nerve being stimulated.

3. Co‐transmitters can act synergistically with each other.

119

VM522P / VM7522 Pharmacology WIMU Fall

L18: Overview of the Autonomic Nervous System (ANS)

Reading assignment: Riviere and Papich, Chapter 5, page 101 An alternative would be:  Rang et al., Chapter 9, pp 131‐134.

Objectives:

1. Understand the divisions and structure of the autonomic nervous system (ANS) so you can identify the site of action of various autonomic drugs.

2. Understand the nature of the various chemical mediators used within the ANS and the receptor types activated by these transmitters so you can identify drug targets within this system.

3. Understand the effects of autonomic activation on various end organs so you can predict the actions of various autonomic drugs and can identify the component of the ANS that the drug is acting upon.

4. Also understand the structure and chemical nature of the peripheral somatic efferent nervous system so that you can identify drug targets within this system, especially the neuromuscular junction blockers.

Outline:

I. Divisions and Structure: 1. Parasympathetic 2. Sympathetic 3. Enteric

II. Chemical Mediators and Receptors

III. Function

IV. Somatic efferent nervous system (motor neurons)

120

VM522P / VM7522 Pharmacology WIMU Fall

The ANS is one of the primary mechanisms by which the CNS controls homeostatic reflexes, e.g. heart rate, blood pressure, GI function. These are things that happen automatically, hence autonomic. Clinically you will use many drugs that act either directly or indirectly to alter the ANS and influence these functions.

I. Divisions and Structure of the ANS

The ANS is divided into two main branches, the Parasympathetic Nervous System (PNS) and the Sympathetic Nervous System (SNS). In each case the outflow from the CNS is comprised of preganglionic neurons that synapse on postganglionic cell bodies located in autonomic ganglia. The post‐ganglionic fibers then innervate the target organ. However, the PNS and SNS have significant differences, both functionally and anatomically:

1. Parasympathetic Nervous System (PNS)

i. “Rest and digest” branch

ii. “Craniosacral” system—parasympathetic fibers emerge from the CNS and the sacral division of the spinal cord

iii. The cell bodies of preganglionic fibers lie in the brain stem and the sacral section of the spinal cord and project to end organs

iv. Post‐ganglionic cell bodies are typically within the target end‐organ

2. Sympathetic Nervous System (SNS)

i. “Fight or flight” branch

ii. “Thoracolumbar” system—sympathetic fibers emerge from the thoracic and lumbar portions of the spinal cord

iii. Cell bodies of preganglionic fibers reside in the lateral horn of the grey matter of the spinal cord

iv. The post‐ganglionic cell bodies reside in the paravertebral chain of sympathetic ganglia or prevertebral ganglia (celiac and hypogastric) and from these sites project to the end organs

v. Cells of the adrenal medulla are equivalent to post‐ganglionic neurons in the SNS

121

VM522P / VM7522 Pharmacology WIMU Fall

Parasympathetic Nervous System

From Riviere and Papich, Veterinary Pharmacology and Therapeutics. 122

VM522P / VM7522 Pharmacology WIMU Fall

Sympathetic Nervous System

From Riviere and Papich, Veterinary Pharmacology and Therapeutics. 123

VM522P / VM7522 Pharmacology WIMU Fall 3. Enteric Nervous System (ENS)

The enteric NS is located in the GI. It is also considered to be part of the ANS, as again its actions are involuntary or automatic. It coordinates the complex control of the GI system. It is innervated by both parasympathetic fibers and sympathetic fibers, which regulate its function. The ENS is primarily made up of two plexuses:

1. Myenteric plexus—controls muscle tone, rhythmic contractions, peristaltic waves

2. Submucosal plexus—controls secretion, absorption, and some localized contractions

Enteric Nervous System

Images are from Guyton, Textbook of Medical Physiology

124

VM522P / VM7522 Pharmacology WIMU Fall

II. Chemical Mediators and Receptors within the ANS

There are key similarities and differences in the neurotransmitters used in the SNS and PNS:

1. Preganglionic neurons (in both PNS and SNS)—use acetylcholine as their transmitter

i. Post‐ganglionic cells activated by neuronal‐type acetylcholine receptors (nAChR)

ii. nAChR are ligand‐gated ion channels (ionotropic receptors)

a. When ligand binds a pore is opened that allows ions to pass through

b. Highly permeable to sodium ions (some calcium and potassium) when ACh is bound

c. Entry of positive ions like sodium and calcium depolarizes (activates) cells

2. Post‐ganglionic neurons of the PNS—use acetylcholine as their transmitter

i. ACh from PNS neurons activate muscarinic acetylcholine receptors (mAChR)

ii. mAChR are G‐protein coupled receptors

Remember: which produces a faster effect – a ligand-gated ion channel or a G protein coupled receptor?

125

VM522P / VM7522 Pharmacology WIMU Fall 2+ iii. Odd numbered mAChR subtypes activate phospholipase C/ diacylglycerol DAG / IP3 / Ca pathways

Remember these signaling pathways are usually excitatory

a. M1 are primarily neuronal

b. M3 are primarily found on glands and smooth muscle

iv. Even numbered mAChR subtypes decrease cAMP formation and activate K+ channels

Remember these signaling pathways are usually inhibitory

a. M2 — Cardiac

126

VM522P / VM7522 Pharmacology WIMU Fall

3. Post‐ganglionic neurons of the SNS—use norepinephrine (NE) as their transmitter (* see exception below)

i. NE from SNS neurons activates adrenergic receptors in the end‐organ

ii. Adrenergic receptors are G‐protein coupled receptors

iii. Main divisions are ‐adrenergic receptors and ‐adrenergic receptors

2+ a. 1‐adrenergic receptors activate phospholipase C / diacylglycerol (DAG) IP3 / Ca pathways. (tends to be excitatory)

+ b. ‐adrenergic receptors decrease cAMP and activate K channels (tends to be inhibitory) c. all ‐adrenergic receptors increase cAMP (tends to be excitatory)

* The exception is that post‐ganglionic sympathetic fibers innervating the sweat gland are cholinergic (i.e. they release acetylcholine, ACh). The receptor activated by both the SNS and PNS in the sweat glands is muscarinic.

The Adrenal medulla receives SNS preganglionic innervations and when activate it releases primarily epinephrine (non‐selective ‐ and ‐receptor agonist) into the general circulation.

ADRENAL MEDULLA

Chromaffin Cells

Epinephrine (+) Dilates Airways (+) Mental Alertness

(+) ACTH & TSH (+) Cardiac Output

(+) Muscle Contraction & Efficiency (+) Glycogenolysis

(+) Fatty Acid Release (-) Intestinal Motility

127

VM522P / VM7522 Pharmacology WIMU Fall

Some tissues the ANS co‐releases non‐adrenergic, non‐cholinergic transmission (NANC)

1. Some examples of NANC transmitters:

a. Serotonin

b. ATP

c. Nitric oxide (NO)

Why should you care! This means that even if you block the actions of the primary ANS neurotransmitters at their receptors (ACh and NE) there will still be a small effect when the ANS is activated due to the actions of these co‐ transmitters.

128

VM522P / VM7522 Pharmacology WIMU Fall III. Function of the ANS

Some functions controlled by the ANS (see table, page 125):

1. Changes in heart rate and force of contraction

2. Changes in blood flow and pressure.

3. Control of bronchioles for airflow (and secretions)

4. Adjusting pupil diameter to changes in light

5. GI function

6. Mobilizing fuels (fat and glucose) in response to exercise

7. Bladder functions

To remember which branch does what think: “flight or fight” – SNS dominates digestion of a meal – PNS dominates

Proviso: There are always exceptions!

129

VM522P / VM7522 Pharmacology WIMU Fall

PNS and SNS function have opposite effects on many end‐organs:

1. Heart: SNS Increase PNS Decrease (Rate and Force)

2. GI tract: SNS Inhibits motility PNS Increase motility and digestion

3. Pupil: SNS Dilate PNS Contract

4. Bronchi: SNS Dilate PNS Contract

5. Bladder: SNS Relaxes Bladder PNS Constricts Bladder Constricts Sphincter Relaxes Sphincter

Some systems are only innervated (or affected) by one system:

1. Most vasculature only have SNS innervation — constricts vessels to skin and viscera dilates vessels to skeletal muscle

but blood vessels do have muscarinic receptors and so will respond to circulating muscarinic agonists

2. Eye – predominately PNS:

a. Stimulation of the PNS releases ACh, which then contracts ciliary muscle of eye via activation of mAChR for near vision

b. Stimulation of the PNS releases ACh, which then increases tear formation in the lacrimal gland of the eye via activation of mAChR.

Some systems respond the same to both systems

1. Salivary glands are activated by both the PNS (ACh through mAChRs) and the SNS (NE through  adrenergic receptors).

130

VM522P / VM7522 Pharmacology WIMU Fall

Some systems require activation of both systems

1. Male sexual function (parasympathetic promotes erection and sympathetic produces ejaculation)

IV. Somatic efferent nervous system (motor neurons)

1. Structure

- Cell bodies lie in the anterior horn of spinal cord grey matter

- Fibers exit through the ventral root

- site of contact between the motor neuron and the muscle is the neuromuscular junction (NMJ)

- One motor neuron can innervate from 3 to several hundred muscle fibers forming a motor unit (depending on how precise vs. strong the muscle control needs to be)

- Each motor unit is innervated by only one ‐motor neuron

2. Chemical neurotransmission

1. Motor neurons use acetylcholine as their neurotransmitter

2. Cause muscle to contract through activation of muscle‐type nAChR

a) Like neuronal‐type nAChR, these are ligand gated‐ion channels that depolarize muscle fibers

b) Muscle contraction also requires opening of voltage‐dependent Na+ channels for the depolarization to spread through the t‐tubules

c) Muscle contraction requires the release of Ca2+ from the sarcoplasmic reticulum

131

VM522P / VM7522 Pharmacology WIMU Fall 3. Function

Movement!

SUMMARY

ACh skeletal (nic) muscle Somatic Efferent Nervous System

heart ACh NE vasculature (nic) viscera ACh ACh sweat (nic) (mus) glands

ACh EPI (nic) NE adrenal medulla System Nervous Central Sympathetic Nervous System

viscera ACh ACh glands (nic) (mus) heart Parasympathetic Nervous System

ANATOMICAL LOCATION PREGANGLIONIC POST‐GANGLIONIC TRANSMITTER TRANSMITTER FIBERS FIBERS (GANGLIA) (ORGAN) Sympathetic Thoracic/Lumbar Short Long ACh NE Parasympathetic Cranial/Sacral Long Short ACh ACh

132

VM522P / VM7522 Pharmacology WIMU Fall

From Riviere and Papich, Veterinary Pharmacology and Therapeutics.

133

VM522P / VM7522 Pharmacology WIMU Fall

L19, 20: Cholinergic Pharmacology

READING ASSIGNMENT:

Riviere and Papich, See Chapter 5, pages 113‐114 and Chapter 7 for a general overview. See chapter 9 for Neuromuscular Blocking Agents. An excellent alternative overview is in Rang et al. (6th edition), Chapter 10, pp 144‐167.

OBJECTIVES:

1. Be able to describe the mechanisms of acetylcholine neurotransmission and the specific actions and locations of nicotinic (nAChR) and muscarinic acetylcholine receptors (mAChR). 2. Be able to explain the systemic effects of cholinergic agonists and antagonists from knowledge of the specific actions and locations of nAChR and mAChR. 3. Know examples of drugs that act at each type of receptor (agonists and antagonists) and drugs that inhibit acetylcholinesterase. 4. Know what types of medical situations use drugs that alter cholinergic function and problems and dangers associated with the use of these agents.

CHOLINERGIC PHARMACOLOGY OUTLINE:

I. Overview of Cholinergic Neurotransmission II. Muscarinic Agonists III. Muscarinic Antagonists IV. Neuronal Nicotinic Receptor Drugs: Ganglionic Blockers V. Muscular Nicotinic Receptor Drugs: Neuromuscular Junction Blockers VI. Acetylcholinesterase Inhibitors

I. OVERVIEW OF CHOLINERGIC NEUROTRANSMISSION

1) Acetylcholine (Ach) is synthesized from choline and acetylCoA via choline acetyl transferase (CAT)

2) ACh is then packaged in vesicles and released upon nerve terminal depolarization

134

VM522P / VM7522 Pharmacology WIMU Fall

3) ACh acts on: i. nicotinic AChR (ligand‐gated ion channel). Remember these receptors are found on the postganglionic neurons of both the PNS and SNS (neuronal nAChR) as well as at the neuromuscular junction (muscle nAChR) and in the CNS (neuronal nAChR).

ii. muscarinic AChR G‐protein coupled receptor) These receptors are found on the target organs of the PNS and in the CNS.

4) Termination of ACh action is by acetylcholine esterase (AChE)

choline acetate

M1/3 Gq 2+ M DAG / IP3 Ca 2 (smooth muscle contraction choline ACh AChE mAChR or glandular secretion) Gi AcetylCoA CAT cAMP (in heart: decrease rate and force) CoA ACh ACh Na+ K+ depolarization (nerve or muscle activation) nAChR voltage-dependent sodium channel cholinergic nerve terminal Na+ e.g. all pre-ganglionic nerves in the ANS, or postsynaptic cell (nerve, muscle, or gland) all post-ganglionic nerves of the PNS

Therefore drugs can regulate cholinergic neurotransmission in several ways: 1. Agonists at muscarinic and nicotinic ACh receptors will mimic the actions of ACh. Muscarinic agonists will mimic the effects of activating the PNS. Nicotinic agonists will have more complex effects as nAChRs are found on post‐ganglionic cell bodies of both the PNS and SNS, as well as at the neuromuscular junction.

2. Antagonists at muscarinic and nicotinic ACh receptors will block the actions of ACh Muscarinic antagonists will block the effects of activating the PNS. The size of the effect will depend on the tone of the PNS, i.e. how much the PNS is activated in that animal. Nicotinic antagonists will also have more complex effects as nAChRs are found on postganglionic cell bodies of both the PNS and SNS, as well as at the neuromuscular junction. However, there are antagonists that can select for muscle nAChRs vs. neuronal (or ganglion) nAChRs.

3. Inhibitors of AChE (the enzyme that breaks down ACh), will increase ACh levels. Inhibiting the breakdown of ACh will prolong the time it is in the synapse, increasing its concentration and its effects. At high enough concentrations of AChE inhibitors ACh concentrations are likely to rise high enough to spill out from the synapse and get into the blood stream.

135

VM522P / VM7522 Pharmacology WIMU Fall

As well as muscarinic and nicotinic, you may also hear people refer to drugs that mimic the actions of ACh as cholinergic or parasympathomimetic.

Cholinergic: describes ACh‐like effects without distinction of anatomical sites Parasympathomimetic: Specifically describes an ACh‐like effect on parasympathetic NS targets.

II. MUSCARINIC AGONISTS

Bind to muscarinic receptors! The muscarinic drugs currently used clinically are fairly non‐specific for all the muscarinic receptor subtypes described above. Of course more specific drugs may be developed in the future.

Remember muscarinic receptors are predominately on the targets organs of the PNS including in nonvascular smooth muscle, glands, heart and eye as well as the CNS. Muscarinic receptors are also found in blood vessels, even though these do not receive PNS innervation. Activating these muscarinic receptors is therefore going to mimic the actions of ACh in the PNS (and have effects on blood vessels). Think rest and digest!

A. Examples

Pilocarpine, Bethanocol

B. Effects

1. Non‐vascular smooth muscle ‐ contraction: a. increase intestinal motility b. contraction of bladder and uterus c. contraction of bronchial smooth muscle d. contraction of smooth muscles in the eye i) ciliary muscle (accommodation of the lens for near vision) ii) constrictor pupillae (causes meiosis – pin‐point pupils)

2. Exocrine glands ‐ secretion: a. lacrimation b. salivation c. sweating d. bronchial secretions

One way to remember the non‐cardiovascular effects of muscarinic agonists is SLUDD (Salivation, Lacrimation, Urination, Digestion and Defecation)

136

VM522P / VM7522 Pharmacology WIMU Fall

3. Cardiovascular ‐ inhibition of heart rate and force ‐ decrease blood pressure

a. Heart: Remember the heart expresses odd number mAChRs, which are inhibitory (decrease cAMP, open potassium channels), therefore muscarinic agonists will slow heart rate and decrease the force of contraction to reduce cardiac output ‐ this is mostly action on atria as ventricles have no parasympathetic innervations b. Blood vessels: Remember blood vessels receive no PNS innervations, BUT they do have muscarinic receptors; so giving a muscarinic agonist will cause vasodilatation and a drop in blood pressure. This mechanism is indirect: i) Activation of mAChR stimulates NO release from the endothelium lining the blood vessels

ii) Not stimulated by parasympathetic activation as there is no innnervation, but receptors are there and therefore will be activated by cholinergic agonists

C. Therapeutic uses:

1. Urinary Bladder Atony — bethanechol (Urecholine®) ‐ Muscarinic agonists increase the tone of the urinary bladder muscle ‐ do NOT use in patients with suspected GI or urinary obstruction

2. Glaucoma (too much pressure from fluid build‐up in the eye) — pilocarpine (Diocarpine®) ‐ Muscarinic actions improve ocular drainage (due to contraction of ciliary muscle) ‐ Another muscarinic agonist, carbachol, has been used to treat colic or GI atony as muscarinic agonists increase movement in the GI. However, do NOT use in patients with suspected GI or urinary obstruction

D. Adverse Effects:

These could be due to an overdose of a clinically used drug, or a patient could present with these symptoms after ingesting a plant with muscarinic agonist activity, such as mushroom poisoning or some alkaloid plants.

Constriction of the pupil, lack of accommodation for vision Excess inhibition of the heart Excess vasodilation and hypotension Diarrhea Bronchoconstriction and excess bronchosecretions leading to breathing difficulties Do not use (or use extreme caution) in cases of urinary obstruction, intestinal obstruction, asthma or bronchoconstriction, pneumonia and cardiac arrhythmias. Should also not be used during pregnancy due to contraction of uterus

Atropine, a muscarinic antagonist can be used to reverse these symptoms.

137

VM522P / VM7522 Pharmacology WIMU Fall

E. Additional Considerations:

Most muscarinic agonists are quaternary amines and thus poorly absorbed

III. MUSCARINIC ANTAGONISTS

A. Examples:

1. Atropine (prototypical muscarinic antagonist; enters CNS) 2. Glycopyrrolate (systemically longer acting than atropine; no CNS actions) 3. Tropicamide (used in the eye, see “OCULAR PHARMACOLOGY”) 4. Others: aminopentamide, propantheline, oxybutynin, ipratropium

B. Effects: (opposite of the agonists!)

1. Non‐vascular smooth muscle—relaxation: a. Inhibition of GI motility b. Relaxation of other smooth muscles: bronchi, bladder, biliary c. Eye i) pupil dilates (mydriasis); unresponsive to light ii) ciliary muscle is paralyzed (cycloplegia); near vision is impaired

2. Exocrine glands ‐ inhibition of secretions: a. salivary, lacrimal, bronchial, and sweat (dry mouth and skin)

3. Cardiovascular

a. Low doses of atropine cause bradycardia!! This seemingly paradoxical effect is because atropine has a higher affinity for presynaptic muscarinic receptors on vagal afferents innervating the heart. These receptors normally act as a negative feedback to decrease ACh release. Blocking them actually will increase ACh release and increase the PNS vagal tone on the heart.

b. Higher doses cause modest tachycardia This is because now atropine is blocking all muscarinic receptors, including the postsynaptic receptors in the heart that would normally slow the heart rate; i.e. atropine is now blocking the parasympathetic tone on the heart. The extent of the tachycardia will depend on what the PNS tone to the heart is, i.e. what the vagal discharge is and how much ACh is being released in the patient.

c. Little change in blood pressure (no cholinergic innervation of many blood vessels) 138

VM522P / VM7522 Pharmacology WIMU Fall

4. CNS (if compound can cross BBB) a. excitation; also b. increase body temperature c. anti‐nausea

C. Therapeutic uses:

1. Antispasmodics, visceral smooth muscle relaxation For example, muscarinic antagonists are often used to depress hypertonicity of the uterus, urinary bladder, ureter, bile duct or GI e.g. for rectal exams.

2. Bronchodilation (atropine, glycopyrrolate, propantheline) While not as effective as epinephrine or other beta adrenergic agonists in causing bronchodilation, they are effective at antagonizing excessive cholinergic stimulation of bronchoconstriction and secretion.

3. Eye exams and eye surgery (atropine, tropicamide) Muscarinic antagonists will cause pupil dilation, which is good for exams. Muscarinic antagonists will also paralyze the ciliary muscle. Their use is contraindicated with increased intraocular pressure, e.g. glaucoma (remember muscarinic agonists are used to treat glaucoma. Even if the patient is not on muscarinic agonists, if there is any PNS tone to the eye blocking this effect could cause a dangerous increase in pressure)

4. Pre‐anesthetic during surgeries, especially involving inhalation anesthesia. (atropine, glycopyrrolate) This use is primarily because of their actions to decrease bronchiole and salivary secretions

5. Reduce vagal tone on heart (atropine, glycopyrrolate) The vagal reflex slows the heart, which can be dangerous during surgery. Atropine is also used to treat sinus bradycardia, sinoatrial arrest and AV block

6. Cholinergic toxicity (atropine) For example due to an overdose of a muscarinic drug or poisoning from a plant with cholinergic activity or excessive acetylcholinesterase inhibition, e.g. from pesticide exposure

139

VM522P / VM7522 Pharmacology WIMU Fall

D. Adverse Effects:

These could be due to an overdose of a clinically used drug, or a patient could present with these symptoms after ingesting a plant with muscarinic antagonist activity.

‐ Tachycardia ‐ Photophobia ‐ Dry mouth ‐ Ileus and constipation ‐ Urine retention ‐ Elevated temperature ‐ Restlessness, disorientation, CNS stimulation.

Symptoms from atropine poisoning can be reduced with an AChE inhibitor (Atropine and other clinically used muscarinic antagonists are competitive antagonists. Remember inhibiting the breakdown of ACh will increase its concentration, allowing it to compete off the antagonist from the muscarinic receptor and therefore reverse some of its effects).

E. Other Considerations:

Several other classes of drugs have significant anti‐cholinergic activity which can either contribute to their therapeutic effects or side effects.

a. Phenothiazine—tranquilizers/anti‐emetics (acepromazine, promazine, triflupromazine) b. Antihistamines—anti‐emetics (diphenhydramine, mecilizine, doxylamine) c. Tricyclic antidepressants—behavior modification (amitriptyline, imipramine)

IV. GANGLIONIC STIMULANTS AND BLOCKERS (Neuronal‐type nAChR)

The effects of Ganglionic Stimulants and Blockers are complex due to either activation of both PNS and SNS or block of both PNS and SNS. For this reason they are no longer used clinically in veterinary medicine. The best known nicotinic agonist is nicotine. Hexamethonium is a ganglionic nAChR antagonist.

Nicotine poisoning

While nAChR agonists, such as nicotine, are not used clinically you may see a client with an animal suffering from nicotine poisoning from ingestion of certain plants, insecticides or even cigarettes. The acute toxicity will have stimulatory or excitatory signs from activation of both the PNS and SNS, e.g. excitement, hyperpnea, salivation, pulse rate irregularities, diarrhea. However, the nAChR will desensitize if nicotine levels remain high and so you will then see a depressed state, e.g. incoordination, tachycardia, dyspnea, coma and death from respiratory paralysis.

140

VM522P / VM7522 Pharmacology WIMU Fall

V. NEUROMUSCULAR BLOCKERS (Muscle‐type nAChR)

These drugs are used during surgery to relax muscles. They act by blocking the actions of ACh at the neuromuscular junction (NMJ). This blocks motor neuron activation of muscle fibers and therefore muscle contraction. There are two main classes of these drugs competitive antagonists (non‐ depolarizing) and depolarizing agents.

Depolarizing vs. Non‐depolarizing Blockers

1. Non‐depolarizing blockers

Mechanism of Action: These are classic competitive antagonists at the muscle nAChR. They will compete with ACh for binding at the receptor and at high enough concentrations prevent ACh from binding to and activating the nAChR so blocking muscle contraction.

Examples are: Atracurium Pancuronium D‐tubocurarine (curare)

2. Depolarizing blocker

Mechanism of Action: These are actually agonists at the muscle nAChR. Therefore they initially cause a depolarization, exactly as ACh would, hence depolarizing blockers. However, whereas ACh is broken down and removed from the synapse in less than a millisecond (msec), these agonists remain present and cause extended (minutes) activation of the nAChR. Extended activation of the nAChR causes desensitization of the receptor (see page 29) and repolarization of the muscle.

These actions of depolarizing blockers are often described as 2 phases:

Phase I: depolarizing phase (hence, depolarizing blockers); receptor is activated leading to depolarization and fasciculation (twitching)

Phase II: non‐depolarizing phase (or desensitized phase); the muscle is re‐polarized, but cannot contract in response to ACh (exact mechanism is not well understood)

Example:

The best known example is succinylcholine.

Another mechanism of producing NMJ block is by Botulinum toxin (very long‐acting). As described on page 39, botulinium toxin cleaves proteins required for release of synaptic vesicles and therefore inhibits acetylcholine release. Interestingly, it preferentially affects the NMJ. Its effects are long lasting as the proteins need to be replaced before ACh release, motor neuron and muscle function are restored.

141

VM522P / VM7522 Pharmacology WIMU Fall

Therapeutic Uses of NMJ Blocking agents:

1. Skeletal muscle relaxation for procedures during surgeries (less anesthetic required)

Selection of agent depends on consideration of length of procedures and other potential contraindications, based on unique profile of each agent (degree of histamine release, mechanism of inactivation, vagolytic actions, muscarinic actions, sympathomimetic actions)

COMPOUND DURATION GANGLIONIC SYMPATHETIC HISTAMINE MUSCARINIC (MIN) EXCRETION BLOCK ACTION RELEASE ANTAGONIST AGONIST 2-8 (E) hydrolysis by plasma Succinylcholine 4-6 (F) – – – –  esterase 22-29 (C)

100 (C) Tubocurarine unchanged in urine  –  – – 20 (F)

8-12 (E) Atracurium hydrolysis by p- – –  – – 17-29 (F/C) esterase and spontaneous 20-35 (E) 50% unchanged Pancuronium   rare  – 30-100 (F/C) 50% hepatic

KEY: (E) Equine; (C) Canine; (F) Feline

Adverse Effects:

Overdose results in death due to paralysis of the diaphragm

‐ AChE inhibitors can be used to reverse non‐depolarizing block, but not depolarizing block

‐ artificial respiration may be needed until recovery

Also may see histamine release, sympathomimetic and muscarinic effects depending on the drug used (see table)

142

VM522P / VM7522 Pharmacology WIMU Fall Additional Considerations:

1. NMJ blocking agents DO NOT CAUSE PAIN RELIEF!!

2. Non‐depolarizing block is enhanced by:

a. Aminoglycoside Antibiotics (neomycin‐streptomycin group)

These antibiotics reduce calcium entry into the presynaptic nerve terminal and so inhibit ACh release. Normally there is sufficient excess ACh for it not to be noticed. However, NMJ nAChR antagonists are competitive antagonists and the less ACh around, the less antagonist you will need to block it. Therefore, a smaller dose of non‐depolarizing blockers in the presence of aminoglycosides may cause the same effect as a larger dose given alone.

b. Volatile anesthetics

Volatile anesthetics are thought to increase the binding affinity of the antagonists for the nAChR.

c. Hypomagnesemia, hypocalcemia and hypokalemia

3. Other considerations about succinylcholine (depolarizing block)

a. Its short duration of action is due to inactivation by cholinesterases i) considerable species and individual variation ii) cholinesterase inhibitors not only make block worse, but prolong action

b. Painful contraction may occur in response to early activation

c. Causes bradycardia and enhanced bronchial and salivary secretion due to direct muscarinic actions

d. Depolarization of muscle results in elevated K+ release and potential for hyperkalemia and potential ventricular arrhythmias

e. Malignant hyperthermia has been observed in some individuals

f. Increases in intraocular and intracranial pressure can occur

g. Birds have unique muscle innervation, with multiple junctions on each fiber causing widespread depolarization of muscle fiber and maintained contracture. Therefore, depolarizing blockers should never be used in birds (even topically)

143

VM522P / VM7522 Pharmacology WIMU Fall Nematocides (anti‐parasitic)

Examples: levamisole, butamisole, pyrantel, morantel

1. Cause a depolarizing block of nAChR in nematodes (like succinylcholine)

2. Also act on nAChR of mammals (very narrow therapeutic window)

3. Note all the dangers associated with succinylcholine use (especially potential for augmentation of effects by insecticides that inhibit AChE)

VI. ACETYLCHOLINESTERASE (AChE) INHIBITORS (Indirect Cholinergic Mimetics)

A. Examples:

Clinically used Drugs:

Neostigmine—quaternary amine; charged; not readily absorbed; does not cross BBB

Edrophonium—quaternary amine; short‐acting; used diagnostically

Demecarium—2 neostigmine molecules attached together; used in the eye

Physostigmine—tertiary amine; readily absorbed; crosses BBB

For reasons that are unclear neostigmine and edrophonium are more active at the NMJ – whereas the others are more effective at postganglionic muscarinic sites.

Some insecticides are also AChE inhibitors:

Carbamate‐type AChE inhibitors—reversible

Example: carbaryl

Organophosphate‐type AChE inhibitors—irreversible

Examples: malathion, parathion, dichlovos

144

VM522P / VM7522 Pharmacology WIMU Fall B. Effects

Acetylcholinesterase (AChE) inhibitors block the primary enzyme that metabolizes ACh. This will result in a higher concentration of ACh, primarily at synapses. However, if the concentration of AChE inhibitors are high enough ACh may spill out into the blood. All ACh synapses have the potential to be effected (depending on whether the drug crosses the BBB you may or may not see CNS effects). This means that these drugs will cause a combination of nicotinic (primarily muscle nAChR) and muscarinic effects.

1. Enhancement of muscarinic effects

These include all the effects of the muscarinic agonists, EXCEPT as there is no innervation of blood vessels by the PNS you won’t see the vasodilation effects unless the AChE inhibitor concentration is very high and ACh gets into the blood.

So the primary effects at therapeutic concentrations will be: - Bronchoconstriction - Bradycardia

- Hypotension (due to decreased heart rate and at higher concentrations some vasodilation)

- Pupillary constriction

- SLUD: salivation, lacrimation, urination, defecation

2. Enhancement of Ach action at the muscle nAChR.

Increase in ACh will augment and prolong muscle contraction. Higher doses lead to muscle paralysis due to postsynaptic depolarizing block (see depolarizing block on page 64).

3. CNS effects

excitation, convulsions, depression, unconsciousness

4. Higher doses also produce depolarizing block of ganglionic transmission and its associated complex effects.

145

VM522P / VM7522 Pharmacology WIMU Fall

C. Therapeutic Uses

1. Glaucoma (demecarium)

Constricts ciliary muscle – improves drainage Constricts sphincter muscle also – may improve drainage

2. Paralytic Ileus (physostigmine/neostigmine)

Constricts muscle in GI – improves movement

3. Atony of the Bladder (neostigmine)

Constricts muscle in bladder – improves muscle tone

4. Myasthenia Gravis (neostigmine, edrophonium diagnostically)

an autoimmune disease that attacks nAChRs – can diminish symptoms by increasing Ach

5. Reverse an overdose of atropine or a non‐depolarizing (nAChR antagonist) NMJ blocker (neostigmine)

Inhibiting AChE will increases ACh levels to overcome a competitive antagonist, such as atropine and atracurium.

D. Adverse effects:

Similar to muscarinic agonists, but with additional muscle twitching and weakness and less blood pressure effects due to no PNS innervation of blood vessels.

‐ miosis ‐ bradycardia ‐ muscle twitching or weakness ‐ bronchoconstriction ‐ diarrhea and increased secretions ‐ constrictions of ureter

As with muscarinic agonists, do not use (or use extreme caution) in cases of urinary obstruction, intestinal obstruction, asthma or bronchoconstriction, pneumonia and cardiac arrhythmias.

146

VM522P / VM7522 Pharmacology WIMU Fall Treat overdose with atropine (block muscarinic effects). If there is muscle paralysis, provide respiratory support/oxygen, as with NMJ depolarizing blocker overdose. (Also decontaminate (wash) to make sure all pesticides are removed.)

E. Additional Considerations:

Organophosphates inhibit AChE function by phosphorylating and covalently modifying the AChE. If the poisoning is caught within 24 hours this can be reversed by the phosphate scavenger pralidoxime (2‐PAM).

147

VM522P / VM7522 Pharmacology WIMU Fall

L21, 22: ADRENERGIC PHARMACOLOGY

READING ASSIGNMENT:

Riviere and Papich, Chapter 5, pages 110‐113 and Chapter 6, page 125. An alternative overview is in Rang et al., Chapter 118, pp 168‐188.

OBJECTIVES:

1. Be able to describe the mechanisms of norepinephrine neurotransmission and epinephrine release and the specific actions and locations of ‐ and ‐adrenergic receptors.

2. Be able to explain the systemic effects of adrenergic agonists and antagonists from knowledge of the specific actions and locations of ‐ and ‐adrenergic receptors.

3. Know which receptors epinephrine, norepinephrine and dopamine bind; as well as their main effects, therapeutic uses and adverse effects.

4. Know the therapeutic uses and adverse effects of each class of drug, as well as the specific bolded examples.

ADRENERGIC PHARMACOLOGY OUTLINE:

I. Review SNS Actions

II. Overview of Adrenergic Neurotransmission

III. Adrenergic Agonists

IV. Adrenergic Antagonists

148

VM522P / VM7522 Pharmacology WIMU Fall I. REVIEW SNS ACTIONS

DECREASED SALIVATION

PUPILS DILATE

PILOERECTION

↑HR ARTERIES CONSTRICT ‐ ↑BP

AIRWAYS DILATE

INCREASED SWEAT CHOLINERGIC!

DIGESTION/VISCERAL

ACTIVITY SHUT DOWN

CONSTRICTION OF SOME BLOOD VESSELS (E.G., SKIN) ADRENALINE BOOST

DILATION OF OTHERS (E.G., SKELETAL MUSCLE)

GLYCOGEN AND FAT CONVERTED TO FREE FATTY ACIDS AND GLUCOSE

149

VM522P / VM7522 Pharmacology WIMU Fall II. OVERVIEW OF ADRENERGIC NEUROTRANSMISSION

1) Norepinephrine and epinephrine are synthesized from tyrosine as shown on page 75. Dopamine, norepinephrine and epinephrine share a common synthesis pathway.

2) NE or epinephrine is then packaged in vesicles and released upon either nerve terminal depolarization or adrenal medulla stimulation.

3) NE and Epinephrine act on:

i. alpha 1 adrenergic receptors (G protein coupled receptors primarily coupling to PLC/DAG/Ca2+) Tends to be excitatory (see diagram below)

ii. alpha 2 adrenergic receptors (G‐protein coupled receptor that decrease cAMP and inhibit potassium channels) Tends to be inhibitory (see diagram below)

iii. beta 1 adrenergic receptors (G‐protein coupled receptor that primarily increase cAMP) Tends to be excitatory (see diagram below)

iv. beta 2 adrenergic receptors (G‐protein coupled receptor that primarily increase cAMP) Tends to be excitatory (see diagram below)

Remember these receptors are found on the target organs of the SNS and in the CNS

4) Termination of NE and epinephrine action is primarily by reuptake

(from circulation) deaminated ( and  effects) EPI metabolite

2 2: cAMP – smooth muscle relaxation: bronchi, uterus, bladder, GI MAO eye: increase ocular fluid formation uptake 1 uptake 2 COMT arteries: dilate skeletal, coronary, renal NE liver: glycogenolysis, gluconeogenesis  1 1: cAMP - heart: increase rate and force of contraction fat: increase lipolysis NE NE NE kidney: increase renin release NE -

PLC – generate DAG/IP /Ca2+ NE synthesis 1: 3 1 arteries constrict: skin, viscera 2 smooth muscle constrict: uterus, pupil, vas deferens tyrosine sphincters contract GI tract relaxes (hyperpolarizes via I ) 2: cAMP K

decrease transmitter release skin: pilomotor muscle contraction CNS effects salivation (GI: relaxation) liver: glycogenolysis, gluconeogenesis

postsynaptic cell noradrenergic varicosity 150

VM522P / VM7522 Pharmacology WIMU Fall You do not need to memorize all the details of the biosynthesis of catecholamines, but you should know the relationship between DA, NE, and EPI. That is that they are all synthesized from the same precursor (tyrosine) and that they are structurally related.

For the breakdown or metabolism of catecholamines you should know that monoamine oxidase (MAO) and catechol‐O‐methyl transferase (COMT) are the main enzymes.

However remember effects of NE and Epi are primarily terminated through uptake!

III. ADRENERGIC AGONISTS

There are several classes of drugs that increase adrenergic activity

A. Endogenous Adrenergic Agonists, Synthetic ones

B. Selective‐Agonists

C. Selective‐Agonists

D. Indirect Acting Agents

151

VM522P / VM7522 Pharmacology WIMU Fall A. Endogenous Adrenergic Agonists

1. Epinephrine:

a. Mechanism of action:

Non‐selective, but affinity for beta receptors is greater than alpha (1,  2 >1,  2)

Epinephrine is released from the adrenal medulla to circulate in the blood. It is a non‐selective agonist having both  and  receptor actions, but has a higher affinity for beta receptors than alpha. As shown on the diagram on page 72 epinephrine has many effects. Epinephrine has a very short half life.

b. Effects:

Cardiovascular effects include:

i) 1 action on the heart increases heart rate and force of contraction (remember beta receptors increase cAMP, which is stimulatory to both cardiomyocytes and the AV node) Because epinephrine has such strong effects on the heart it can increase the potential for arrhythmias and decrease cardiac efficiency (greater oxygen consumption)

ii) 2 action on blood vessels causes vasodilation and decreases peripheral resistance

iii) 1 action on blood vessels causes vasoconstriction and increases peripheral resistance (blood pressure). This 1 action is what makes epinephrine such a potent vasoconstriction locally.

NOTE: the effect of epinephrine on peripheral resistance, or blood pressure, is going to depend on the dose as epinephrine has a higher affinity for receptors therefore at lower doses the  action to decrease peripheral resistance dominates, while at higher doses the  action to increase peripheral resistance dominates.

Smooth muscle effects

i) Bronchi—relaxation (2)

ii) Uterus—relaxation () or contraction () ‐dominant effect depends on species and whether pregnant

iii) GI tract—relaxation of muscle ( and ), but constriction of sphincters ()

152

VM522P / VM7522 Pharmacology WIMU Fall iv) Urinary bladder—urinary retention (bladder relaxes due to  action, but trigone and sphincter contract via ‐action)

v) Eye – mydriasis—contract radial muscle ()

vi) Pilomotor muscles contract ()

Metabolic effects

i) Blood glucose and FFA increase

 stimulates liver to release glucose ( and 1)

 stimulates fat to release FFA (1)

 inhibits insulin secretion (2) and increases glucagon secretion (2)

c. Therapeutic uses – acute or emergency situations i)

To treat allergic Reactions

(hypersensitivity reactions, anaphylactic shock)

Epinephrine is used in emergency situations for allergic reactions to reverse some of the life threatening symptoms until other help can be given. For example, eipnephrine quickly reverses the precipitous fall in blood pressure (through 1 activation) cardiac irregularities (1) as well as bronchodilating the lungs (2) to reverse the histamine‐induced bronchoconstriction.

ii) Bronchodilation

Epinephrine can be used to give immediate relief in emergency situations for asthma. The bronchodilation is due to 2 effects in the lungs.

ii) Vasoconstriction

e.g., ‐ with local anesthetics to prolong their action as vasoconstriction slows the blood flow and so slows the removal of the local anesthetic

‐ to control hemorrhage

iii) Cardiac Arrest .

Epinephrine will increase heart rate and cardiac output (as well as conduction in the case of an AV block).

153

VM522P / VM7522 Pharmacology WIMU Fall d. Adverse effects

i) Potential for arrhythmias (have a defibrillator handy if using high doses)

ii) Excessive vasoconstriction will cause hypertension

iii) Can cause problems in circulation when used with local anesthetics due to excessive vasoconstriction locally, e.g. digits, tail, ears, penis

‐ can cause tissue necrosis due to intensive vasoconstriction and ischemia

2. Norepinephrine

a. Mechanism of action:

Activates 1, 1 and  2 adrenergic receptors not  2

Norepinephrine is released from SNS terminals. It has a very short half life.

b. Effects

Cardiovascular

i) Increase Blood Pressure

‐ 1 receptor action to constrict blood vessels and increase peripheral resistance.

ii) Slows Heart Rate

‐ 1 receptor action should increase heart rate, but due to the very larger increase in blood pressure from the 1 receptor mediated vasoconstriction the baroreceptor reflex is activated, which slows the heart (vagal reflex, blocked by atropine)

Other actions:

i) All smooth muscle actions of epi except 2 effects

 the effects are more modest than EPI (less potent)

 most importantly no relaxation of bronchi

c. Therapeutic uses i) None

154

VM522P / VM7522 Pharmacology WIMU Fall 3. Dopamine

a. Mechanism of action:

‐ primarily acts on DA receptors

‐ some actions on 1

‐ dopamine also stimulates the release of NE at high doses = 1 receptor actions

‐ very short half life, given IV

B. Effects:

Cardiovascular

i) Increases heart rate ‐ positive inotropic effect on heart is via 1 action ii) Decreases blood pressure at lower doses action on DA receptors dilates renal, mesenteric, and coronary arteries iii) Increases blood pressure at higher doses action on 1 receptors, potentially due to NE release from terminals

One thing that is important to remember is that even at higher doses when dopamine is causing an increase in blood pressure due to 1 actions, the vasodilation of renal and coronary arteries due to activation of DA receptors is still occurring, which means you have increased perfusion of those organs. It is just that the contribution to blood pressure of the vasodilation is outweighed by the vasoconstriction due to 1 effects so the mean or overall blood pressure is increased.

CNS:

Dopamine is known to have important effects in the CNS. It is required for the initiation of movement (L‐DOPA is used to treat human parkinson patients), as well as for motivation, reward and mood.

c. Therapeutic uses – to stimulate myocardium , given IV

i) Cardiogenic and septic (endotoxic) shock

Dopamine will stimulate the heart due to its 1 action. At low doses dopamine will cause hypotensive effects (DA receptors) and at higher doses hypertensive effects (1 receptors). The actions of dopamine to dilate renal and coronary arteries can be particularly useful during systemic vasoconstriction, e.g. due to shock. However, if the blood pressure is falling too fast the hypotensive effects of dopamine could be dangerous. At low doses dopamine will cause hypotensive effects (DA receptors) and at higher doses hypertensive effects (1 receptors).

155

VM522P / VM7522 Pharmacology WIMU Fall

d. Adverse effects

i) can cause tachycardia and ventricular arrhythmias

ii) avoid leakage from the vein as DA can cause tissue necrosis

Synthetic agonists

1. Dobutamine (Synthetic ‐Agonist)

a. Mechanisms of Action:

Dobutamine produces its effects through activation of primarily  adrenergic receptors 1 > 2 (it can have some  effects at higher doses). Dobutamine has a very short half life and is usually given IV.

b. Effects:

Dobutamine has a greater (more selective) effect to increase contractility vs. heart rate compared to dopamine or epinephrine.

c. Therapeutic uses:

i) Heart failure (short term – emergency room situation)

Stimulates the heart with only small change in peripheral resistance. Also the relative inotropic selectivity of dobutamine can be an advantage over epinephrine or dopamine for some cases. E.g.: Short term rescue of myocardial failure due to dilated cardiomyopathy (emergency situation)

Positive ionotrophic effect of dobutamine can be useful for emergency therapy or rescue, not for long‐ term treatment.

d. Adverse effects:

i) Increased potential for arrhythmias (but less of a problem than epinephrine)

ii) Tachycardia and increased oxygen consumption

156

VM522P / VM7522 Pharmacology WIMU Fall

B. Selective beta 2 (2) Agonists:

a. Examples: Terbutaline, albuterol, clenbuterol

b. Effects: bronchodilation short‐term decrease in blood pressure

c. Therapeutic uses:

i) bronchodilation

2 selective action especially useful in animals with heart disease, hyperthyroidism, or hypertension.

d. Adverse effects:

i) Tachycardia

ii) Blood pressure changes.

Cautions: Patients often become refractory to the bronchodilatory effects of 2‐Agonists due to desensitization and downregulation of the receptors.

Clenbuterol is used in horses but there is a FDA ban in food animals

C. Selective alpha agonists C.1. Selective alpha 1 () agonists

a. Example: phenylephrine

b. Effects: Constricts smooth muscles:  Vasoconstriction  Constriction of radial muscle in the eye (helps dilate the pupil)  Can also affect bladder, uterus and GI

157

VM522P / VM7522 Pharmacology WIMU Fall c. Therapeutic uses:

i) To treat Hypotension (especially when a lack of direct cardiac effect is required)

ii) To cause vasoconstriction, e.g. to decrease bleeding during surgery

iii) As a Mydriatic (dilate pupil) during eye surgery.

Not as effective as muscarinic blocker, but can augment muscarinic blockers or be used when muscarinic blockers are contraindicated. Also vasoconstriction action useful during eye surgery.

iv) Nasal decongestion

d. Adverse effects:

Vasoconstriction and hypertension

C.2. Selective alpha 2 () agonists

a. Example: Xylazine (and medetomidine, detomidine, romifidine)

b. Effects: CNS effects i) Sedation ii) Pain sensation iii) Arousal

c. Therapeutic uses:

i) Sedation and analgesia

d. Adverse effects:

i) May cause vomiting

ii) Use cautiously in animals with compromised cardiac or respiratory function

158

VM522P / VM7522 Pharmacology WIMU Fall

D. Indirect‐Acting Agents

These drugs act primarily by releasing NE and epinephrine from terminals. That is they indirectly activate adrenergic receptor by increasing the release of the endogenous agonists.

a. Example: Phenylpropanolamine and ephedrine

b. Effects:

1. Cardiovascular effects—increased blood pressure

i) cardiac effects via 1 effect

ii) peripheral resistance increases via 1 effect

2. Bronchodilation via 2 effects

3. Constriction of sphincters—of note is urinary bladder sphincter

c. Therapeutic uses:

i) Primary urinary bladder sphincter incompetance

d. Adverse effects:

‐ Hypertension,

‐ Tachycardia

‐ CNS stimulation—insomnia, nervousness, agitation

e. Other considerations:

Tachyphylaxis frequently occurs—loss of NE from terminals

Amphetamine is also an indirect acting agonist as it causes the release of norepinephrine and dopamine.

159

VM522P / VM7522 Pharmacology WIMU Fall

IV. ADRENERGIC ANTAGONISTS

There are several classes of drugs that counteract adrenergic activity

A. Non‐selective ‐adrenergic antagonists

B. Selective‐antagonists

C. Alpha 1 receptor‐antagonists

D. Alpha 1 receptor‐antagonists

A. Non‐selective ‐adrenergic antagonists

a. Examples: Sotalol, propanolol, Timolol

b. Effects:

Cardiac effects

i) Reduced sympathetic tone

Under normal circumstances  antagonists have very little effect. However, under conditions of increased sympathetic tone (e.g., exercise, compensated cardiac failure) these drugs will reduce cardiac rate and contractility

ii)  Propensity for arrhythmias

‐ Remember that ‐agonists had increased propensity for arrhythmia. So it should be no surprise that blocking receptors will reduce the propensity for arrhythmias.

‐ In addition to  block, sotalol inhibits potassium channels to prolong repolarization,  the QT interval and  automaticity

+ ‐ Propranolol also has a membrane stabilizing effect not associated with  block (Na channel block)

Vascular effect

i) transient small  in peripheral resistance (2‐block on vasculature)

ii) prolonged in blood pressure

Why beta blockers cause a long‐term decrease in blood pressure is not completely understood. It is thought to be due to:

a) Blocking 1 receptors in the kidney that normally increase renin release. So with beta blockers you see decreased renin release from kidney, decreased activation of the renin‐angiotensin system and diuresis, which would lead to deceased blood pressure

b) A small amount of ‐receptor block c) Some unknown non‐adrenergic action 160

VM522P / VM7522 Pharmacology WIMU Fall

Pulmonary effects

i) Normally these drugs have little effect, however in patients with asthma or COPD blocking the 2 receptors, which mediate SNS tone to cause bronchodilation, can be life threatening

Metabolic effects

i) ‐block reduces ability of liver to respond to hypoglycemia

ii) Reflex increase in heart rate associated with hypoglycemia is also blocked

Eye effects

i) ‐block reduces fluid formation in the eye (see Ocular Pharmacology in Spring)

c. Therapeutic uses:

i) To control Hypertension (Sotalol, propanolol)

ii) As antiarrhythmics (Sotalol, propanolol)

‐ controls cardiac dysrhythmias due to overstimulation of the SNS

‐ decreases AV conduction in patients with atrial fibrillation or flutter

‐ controls atrial and ventricular arrhythmias (e.g. due to excess digitalis)

iii) To treat Glaucoma: decreases ocular fluid formation (Timolol only)

d. Adverse effects:

i) Negative inotropic effects and decreases in cardiac output – could be a problem in animals with poor cardiac function

iii) Bronchoconstriction can be fatal in susceptible animals (asthma, COPD)

iii) The reduced ability to respond to hyperglycemia can be dangerous in diabetic patients

ii) Watch out for rebound hypertension and tachycardia upon discontinuation

‐ due to receptor up‐regulation

iii) With ocular use side effects are rare, but timolol can cause irritation

161

VM522P / VM7522 Pharmacology WIMU Fall

B. Selective 1‐antagonists

a. Examples: Atenolol, metoprolol, esmolol (short‐acting)

b. Mechanism of action: Selective 1‐antagonists

c. Effects:

Selective beta1 antagonists should have all the cardiovascular effects of non‐selective blockers without effects in the lung

d. Therapeutic uses:

i) Treat supraventricular arrhythmias

ii) Treat hypertension

e. Adverse effects: i) Cardiac depression– could be a problem in animals with poor cardiac function iii) Still have a reduced ability to respond to hyperglycemia that can be dangerous in diabetic patients

ii) Watch out for rebound hypertension and tachycardia upon discontinuation

C. Alpha 1 (1) receptor antagonists:

a. Examples:

Prazosin ‐ reversible

Phenoxybenzamine – irreversibly binds to alpha1 receptors (covalent bond is formed), therefore long lasting effect.

b. Effects:

i) CV: relaxes vascular smooth muscle so see a drop in blood pressure and can get reflex tachycardia

ii) Other: relaxes other smooth muscle e.g. urethral, radial muscle in the eye (blocks dilation)

c. Therapeutic uses:

i) To reduce vasoconstriction, useful to alleviate:

‐ Hypertension (especially associated with pheochromocytoma)

‐ Peripheral vasospasm

‐ Visceral ischemia

‐ laminitis 162

VM522P / VM7522 Pharmacology WIMU Fall

ii) Hypertonus of the urethral sphincter

d. Adverse effects:

i) Vasodilation and hypotension (particular orthostatic)

ii) Reflex tachycardia

caution with animals with compromised cardiac function

iii) Other expected ‐block actions

blocks pupil dilation and lid retraction

nasal congestion

diarrhea in horses

inhibits ejaculation

intraocular pressure

D. Alpha 2 (2) receptor antagonists:

a. Example: Yohimbine, atipamezole

b. Effect:

i) Will block all alpha2 effects.

c. Therapeutic uses:

i) To reverse 2‐agonist sedation/anesthesia (xylazine, medetomidine)

d. Adverse effects:

i) High doses can cause tremors, seizures

ii) Caution: monitor heart rate and rhythm when reversing the effects of an 2‐ agonist

163

VM522P / VM7522 Pharmacology WIMU Fall L23: Integrated Overview of Cardiovascular Drugs

Cardiovascular Effects of epinephrine and adrenergic drugs

1. 1 action—positive inotropic and chronotropic effects on heart a.  in force of contraction b.  HR

2. 2 action—relax coronary and skeletal arteries a.  in peripheral resistance

3. 1 action—constrict arteries to skin and viscera a.  in peripheral resistance

The effect of adrenergic drugs on heart rate and blood pressure is going to depend on the relative selectivity of a given drug for each receptor subtype. To understand the complex outputs of adrenergic drugs examine the figure below.

systolic mean

diastolic

Note:  Mean Arterial Pressure (dashed line) is determined by Cardiac Output and Peripheral Resistance. The upper line in the arterial pressure trace represents systolic pressure, i,e the pressure during contraction, this is more affected by cardiac output The lower line represents diastolic pressure during relaxation, i.e. while heart fills with blood, this is most affected by peripheral resistance  Cardiac Output is determined by Force of Contraction and Heart Rate (HR)

164

VM522P / VM7522 Pharmacology WIMU Fall

This example shows a low dose of epinephrine. Epinephrine has a higher affinity for receptors and, therefore, at this low dose  actions will dominate over action

Explanation of effects:

Epinephrine: stimulates 1,  and 1. As mentioned above, in this figure a low dose of epinephrine is used so  The decrease in peripheral resistance is due to  action to dilate peripheral arteries and decrease peripheral resistance. 1 receptors will also be activated to increase peripheral resistance but the 2 action dominates at low doses. The increase in heart rate is via  action to directly increase heart rate. The

decrease in diastolic pressure is due to  action to dilate peripheral arteries and decrease peripheral resistance, which affects diastolic pressure. In contrast, the increase in systolic pressure is due to  action to directly increase heart rate and force of contraction, increasing cardiac output and so increasing systolic pressure.

Norepinephrine: stimulates  and  (very little ). The increase in peripheral resistance is due to  action to constrict peripheral blood vessels and so increases peripheral resistance. The increase in diastolic pressure is again due to  action to constrict peripheral blood vessels and so increase peripheral resistance, which affects diastolic pressure. Due to this dramatic increase in mean arteriole pressure the baroreceptor reflex (see below) is activated, leading to a reflex decrease in heart rate through activation of the PNS that overrides the  receptor stimulated increase in heart rate. This decrease in heart rate can be blocked by the muscarinic receptor antagonist atropine, which would block the PNS effect to slow the heart rate and unmask a  action to directly increase heart rate.

Isoproteronol: this is a non‐specific beta agonist that stimulates 1 and 2 only. The decrease in peripheral resistance is due to 2 action to dilate peripheral arteries and decrease peripheral resistance. The increase in heart rate is via 1 action to directly increase heart rate. The decrease in diastolic pressure is due

to  action to dilate peripheral arteries and decrease peripheral resistance, which affects diastolic pressure. In contrast, the increase in systolic pressure is due to 1 action to directly increase heart rate and force of contraction, increasing cardiac output and so increasing systolic pressure. As the net is a decrease in mean arteriole pressure the baroreceptor reflex is not activated.

Note that while the response of isoproteronol is similar to epinephrine, there are four differences all due to the fact isoproteronol does not stimulate  receptor. 1) Isoproteronol causes a larger decrease in

peripheral resistance because the 2 action to decrease peripheral resistance is not opposed by any  receptor action to increase peripheral resistance. 2) Isoproteronol causes a larger decrease in diastolic

resistance again because the  action is unopposed. 3) The increase in systolic pressure caused by Isoproteronol is less as there is no 1 action to increase peripheral resistance. This results in a decrease in mean arteriole pressure. 4) Isoproteronol causes a larger increase in heart rate than epinephrine as the mean

arteriole pressure is lower. In the case of epinephrine the added activation of 1 is sufficient to cause an increase in mean arteriole pressure and so the baroreceptor reflex is activated and there is an increase in PNS drive to decrease the heart rate. This is insufficient to completely overcome the direct  action however it blunts the increase so that the composite result is that epinephrine does not increase heart rate as much as isoproterenol.

Reminder:

Baroreceptor Reflex: activated by  Mean arteriole pressure; activates stretch receptors (carotid sinuses, aortic arch)   afferent nerve discharge to CNS 165

VM522P / VM7522 Pharmacology WIMU Fall   SNS and  PNS   HR and  peripheral resistance   BP

Something to think about:

What would be the effects of a high dose of epinephrine that would activate alpha1 receptors as well? How exactly is this different to a low dose and why?

Dobutamine (1 > 2 >1)

Heart:– Increase HR and Force (1). Greater effect to increase contractility vs. heart rate compared to epinephrine and norepinephrine

Blood pressure – stimulates heart with only a small effect on blood pressure as 1 receptor activation is > than 2

– At higher doses you will get vasodilation of arteries through activation of 2 receptors, decreasing peripheral resistance. – At even higher doses still you can get some vasoconstriction as at very high doses dobutamine will activate 1 adrenergic receptors, causing an increase in peripheral resistance

Dopamine (dopamine receptors, 1 and some 1 effects at high concentrations)

Heart: – Increase HR and Force 1)

Blood pressure – low doses you see a decrease in blood pressure, higher doses you see an increase in blood pressure – Low doses you get vasodilation of coronary and renal arteries through activation of dopamine receptors, decreasing peripheral resistance.

– At high doses you get vasoconstriction as at high doses dopamine releases NE and you see 1 adrenergic effects, causing an increase in peripheral resistance

Cholinergic Summary:

M2 receptors: Decrease HR and Force, (Decreases conduction velocity, particularly at AV node)

M3 receptors: Vasodilation (via NO) Vasculature not innervated by PNS Agonists cause vasodilation Antagonists no effect unless there is an agonist present

166

VM522P / VM7522 Pharmacology WIMU Fall Part 4 – Autacoids (S. Appleyard, R. Meyer) L24 ‐26: AUTACOIDS ‐ Additional chemical mediators

READING ASSIGNMENT:

Riviere and Papich, Chapter 16, page 411‐438. Alternative: Rang et al., Chapters 12 and 13.

OBJECTIVES OF PART 4, section 1:

1. Be able to explain the sources of and physiological functions of histamine, serotonin, angiotensin, bradykinin, and purines.

2. For histamine, know the effects mediated by H1 and H2 receptors, a prototypical drug for each class, and the therapeutic uses of drugs from each class.

3. For serotonin, know the general effects mediated by serotonin receptors, and in particular know the significance of serotonin in the actions of metoclopramide (5‐HT4 receptor), ondansetron (5‐HT3 receptor) and the CNS acting agents buspirone, fluoxetine.

4. For angiotensin, know the effects of ACE inhibitors on the renin‐angiotensin and kallikrein‐bradykinin systems and understand its role in volume regulation in concert with aldosterone.

CHEMICAL MEDIATORS

Chemical communication occurs in all tissues/systems. Nerves communicate with each other and to target organs by neurotransmitters, such as in the autonomic nervous system. Another type of long‐ distance communication is hormonal, where a hormone is released from one site, the site of production, circulates in the blood (or lymph) and has an effect at a distant target site. There is also local communication including both paracrine (affecting the cells around the cell the mediator was released from) and autocrine (affecting the cells that released the mediator) mechanisms. There are many different types of chemical mediators; including small molecule mediators such as histamine, serotonin and purines.

OUTLINE:

I. Types of autacoids – overview

II. Histamine and antihistamine drugs

III. Serotonin (5‐HT)

IV. Purines

V. Eicosanoids (prostaglandins, leukotriens)

VI. Peptides (neurokinin, angiotensin, bradykinin)

167

VM522P / VM7522 Pharmacology WIMU Fall I. TYPES OF AUTACOIDS ‐ OVERVIEW

A large variety of different types of chemicals can have effects on cells:

i. Gases (nitric oxide or NO) – not covered here ii. Small molecule (histamine, serotonin, purines) iii. Lipid mediators iv. Peptides and proteins

Chemical mediators can have many different actions in the body, e.g. as a neurotransmitter or a hormone.

Neurotransmitters e.g. Acetylcholine, norepinephrine, dopamine, serotonin, etc.

i. Found in neurons

ii.. Synthesized de novo and packaged into synaptic vesicles

iii. Released from synapses (rapid onset, rapid termination, localized action)

Hormones e.g. Insulin, cortisol, thyroid etc.

i. Found in endocrine organs and a few other tissues

ii. They can be released into the blood and have actions at long distances from the site of release, e.g. insulin, or they can have actions locally, within the organ of release, e.g. prostaglandins.

ii. Variety of synthetic pathways ‐ peptides/proteins, e.g. insulin ‐ steroids, e.g. cortisol ‐ others (thyroid hormone, Vitamin D)

A mediator can belong to more than one category:

Examples include:

Histamine is released from both mast cells, where it has paracrine actions, and neurons, where it is a neurotransmitter.

Endogenous opioids can be released from 1) neurons, where they are neuromodulators, 2) the pituitary, where they have hormonal actions, and 3) from immune cells, where they have autocrine and paracrine functions.

168

VM522P / VM7522 Pharmacology WIMU Fall

II. HISTAMINE AND ANTIHISTAMINE DRUGS 1. Basic Background

A. Chemistry 1. 2‐(4‐imidazolyl)ethylamine 2. Hydrophilic vasoactive amine 3. Histamine is found in all tissues of plants and animals, but concentration varies: species‐ and tissue‐specific. High concentration in mast cells & select other tissues e.g. stomach mucosa (enterochromaffin like cells, ECL). a) These tissues contain large amounts of enzymes responsible for the formation and metabolism of Histamine (see Figure 1)

B. Histamine released from tissue sites by: 1. Injury to tissue (e.g. trauma) 2. Physical agents: heat, cold, x‐rays, etc. 3. Chemical compounds: many drugs used clinically will release histamine (e.g. morphine, tubocurarine, can displace histamine in granules => release) 4. Immunological: FcεRI, or Fc epsilon RI: high‐affinity receptor for immunoglobulin E (IgE, antibody isotype involved in allergy and, arguably, resistance to parasites). FcεRI is expressed predominantly on mast cells and basophils. Crosslinking of FcεRI via IgE‐allergen complexes => energy & calcium dependent degranulation of the cells => histamine release. 5. Release of histamine and other factor from rapid, massive degranulation of mast cells has systemic effect (anaphylaxis).

C. Histamine Formation and Metabolism (see Figure 1) 1. The amino acid histidine is decarboxylated by “histidine decarboxylase” to form histamine. 2. There are two major pathways by which histamine is metabolized in humans: a) Methylation by N‐methyl transferase followed by oxidative deamination (monoamine oxidase B, =MAO B) to form N‐methylimidazole acetic acid b) Deamination and oxidation (diamine oxidase, histaminase) followed by ribosylation to form imidazole acetic acid riboside 3. These pathways exist in most animal species, but their relative activities may vary

2. Histamine Receptors (see Table 1)

A. H1 Type Receptor 1. This is the “classical” receptor which has been recognized for many years. 2. Effector pathway: The H1 receptor is a G protein‐coupled receptor (GPCR) (like all histamine receptors) but H1 specifically linked to phosphatidylinositol pathway (increasing IP3, mobilizing intracellular Ca2+ and increasing cAMP) B. H2 Type Receptor 1. In contrast to H1 receptor, H2 type receptor not antagonized by pyrilamine (a.k.a mepyriamine) or ® diphenhydramine (Benadryl ). Binding of different molecular property of histamine than in H1 receptor. 169

VM522P / VM7522 Pharmacology WIMU Fall 2. Powerful stimulant of gastric acid secretion, increases production of pepsin, intrinsic factor (see lecture “Gastrointestinal Drugs”) a) Therefore, pyrilamine does not block action of histamine on gastric secretion, inhibition of uterine motility, or its chronotropic effect on the heart. 3. Effector pathway: GPCR with activation of adenylate cyclase leading to upregulation of cAMP

C. H3 Type Receptor 1. Presynaptic autoreceptor that provides Ca2+ dependent negative feedback to modulate/dampen release of histamine as a neurotransmitter. a) The level of histamine release is higher after stronger electrical stimulation and this can be blocked by selective H3 agonists b) In addition to the release, the rate of histamine synthesis in histaminergic neurons is also controlled by extra‐neuronal histamine, by binding to the H3 receptor. c) H3 receptors also negatively regulate release of other neurotransmitters such as serotonin, dopamine and acetylcholine (heteroreceptor). 2. Expression widespread in cerebral cortex and subcortex and elsewhere, low density of expression 3. May be involved in regulation of satiety, targeted experimentally for treatment of obesity, sleep disorders, schizophrenia and other disorders 4. No commercially available drugs, yet. 5. Effector pathway: GPCR coupled to Gi type G protein, i.e. downregulating adenylate cyclase which lowers cAMP levels.

D. H4 Type Receptors 1. Involve in mast cell chemotaxis 2. Expressed at high levels in bone marrow, where it may be involved e.g. in neutrophil release 3. No drugs targeting this receptor are currently available 4. Effector pathway: GPCR, Gi Protein‐coupled therefore lowering Camp

3. Physiological Effects of Histamine

A. Smooth Muscle 1. Histamine produces contraction of most smooth muscle but there are species differences. These differences may be related to differences in distribution of H1 and H2 receptors. 2. Bronchoconstriction: Guinea pigs/H.s.: bronchoconstriction (H1 effect). Cause of death in guinea pigs from histamine toxicity. In humans asthma patients 100‐1000fold sensitivity. Rabbits and some other species: bronchodilation (H2>H1) 3. GI tract: a) Guinea pigs/H.s.: contraction of intestinal smooth muscle b) Contraction of guinea pig ileum standard bioassay for histamine c) H1 receptor effect

170

VM522P / VM7522 Pharmacology WIMU Fall

B. Exocrine glands 1. Histamine produces marked increase in gastric secretion by H2 receptor activation on gastric 2+ parietal (oxyntic) cells => cAMP and Ca increased, blocking by H2 drugs a) Increased secretion of pepsin and intrinsic factor accompanies that of HCl in man. b) Gastric secretion cannot be blocked by H1 antihistamine drugs 2. Stimulates salivary, pancreatic, intestinal, bronchial and lacrimal secretion. 3. May produce a release of catecholamines from chromaffin tissue of the adrenal medulla. a) Particularly relevant to patients with pheochromocytoma (tumor of chromaffin cells of the adrenal medulla) who may exhibit a rise in blood pressure following histamine release/administration by catecholamine “flooding”.

C. Anaphylaxis and Tissue pathology 1. “Triple response” of Lewis. a) Small localized red spot at histamine injection site (local dilation of blood vessels) b) Large red “flare” around original red spot (prostaglandin effect?) c) Wheal at site of red spot (edema due to increase of vascular permeability, separation of epithelial cells) 2. This response inhibited by prior administration of H1 antagonists. 3. Activation of mast cells after mosquito bite, histamine is compound of some venoms and stinging secretion. 4. Histamine release from mast cells irritates nerve endings, causing itching or pain.

D. Neurotransmitter 1. Sleep regulation: d) Histamine is released as a neurotransmitter from cell bodies of neurons in the posterior hypothalamus. From here, these histaminergic neurons project throughout brain incl. the cortex. e) Classically, antihistamines (H1 histamine receptor antagonists) produce sleep. Likewise, destruction of histamine releasing neurons, or inhibition of histamine synthesis leads to an inability to maintain vigilance. f) H3 receptor antagonists (which stimulate histamine release) increase wakefulness. 2. Energy production, water regulation (intake) in rats and satiety 3. Modulator influencing release of other neurotransmitters (H3 effect?) a) Inhibits serotonin release in vitro from rat brain cortical slices 4. Role in etiology of mental illness, schizophrenia and depression (?)

E. Cardiovascular effects 1. Injection: a) Decrease in systolic/diastolic blood pressure (H.s.) b) Vasodilation of arterioles, NO release c) Increased heart rate (stimulates heart and reflex tachycardia, increased contractility, but not atrial muscle) d) Flushing, sense of warmth, headache (vasodilation)

2. Some cardiovascular symptoms involved in anaphylaxis due to histamine but other factors more important

171

VM522P / VM7522 Pharmacology WIMU Fall

F. Histamine Toxicity 1. Example scombroid fish poisoning – conversion of fish flesh histidine by bacterial histidine decarboxylase to histamine. Symptoms (dose related) include: a) Flushing b) Hypotension or hypertension c) Tachycardia d) Headache e) Wheals / hives / urticaria f) Bronchoconstriction (Homo, see A.2.) g) Gastrointestinal upset (H2 effect)

4. Representative Drugs and Their Uses

A. Classes of histamine antagonists 1. Physiological antagonists: epinephrin: smooth muscle action opposite of histamine, different receptor, clinically: life‐saving in acute anaphylaxis and for rapid bronchodilation (H1 receptor antagonists only suitable prophylactically, use epinephrin (adrenalin) for acute cases) 2. Release inhibitors: reduce immunologically triggered mast cell degranulation: a) Cromoglicate, nedocromil: asthma treatment in man b) Beta2 adrenoreceptor agonists also reduce histamine release 3. Receptor antagonists (Antihistamines) 4. Histidine decarboxylase inhibitors are still under development.

B. H1 receptor antagonists 1. General pharmacology, usage a) Will help prevent bronchoconstriction when taken prophylactically b) These drugs do not have bronchodilator action of their own like epinephrine and are not useful to achieve rapid bronchodilation in an emergency situation. Epinephrin should be used to induce rapid bronchodilation in these situations. c) H1 receptor antagonists do not block gastric secretion. d) Multiple uses, e.g. for diphenhydramine (Benadryl®): ‐ bug bite/allergies in small animals (D,C), bee stings ‐ (canine) atopy/dermatitis/urticaria ‐ reverse sneeze syndrome (D) ‐ agitation assoc. w./ pruritus/allergies (sedation, anticholinergic): D, C; adjunctive: feather picking due to pruritus in birds (psittacines) ‐ transient behavioral management (car travel, etc.) ‐ prevention of motion sickness/antiemetic (D, C) ‐ adjunctive treatment aseptic laminitis (cattle) ‐ adjunctive therapy of anaphylaxis (cattle, horses) 2. Toxicity and common side effects of H1 type drugs: a) Excellent safety record b) Sedation, drowsiness c) Dryness of mucous membranes (mouth, nose, throat) d) Skin allergies possible after topical application 172

VM522P / VM7522 Pharmacology WIMU Fall e) Gastrointestinal motility affected f) Effects on muscarinic choline receptor, serotonin, local anesthetic receptors 3. First Generation H1 antagonist drugs: strong sedative side effects, lipophilic a) Ethanolamines: e.g. diphenhydramine HCl (Benadryl®) b) Ethylaminediamines: e.g. pyrilamine (Histall®, E) c) Piperazine derivatives: Hydroxyzine (Ifosfamide, Ifex®) 4. Second Generation H1 antagonists: less lipophilic =>enter CNS less efficiently => less drowsiness or sedative side effects: a) Ethylenediamine: Tripelennamine (TBZ®, Fenistil®), halogenated: Chlorpheniramine (Chlorphenamine, often used in small animal practice) also serotonin/epinephrin reuptake inhibitor (SNRI): mild antidepressant. b) Loratadine (Claritin®), Cetirizine (Zyrtec®), expensive c) Fexofenadine (Allegra®), newer alternative to Terfenadine (Seldane®) which has been taken off the market

C. H2 receptor Blocking Drugs: 1. General pharmacology and usage in reduction of GI acid. Sample drugs: cimetidine (Tagamet®), ranitidine (more effective than cimetidine) for treatment/prophylaxis of: a) Duodenal ulcers (up to 8 wks), b) Uremic gastritis, stress‐related erosive gastritis (gastric) ulcers (stress‐induced in horses/very common, esp. race horses + neonatal foals, Dogs), gastrinomas c) Duodenal gastric reflux =>esophagitis, prokinetic effects promoting gastric emptying, GI motility (D, C) d) Pathological hypersecretory conditions (rare): i. Zollinger‐Ellison Syndrome (gastrin producing pancreatic tumors) ii. Systemic mastocytosis iii. multiple endocrine adenomas 2. Toxicity and common side effects of H1 type drugs: a) Excellent safety record (ranitidine side effects/toxicity rare, drugs rel. safe even if overdosed b) H2 antagonists generally well‐tolerated, except for cimetidine (infrequent adverse drug reactions): headache, tiredness, dizziness, confusion, diarrhea, constipation, rash. c) Long‐term use of H2 blockers may increase the risk of cognitive decline in geriatric patients. d) Caution: hepatic or renal insufficiency e) H2 antagonists have been largely, but not entirely, superseded by proton pump inhibitors (e.g. omeprazole, Prilosec®)

173

VM522P / VM7522 Pharmacology WIMU Fall Figure 1: MAJOR PATHWAYS OF HISTAMINE METABOLISM

H H HISTIDINE CH2 C COOH CH2 C NH2 (Orally ingested DECARBOXYLASE H histamine HN N NH HN N 2 is quickly metabolized and eliminated) HISTIDINE HISTAMINE CO2

DIAMINE OXIDASE, N-METHYLTRANSFERASE HISTAMINASE *

H CH2 C NH2 CH2 COOH H NN HN N H3C N-METHYLHISTAMINE IMIDAZOLEACETIC ACID

MONOAMINE RIBOSE OXIDASE ( MAO-B)

CH COOH CH COOH 2 2 H C N N Ribose 3 N N N-METHYLIMIDAZOLE- IMIDAZOLEACETIC ACID ACETIC ACID RIBOSIDE * major pathway in human but relative activities of the two pat hways in animal species may vary Elimination (H.s.): Renal; 2 to 3% excreted as free histamine, 4 to 8% as N-methylhistamine, 42 to 47% as N-methyl imidazole acetic acid, 9 to 11% as imidazole acetic acid, and 16 to 23% as imidazole acetic acid riboside.

Table1: HISTAMINE RECEPTOR SUBTYPES

H1 H2 H3 H4

Size (amino a.) 487 359 373, 445, 365 390

G protein Gq Gs G i Gi coupling (2nd (IP3 , DAG, (cAMP ) (cAMP ) (Ca2+ , cAMP ) 2+ messengers) Ca , cAMP ) Distribution Smooth muscle Gastric parietal CNS: Eosinophils, Endothelial cells, cardiac Presynaptic, neutrophils, cells, CNS muscle, mast myenteric CD4 T cells cells, CNS plexus Representative 2-Methyl- Dimaprit, (R)--Methyl- Clobenpropit agonist histamine impromidine, histamine imetit, clozapine anthamine (chir.), imetite, immepip Representative Pyrilamine Ranitidine Thioperamide, Thioperamide antagonist Cimetidine, clobenpropit, iodophenpropit

174

VM522P / VM7522 Pharmacology WIMU Fall III. SEROTONIN (5‐hydroxytryptamine, 5‐HT)

1. Historical Background

A. Vasoconstrictor activity in serum after clotting of blood 1. Isolated in 1948 and named serotonin 2. Synthesized in 1951 and shown to be 5‐HT

B. Gut stimulating factor (enteramine) 1. Present in invertebrates and invertebrates as indole alkylamine 2. Identified as 5‐HT in 1952.

C. Modern Era: 1. Widespread distribution and functions 2. Role as neurotransmitter and psychopharmacology

2. Distribution and Chemical Properties of 5‐HT

A. Background 1. Widely distributed: b) Plants including fruits, vegetables and various nuts, mushrooms c) Animals including humans. d) Stings and venoms 2. Distribution in mammals a) 90% located in the enterochromaffin cells of GI tract b) Platelets c) CNS: neurotransmitter d) Mast cells: e.g. rodents, cattle, but not humans 3. Present in diet but rapidly metabolized, active 5‐HT is synthesized in situ

A. Structure and Synthesis: See Figure 2 1. Structure – general features a) Indole group b) Terminal amine 2. Synthesis – except platelets which acquire 5‐HT by passage through intestinal blood vessels (active carrier through platelet membrane) a) Amino acid precursor: Tryptophan (Trp, W) b) Initial step, which is also rate‐limiting, is hydroxylation of tryptophan to 5‐ hydroxytryptophan (5‐HTP) by tryptophan hydroxylase. c) Second step is rapid decarboxylation of 5‐HTP to 5‐HT by non‐specific aromatic amino acid decarboxylase. 3. Storage and release: a) Storage in secretory granules, complex with ATP b) Exocytosis (“release reaction” of platelets) 4. Metabolism: a) Deamination by monoamine oxidase (MAO) followed by oxidation or reduction and excretion in the urine b) High affinity uptake in nerve terminals and platelets (terminates 5‐HT action)

175

VM522P / VM7522 Pharmacology WIMU Fall

3. Physiological Effects of 5‐HT

A. Central Nervous System – raphe nuclei of brainstem 1. Pain perception 2. Sleep 3. Autonomic function: temperature, blood pressure (species specific: humans, dogs: increase ‐ cats: decrease 4. Mood, sexuality, i.e. many drives, low levels associated with bipolar disorder, anxiety, depression 5. Food intake 6. Emesis

B. Neuroendocrine Effects 4. Pineal gland ‐ precursor of melatonin 5. Hypothalamic‐hypophysial function 6. Endocrine glands, co‐storage and release (?) with polypeptide hormones c) Thyroid d) Pancreas

B. Gastrointestinal System 5. Peripheral nervous system. 6. Enterochromaffin cells g) stimulates GI smooth muscle => facilitates tone, peristalsis, motility enhancing effect by 5‐HT2 agonists (e.g. Cisapride). h) basal release augmented by mechan. stimulation, hypertonicity, acidity, norepinephrine and vagal tone i) Serotonin overproduction in carcinoid tumor => severe diarrhea, abdominal cramps

C. Cardiovascular System 5. contraction of vascular smooth muscle (mainly 5‐HT2 receptor) => vasoconstriction, but not skeletal muscle & heart (vasodilation)

D. Platelets 3. Bleeding time , clotting time and capillary resistance unaffected by serotonin depletion 4. Regulation of vascular tone – antihypertensive action of 5‐HT antagonists unaffected by serotonin depletion

4. Receptor Diversity for 5‐HT

A. Subtypes of 5‐HT receptors 5. Seven main classes identified: 5‐HT1 to 5‐HT7, 6. Identified based on affinities for 5‐HT, selective antagonists, distribution, species‐specificity. 7. Several subtypes, total of 25 postulated receptors 8. Most have been cloned, not all characterized

B. Mechanisms of Receptor‐Effector Coupling

176

VM522P / VM7522 Pharmacology WIMU Fall 5. Receptor types: G protein coupled receptors and one ligand‐gated ion channel (5‐HT3: monovalent cation channel that mediates fast depolarization responses) 6. Second messenger systems: adenylate cyclase (cAMP) and phosphoinositide hydrolysis (IP3)

5. Drugs that affect the action of 5‐HT

A. Drugs that alter endogenous 5‐HT levels by affecting its synthesis 1. The precursor tryptophan – may be of use in phenylketonuria. Competition for transport across blood/brain barrier with phenylalanine and tyrosin (aromatic amino acid transporter). 2. Inhibitors of 5‐HT synthesis: toxic (e.g. p‐chloroamphetamine) 3. 5‐HT (presynaptic) uptake inhibitors: increase 5‐HT activity by prolonging 5‐HT location in the synaptic cleft (=> higher synaptic concentration) a) Tricyclic “antidepressants” (TCAs) ‐ will also partially inhibit norepinephrin reuptake by inhibiting monoamine pump, for treatment of obsessive compulsive disorders in pet behavioral management: ‐ separation anxiety (D), excessive grooming ‐ Spraying, self‐mutilation, anxiety (C) ‐ Pruritus (adjunctive, D, C) ‐ neuropathic pain (D, C) ‐ feather plucking (adjunctive, B) ‐ urine marking, mounting, inter‐male aggression (D) ‐ cataplexy (attacks suffered in narcolepsy) D, C, H ‐ urinary incontinence, D, C ‐ ejaculatory dysfunction / pharmacol. induced ejaculation, Horse i. Amitryptiline ii. Clomipramine (Clomicalm®) iii. Imipramine b) SSRI (selective serotonin reuptake inhibitors): little or no effect on other neurotransmitters (e.g. dopamine, norepinephrine), usage like TCA i. Fluoxetine (Prozac®, Reconcile® for dogs) ii. Paroxetine HCl (Paxil®) iii. Sertraline (Zoloft®) iv. (Chlorpheniramine, H1 receptor antihistamine is also SSRI) 4. MAO inhibitors (MAOI): prevent monoamine neurotransmitter breakdown, MAO A: mostly serotonin/norepinephrin degradation5‐HT location in the synaptic cleft (=> higher synaptic concentration) a) Clorgyline®: irreversible inhibitor b) Moclobemide (Aurorix®): reversible inhibitor of MAO A (RIMA) c) All other MAOI non‐selective inhibitors of MAO A and MAO B

B. Drugs that interact with 5‐HT receptors 1. 5‐HT receptor agonists: a) Cisapride: 5‐HT4 agonist, formerly used in treatment of gastro‐esophageal reflux, GI stasis (motility disorders, constipation), toxic b) Sumatriptan, other “triptans” (5‐HT1D and 5‐HT1B agonists): Expensive, exp. stage as veterinary medicinal product (VMP) c) Buspirone (5‐HT1A agonist), nonbenzodiazepine anxiolytic, used for behavior disorders in D, C related to fear/phobia

177

VM522P / VM7522 Pharmacology WIMU Fall d) Ergot alkaloid derivatives (Claviceps purpurea), e.g. ergotamine, partly agonists and antagonists at numerous 5‐HT receptor subtypes, effective drugs for prophylactic and acute treatment of migraine, e.g. methysergide (LSD congener without hallucinogenic properties) 2. 5‐HT antagonists: a) Phenoxybenzamine: a adrenoreceptor blocker, 5‐HT2 antagonist, used to treat: ‐ functional urethral obstruction: decreases sympathetic‐mediated urethral tone (D, C) ‐ hypertension (D, C), short‐term treatment ‐ laminitis, developmental phase, Horse b) Cyproheptadine: also H1 antagonist (antihistamin, D) (5‐HT2 antag., ) for treatment of ‐ skin allergies/pruritus (D, C) ‐ appetite stimulant (C) ‐ equine Cushing’s disease (cortisol overproduction) c) Ketanserin: highest affinity for 5‐HT2A receptor, but also binds less potently to the 5‐ HT2C, 5‐HT2B, 5‐HT1D, alpha‐adrenergic, and dopamine receptors. Experimental antihypertensive drug. d) Dolasetron Mesylate: 5‐HT3 antagonist, antiemetic esp.in chemotherapy (D, C), also: Ondansetron: 5‐HT3 antagonist, potent antiemetic, limited use in severe nausea/vomiting, Zofran®, no VMP, yet.

6. 5‐HT Toxicity

A. Serotonin syndrome 1. Serotonin poisoning (iatrogenic toxic syndrome), potentially life‐threatening condition, caused by combination of drugs that have additive or synergistic effects on 5‐HT levels, e.g. combinations of “antidepressants” (TCA, SSRI, MAOI) and each of these drug classes alone in combination with: a) Opioids: tramadol, pethidine, oxycodone, morphine... b) Illicit drugs: methylenedioxymethamphetamine (MDMA or ecstasy), lysergic acid diethylamide (LSD), cocaine, heroin c) CNS stimulants: phentermine, amphetamines, sibutramine, etc d) 5‐HT1 agonists: triptans e) others: selegiline, tryptophan, lithium, linezolid, dextromethorphan (DXM), 5‐HTP, chlorpheniramine 2. Treatment: benzodiazepines (diazepam (Valium®), clonazepam): 5‐HT antagonists by depression of subcortical levels of CNS, exact mechanism unknown

178

VM522P / VM7522 Pharmacology WIMU Fall

Figure 2: STRUCTURE AND METABOLISM OF 5‐HT

H COOH C C NH Tryptophan 2 H H N H (* rate limiting step) tryptophan-5-hydroxylase H COOH HO * C C NH 2 5-Hydroxytryptophan H H (5-HTP) N H aromatic L-amino acid decarboxylase H H H H H O HO HO C C NH2 C C N C CH3 5-Hydroxytryptamine H H 5-HT N-acetylase H H (5-HT) N N H H monoamine oxidase (MAO-A) H H HO C C hydroxyindole 5-Hydroxyindole- O-methyltransferase acetaldehyde H O N aldehyde H aldehyde dehydrogenase NAD NADH reductase H OH H H H H H O HO HO H3CO C C C C H C C N C CH3 H O H OH H H N N N 5-Hydroxyindole- 5-Hydroxytryptophol Melatonin acetic acid (5-HIAA)H (5-HTOL) H Excretion in the urine H

179

VM522P / VM7522 Pharmacology WIMU Fall IV. PURINES (Adenosine, and Purine Nucleotides, ATP and ADP)

1. Adenosine i. Released from most cells as a breakdown product of ATP ii. Short lived in the extracellular fluid iii. Thought to relate blood flow to tissue metabolism iv. Multiple adenosine receptors (all G‐protein coupled)

a. Effects:

i) A1 ‐ heart and CNS o in heart it depresses cardiac conduction o in CNS inhibits glutamate release (neuroprotection in ischemia?)

ii) A2 ‐ vasodilatation, inhibit platelet aggregation

iii) A3 ‐ activation of mast cells – bronchoconstriction

b. Therapeutic use: To terminate supraventricular tachycardia (short acting) Given as an IV bolus as short‐lived c. Adverse effects: Very transient asystole. Bronchospasm and atrial fibrillation are rare effects.

2. Adenosine A1/A2 receptor antagonists

a. Examples: Methylxanthines such as theophylline Methylxanthines also inhibit phosphodiesterase and so inhibit cAMP breakdown

b. Therapeutic use: Bronchodilator Theophylline is a weaker bronchodilator than beta 2 agonists, but it also inhibits mast cell degranulation and therefore mediator release (such as histamine).

c. Adverse effects: i.CNS excitation (restlessness, tremors and seizures) ii.Nausea and vomiting, iii.Diuresis iv.Cardiac stimulation

Caffeine is also a methylxanthine

180

VM522P / VM7522 Pharmacology WIMU Fall

V. EICOSANOIDS (PROSTAGLANDINS & LEUKOTRIENS)

READING ASSIGNMENT:

Riviere and Papich : Chapter 18, page 439‐455.

Rang et al., Chapter 13, pages 215‐225 and Chapter 16.

OBJECTIVES OF PART 4, section 2 :

1. Be able to explain the sources of and physiological functions of eicosanoid mediators.

2. Know what therapeutic situations involve the use of a prostaglandin (or synthetic derivatives of a prostaglandin) and situations in which the inhibition of prostaglandin generation is a key component of the therapeutic mechanism.

3. For peptide and protein type mediators know common aspects in the generation, action, breakdown and therapeutic applications of these types of agents. Because there are so many different specific agents in this class, and specific applications will be discussed latter in clinical pharmacology, at this time we will not focus on the physiological actions of these types of mediators.

OUTLINE: I. Prostglandins II. Leukotrienes III. Endocannabinoid

Lipid derived chemical mediators, e.g. eicosanoids

I. PROSTAGLANDINS

1. Eicosanoids are generated in almost all tissues from phospholipids by cyclooygenase (CO) and lipoxygenase (LO).

Biosynthesis and metabolism of prostaglandins (PGs) and other eicosanoids

PAF (platelet activating factor) phospholipids

O O O PLA2 R1 COOH R2 R1 arachidonic acid R 2

lipoxygenase cyclooxygenase

TXA PGH PGI leukotrienes prostacyclins thromboxanes O COOH

PGE PGF prostaglandins

O COOH O 181

VM522P / VM7522 Pharmacology WIMU Fall

2. They are not stored in tissue, but rather synthesized rapidly on demand

This is why so many drugs used therapeutically interfere with the synthesis of eicosanoids, e.g. aspirin inhibits CO

Many stimuli can generate PG’s. These include factors released during trauma and infections. Some specific examples:

i) Thrombin in platelets

ii) Antigen‐antibody reactions in mast cells

iii) Bradykinin in fibroblasts

iv) C5a in neutrophils

v) Cytokines, e.g. TNF, IL1

3. In general, they act locally within the tissue they are generated

4. Their systemic half‐life is in the range of seconds to at most a minute or two. The lung is the major site of degradation, with 95% inactivated at 1st pass through lung

5. Local concentrations may be very elevated and last considerably longer than system levels

a. Mechanism of action of prostaglandins:

1. PGs produce their effects by binding to specific receptors. These are listed in the table below. However, this is for your information only. You do not need to memorize them at this time. You also do not need to memorize all the transduction mechanisms for the exam.

RECEPTOR TYPE ENDOGENOUS LIGAND TRANSDUCTION MECHANISM

DP PGD2 increases cAMP EP1, EP2, EP3 PGE2 EP1 activates PLC EP2 Increases cAMP EP3 decreases cAMP FP PGF2 activates PLC IP PG12 increases cAMP TP TXA2 activates PLC

2. One thing to keep in mind is that most PGs don’t just bind to one receptor subtype; there is a lot of cross‐reactivity at other receptors

3. Some cells may express more than one type of receptor

182 VM522P / VM7522 Pharmacology WIMU Fall

b. Effects of prostaglandins:

PGs have mixed effects on different tissues. For example some cause constriction and some relaxation of smooth muscle. There is also species variation of PG effects. The main effects are summarized below:

i. Smooth muscle:

‐ Bronchial: constriction: EP1, TP, FP in some species (cats and dogs) relaxation: EP2

‐ Intestinal: contract: EP1, EP3 relax: EP2, DP

‐ Uterine: contract: FP (PGF2) FP (PGF2) also causes luteolysis in some species, e.g. cattle, sheep, pigs, horses ‐ see next page. ‐ Blood vessel: constriction TP (TXA2 – platlets) dilation: EP2, DP, IP (PGI2 endothelial cells lining BV)

ii Platelet aggregation

activate: TP (TXA2 ‐ platlets), inhibit: DP (PGD2), IP (PGI2 – endothelial cells of vasculature)

iii. GI secretion

EP3 ‐ inhibits gastric acid secretion ‐ increases mucosal secretion EP2 ‐ increases intestinal secretions

iv. Inflammation

‐ PGs are profound vasodilators and synergize with other vasodilators (histamine, bradykinin) ‐ Increased capillary permeability mediated by histamine and bradykinin ‐ Sensitize pain fibers

‐ In the CNS (hypothalamus) generation of PGE2 is associated with fever

v. Eye

Increases ocular fluid drainage

183 VM522P / VM7522 Pharmacology WIMU Fall vi. Reproductive Effects:

PGF2 is produced by the uterus in some non‐primates, e.g. mares, cows, sows, ewes. In non‐ pregnant females PGF2 is released about day 14 or 15 in the estrous cycle and the corpus luteum degenerates. Pregnancy inhibits release of PGF2, the corpus luteum persists and the fetus develops.

c. Therapeutic uses of prostaglandins and prostaglandin analogues:

1. PGE1 analog

a. Examples: misoprostol

b. Effects:

Inhibits gastric acid secretion and protects gastric mucosa

c. Therapeutic uses:

GI ulcers (especially prevention of NSAID induced ulcers) (dog)

d. Adverse effects:

i. GI discomfort, vomiting and diarrhea ii. Abortifacient (contradicted in pregnancy)

2. PGF2 agonists

a. Examples: dinoprost and latanoprost (eye)

b. Effects:

i) Remember that activation of the FP receptor causes contraction of the uterus as well as luteolysis in some species, e.g. cattle, sheep, pigs, horses .

ii) Activation of the FP receptor causes increased ocular fluid drainage.

Therefore there are two main therapeutic uses of PGF2 agonists. First as regulators of uterine function. Second as a treatment for glaucoma. However the drugs used are different.

184 VM522P / VM7522 Pharmacology WIMU Fall

c. Therapeutic uses:

i) Reproduction and uterine function:

Used clinically: PGF = dinoprost tromethamine (LUTALYSE)

synthetics (cloprostenol, fenprostalene)

Estrus synchronization (cattle, mares, swine, sheep, goats)

Luteolysis (CL regression) (cows, mares: in animals who are acyclic due to persistence of corpus lutea, e.g. due to ovarian cysts)

Pyometra (uterine infection) (dog, cats, cows)

Abortifacient (cows, mares, swine, sheep, goats, dogs, cats)

Induce parturition, i.e birth (cows, swine, sheep, goats)

ii) Glaucoma:

Used clinically:Latanoprost (PGF2analog XALATAN)

Increases ocular fluid drainage by the alternative route (see ocular pharmacology, pages 111‐113)

d. Adverse Effects:

Dinoprost: Smooth muscle contraction ‐ diarrhea, abdominal discomfort ‐ bronchoconstriction ‐ hypertension ‐ may cause retention of the placenta Should not be handled by pregnant women or people with bronchial disease (e.g. asthma)

Latanoprost: Eyes ‐ include redness, discomfort, blurred vision Has the same potential side effects as dinoprost, but because latanoprost is administered to the eye, these side effects are not normally seen unless it gets into the bloodstream.

185 VM522P / VM7522 Pharmacology WIMU Fall

3. Cyclooxygenase Inhibitors

Examples: Non‐steroidal anti‐inflamatory drugs (NSAIDs)

These will be covered next semester.

II. Leukotrienes (examples: LTA4, LTB4, LTC4, LTD4, LTE4, LTF4)

1. Produced by leukocytes

2. LTC4, LTD4, and LTE4—constitute “slow‐reacting substance of anaphylaxis (SRS‐A)

3. LTB4—powerful chemotactic agent for both neutrophils and macrophages

4. Local vasodilators and increase vascular permeability

5. Lipoxygenase inhibitors are being developed as drugs

6. Leukotriene receptor antagonists are marketed for human asthma

III. Endocannabinoids (Anandamide, 2‐arachidonoylglycerol (2‐AG))

1. Are generated in almost all tissues

2. They are not stored, but are synthesized rapidly on demand through a calcium/cAMP sensitive mechanism

3. In general, they act locally within the tissue they are generated

4. Produce effects through two G protein coupled receptors (CB1 and CB2)

5. Currently this system is a target of pharmaceutical companies (obesity).

186 VM522P / VM7522 Pharmacology WIMU Fall

VI. PEPTIDES (NEUROKININ, ANGIOTENSIN, BRADYKININ)

I. Peptides and Protein mediators

This class includes a large variety of mediators

1. Hormones (e.g., insulin, GnRH, ACTH, oxytocin, atrial natriuretic peptide)

2. Cytokines and chemokines (e.g., TNF, IL‐1)

3. Neuromodulators (substance P, enkephalins, endorphins, neuropeptide Y)

4. Paracrine and autocrine factors (e.g., bradykinin)

5. Growth factors (e.g., vascular endothelial growth factor, platelet‐derived growth factor)

6. Others (e.g., angiotensin, vasoactive intestinal peptide)

Biosynthesis and actions

1. All of these (even the tiny tripeptide TRH) are coded for by specific genes

2. Synthesized by standard protein synthetic pathways i. gene product may have multiple copies of peptide, and multiple peptide fragments ii. some may need post‐translational modification in order to have biological activity (e.g. glycosylation, acetylation)

3. Release i. standard regulated vesicular release (e.g., circulating hormones from the pituitary or pancreas) ii. active component generated by peptidergic cleavage of a precursor (e.g., angiotensin, bradykinin)

4. They all work on cell membrane receptors (do not cross lipid membranes)

5. Actions may be rapid (a few seconds to minutes), to delayed (hours, days, weeks)

187 VM522P / VM7522 Pharmacology WIMU Fall

Pharmacological considerations of using peptide drugs:

1. They do not readily cross membranes and, thus, are not readily absorbed

i. need to be given by parenteral mechanisms (IV, IM, SC injections)

ii. some can be given across nasal epithelium iii.

don’t cross the BBB

2. They are not stable in the stomach and intestines (treated as any other protein substance destined for digestion)

3. They tend to have short half‐life’s if not stabilized ‐ Can stabilize by either adding D‐amino acids into the structure or changing nature of the peptide bond

4. For most synthesis inhibitors are not successful because it would disrupt general protein biosynthesis ‐ exception is those in which a specific enzyme is responsible for generating an active fragment from proteolytic cleavage best example: ACE inhibitors—Angiotensin Converting Enzyme

5. For some small molecule inhibitors and mimetic have been generated (e.g., opiates)

6. For others the agent itself (e.g., insulin, growth hormone) have been developed ‐ tend to be very expensive

7. For others antagonistic peptides or antibodies have been developed

1. NEUROKININ (NK1) RECEPTOR ANTAGONISTS

Example: Maropitant

Therapeutic use: Antiemetic These antagonists decrease vagal afferent activation of the nucleus of the solitary tract (NTS) in the hindbrain, by blocking the actions of the neurokinin substance P. Too much vagal activation of the NTS results in nausea. Therefore decreasing this helps reduce nausea and emesis. Adverse effects: Few at the doses used to treat nausea

188 VM522P / VM7522 Pharmacology WIMU Fall 2. RENIN_ANGIOTENSIN SYSTEM

1. Introduction

A. Historical background: 1. Tiegerstedt and Bergman (1898) – Renal extracts contain a pressor substance (renin). 2. Braun‐Menendez et al. and Page et al. (1940): renin is an enzyme which acts on a circulating substrate to produce biologically active peptide (angiotensin II) 3. Gross (1958) – Involvement of angiotensin II in the release of aldosterone 4. Today often exploited for pharmacological targeting of hypertension.

2. Components of the Renin‐Angiotensin system (see Figure 1)

A. Renin 1. Main source: kidney, but also other tissues. 2. Aspartylprotease, generates angiotensin I from circulating substrate, angiotensinogen 3. Plasma concentration of renin rate limiting factor in angiotensin II formation 4. Renin secretion controlled by: a) Reduced renal perfusion pressure (intrarenal stretch receptor) b) Reduction of sodium concentration in early distal tubule c) Sympathetic activation of beta‐adrenergic receptors

B. Angiotensinogen 1. Abundant protein in the plasma (~57 kDa) 2. Angiotensin is cleaved from the N‐terminus initially as a decapeptide

C. Angiotensin Converting Enzyme (ACE) 1. Nonspecific enzyme that cleaves dipeptides from diverse substrates (e.g. bradykinin, syn.: “kininase II”), dipeptidyl carboxypeptidase 2. Ectoenzyme associated with the lumen of endothelial cells, converts angiotensin I to angiotensin II (lung). 3. Preferred substrate: not prolin as penultimate amino acid at the carboxyterminus => does not further process angiotensin II

D. Angiotensin I, II, and III 1. Angiotensin I: very little biological activity 2. Angiotensin II: has most of the biological activity, structure‐activity relationships: a) Most of the information contained in C‐terminal heptapeptide (reason for biological activity of angiotensin III) b) Agonist activity dependent on phenylalanin (F) in position 8 (at least in peripheral tissues but not brain) c) Peptidic antagonists: substitutions in pos 1 and 8 (=> drug compounds less susceptible to degradation by peptidases 3. Angiotensin III: a) Angiotensin III is as potent as Angiotensin II in some systems, e.g. aldosterone release. b) However, in other tissues it is much less potent – e.g. vascular smooth muscle

E. Angiotensinases – Peptidases involved in the inactivation of angiotensins

189 VM522P / VM7522 Pharmacology WIMU Fall 1. Angiotensin half‐life: ~15‐60 seconds; mostly destroyed by single passage through the pulmonary vascular bed 2. Rapid removal from circulation by variety of peptidases (collectively “angiotensinases): a) Aminopeptidases b) Endopeptidases 3. Enzymes not specific to angiotensins

3. Functions of the Renin‐Angiotensin system – Plays a major role in cardiovascular and body fluid homeostasis through the actions of circulating angiotensin II on its various target organs (see Fig. 2)

A. Adrenal Cortex 1. Stimulates synthesis and release of aldosterone from zona glomerulosa cells. 2. This action is augmented by hyponatremia and hyperkalemia

B. Kidney – complex effects 1. Antidiuresis and antinatriuresis by promoting sodium reabsorption by kidney 2. Reduced glomerular filtration

C. Cardiovascular System 1. Direct vasoconstrictor action on vascular smooth muscle (augmented by sympathetic nerve system, see (D.)), increased blood pressure 2. Increases cardiac contractility with little change in heart rate 3. Renin‐Angiotensin S. appears to play a major role in the pathogenesis of hypertension, even when plasma renin activity is not elevated (see (D.)

D. Peripheral autonomic nervous system 1. Sympathetic nervous system 2. Adrenal Medulla ‐ release of catecholamines from chromaffin cells (D.) e) Release of norepinephrin from postganglionic neurons f) Enhanced sensitivity of target tissues to norepinephrin E. Central Nervous System – Circulating angiotensin II can gain access to brain regions lacking a blood/brain barrier and may be produced within the brain. 1. Physiological – Central pressor response resulting from sympathetic activation 2. Endocrine – release of ADH and ACTH 3. Behavioral – thirst and sodium appetite

4. Receptors and Signal Transduction Mechanisms

A. Receptor subtypes – at least 4, only two well characterized 1. Type 1 (AT1) ‐ mediates most known functions of angiotensin a) sensitive to Losartan b) predominates in vascular smooth muscle c) GPCR, activates phosholipase C (=>IP3/diacylglycerol production): smooth muscle contraction (within seconds) 2. Type 2 (AT2) ‐ affinity for angiotensin II similar to AT1 a) present at high density in fetal tissues, later only in adrenal medulla, reproductive tissues, vascular epithelium, parts of the brain b) GPCR, involves serine and tyrosine phosphatases, phospholipase A, NO, cGMP (from stimulation of guanylate cyclase) c) Upregulated in pathological conditions (heart failure, myocardial infarction) 5. Inhibitors of the Renin‐Angiotensin System 190 VM522P / VM7522 Pharmacology WIMU Fall

B. Inhibitors of Angiotensin Converting Enzyme (“ACE inhibitors”) 1. Captopril – Highly specific blocker of conversion of angiotensin I to angiotensin II: although enzyme is not that specific, also cleaves bradykinin (=>) a) Hypertension i. Useful in all forms of hypertension not just high‐renin ii. May be combined with diuretic. b) Chronic Congestive Heart Failure i. Reduces afterload and preload ii. Improves cardiac output and lessens congestion 2. Enalapril – Similar to captopril, many similar drugs exist a) Converted to active compound in serum b) More potent than captopril c) Longer duration of action allowing for single daily dose. 3. Adverse drug effects, toxicity: a) Angioedema, cough b) Renal impairment c) Birth defects, congenital malformation of CNS, cardiovascular system

C. Receptor Antagonists 1. Peptide congeners ‐ Saralasin and others ‐ antagonists with substitutions in positions 1 and 8 of angiotensin II. Limited potential use as intravenously administered antihypertensive agents (orally ineffective). 2. Nonpeptide antagonists: Losartan, Valsartan, and others: antihypertensive drugs with lower side effects than ACE inhibitors, e) interact with AT1 but not AT2 receptors f) Cardioprotection still unclear

Figure 1: FORMATION AND DESTRUCTION OF ANGIOTENSINS 1… …14 NH2-DRVTIHPFHLVIHS-Rest Angiotensinogen ACE = angiotensin conv. enzyme ACE2 = ACE-related carboxypeptidase ACE2*) = alt.: prolylcarboxypeptidase RENIN AP = aminopeptidase E = endopeptidases 11… 14 VIHS-Rest

2 … …10 1… …10 19… … NH2-RVTIHPFHL-COOH NH2-DRVTIHPFHL-COOH NH2-DRVTIHPFH-COOH Angiotensin (2-10) AP Angiotensin I ACE2 Angiotensin (1-9) 1 10 D L E ACE ACE ACE 910 8 9 10 9 10 9 10 HL FHL HL HL 2… …8 1… …8 1… …7 NH -RVTIHPF-COOH NH -DRVTIHPF-COOH NH -DRVTIHP-COOH 2 2 *) 2 Angiotensin III AP Angiotensin II ACE2 Angiotensin (1-7) 1 8 (active) D (active, main) F

Figure 2: THE HOMEOSTATIC ROLE OF THE RENIN‐ANGIOTENSIN SYSTEM 191 VM522P / VM7522 Pharmacology WIMU Fall

Blood pressure Blood pressure rises falls

Perfusion Perfusion of juxta- RENIN of juxta- Blood volume glomerular glomerular Blood volume - RELEASE + increases apparatus apparatus decreases increases decreases

+ Na retention Na+ depletion

Aldosterone ANGIOTENSIN secretion FORMATION - inhibits

stimulates Vaso- + constriction

2. BRADYKININ

1. Introduction

A. Chemical structure 1. Bradykinin: Arg‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe‐Arg (=RPPGFSPFR) 2. Kallidin (=Lysylbradykinin): KRPPGFSPFR, additional lysine at N‐terminus 3. Methionyllysylbradykinin: MKRPPGFSPFR

2. Synthesis and Metabolism

A. Synthesis – A variety of inflammatory events activate reactions that generate bradykinin in plasma (see Figure 1). 1. Kininogens: large alpha‐2 globulins present in plasma, lymph and interstitial fluid that are synthesized in the liver as the precursors of kinins. Substrates of kallikreins: a) High‐molecular‐weight (HMW) kininogen (15‐25%). i. Confined to blood stream ii. Substrate of plasma kallikrein to form bradykinin b) Low‐molecular‐weight (LMW) kininogen. i. Alternative splicing => smaller carboxyterminus => crosses capillary walls ii. Substrate of tissue kallikrein to form kallidin 2. Kallikreins: enzymes are normally inactive and must be activated by proteases. a) Plasma kallikrein: inactive complex with HMW kininogen. b) Tissue kallikrein: ususlly acts locally near its site of origin.

B. Metabolism (see Figure 2)

192 VM522P / VM7522 Pharmacology WIMU Fall 1. Very short half‐life in plasma (<15s); destroyed in pulmonary vascular beds by nonspecific kininases: a) Dipeptidyl carboxypeptidase = kininase II = angiotensin converting enzyme (ACE) b) Arginine carboxypeptidase = kininase I: generates fragments that remain active in damaged tissue (as “des‐Arg kinins”) where they bind to novel receptor. c) Aminopeptidase P: inactivates bradykinin by removing N‐terminal arginine (R) residue

3. Physiological Functions of Kinins – similar to histamine (see Table 1)

A. Inflammation – Involved in a variety of inflammatory disorders (B1) 1. Rhinitis caused by inhalation of antigens/rhinoviral infection, other respiratory problems (asthma, other forms of rhinitis), 2. Endotoxic shock and vascular complications 3. (Hereditary) angioedema – excessive activation of plasma kallikrein

B. Pain: strong algesic agents, lead to release of neuropeptides from primary sensory neurons (e.g. substance P, neurokinin A), B2

C. Renal Function (see Figure 3) 1. Local, paracrine regulation of kidney function: urine volume and composition

D. Cardiovascular System (see Figure 3) 1. Vasodilation, lowers blood pressure (B2). B2 receptor KO mice: normal basal BP but exaggerated BP response to salt loading or activation of the renin –angiotensin system 2. Cardioprotective in hypertensive states 3. Exp. infusion of bradykinin: preconditioning of the heart against ischemia reperfusion injury 4. Antiproliferative, prevents smooth muscle cell proliferation in the presence of endothelial cells by stimulating tissue plasminogen activator (tPA) release.

4. Mechanisms of Action

A. Receptor Heterogeneity 1. B1 receptors ‐ contraction of vascular smooth muscle in damaged tissue. a) Expression induced by tissue damage b) Most sensitive to des‐Arg kinins c) GPCR, activates phosholipase C 2. B2 receptors ‐ opposite effects of angiotensin II AT1 receptor a) Mediate effects of intact kinins (bradykinin, kallidin) i. Vasodilation, smooth muscle cell relaxation (via EDRF) ii. Na+ retention iii. Pain b) GPCR, activates phospholipase A2 c) Production of nitric oxide (NO) in endothelial cells (EDRF)

5. Inhibitors of Kinins and Therapeutic Uses

A. Receptor Antagonists – Limited usefulness due to peptidic nature; non‐selective for B1 and B2 subtypes. 1. Cold symptoms caused by rhinovirus

193 VM522P / VM7522 Pharmacology WIMU Fall 2. Allergic asthma 3. Analgesic with burns

B. Kallikrein Inhibitors: also peptide inhibitors. 1. Aprotinin (Trasylol): acute pancreatitis and carcinoid syndrome (GI neuroendocrine tumors) 2. Newer compounds under development that target other cleavage sites.

Figure 1: FORMATION AND DESTRUCTION OF KININS

Hageman factor Trypsin =Active peptidases Kallikrein Note: kininase II + is identical to ACE Plasma Plasma kallikrein prekallikrein +

HMW kininogen Bradykinin

Kininases Inactive Aminopeptidases I and II fragments

LMW kininogen

Kallidin

+ + + + +

Tissue kallikreins

Figure 2: METABOLISM OF BRADYKININ

Aminopeptidase P Kininase I

R P P G F S P F R Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg

1 2 3 4 5 6 7 8 9

Dipeptidyl- Kininase II (ACE) & peptidase IV Neutral endopeptidase

Table 1: Interactions of the kallikrein‐kinin and renin‐angiotensin systems

194 VM522P / VM7522 Pharmacology WIMU Fall

Tissue or Cell type Histamine Bradykinin Serotonin (5-HT) Receptor Response Receptor Response Receptor Response

1) Arteries H Contraction B Contraction 5-HT2 Contraction 1 1 2) Arterioles H Relaxation (via EDRF) B Relaxation (via EDRF) 5-HT1 Relaxation (via EDRF) 1 2 - Relaxation 5-HT2 Relaxation H2 - Venules Contraction Relaxation (via EDRF) Contraction H1 B2 Relaxation H2 - Veins Contraction Contraction 5-HT Contraction H1 B1 2 - - 5-HT1 Relaxation - - Endothelial cells Release of ERDF Release of ERDF 5-HT1 Release of ERDF H1 B2 1 a)Release of mediators b)contraction Increased permeability - 3) - H B 1 Increased permeability 1 Autonomic nerves - - - - 5-HT1 Decreased release of NE4) Sensory nerves Depolarization Depolarization 5-HT Depolarization H1 B2 3 (itch, pain) (pain) (itch, pain) Mast cells Decreased release of Histamine release - - H2 ? mediators

Platelets - - - - 5-HT2 Stimulate aggregation

1) Synthesis of B1 receptors is induced by tissue damage. The normal response is dilatation via B2 receptors 2) EDRF = endothelium-derived relaxing factor, NE = norepinephrine 3) 5-HT does not markedly increase vascular permeability (in man)

Figure 3: INTERACTIONS OF THE KALLIKREIN‐KININ AND RENIN‐ANGIOTENSIN SYSTEMS

195 VM522P / VM7522 Pharmacology WIMU Fall Part 5 – Diuretics (R. Meyer) L27, L28: Diuretics

1. General Concepts of Diuretics

A. Definition 1. Agents that increase urine flow; actually, all current diuretics increase urinary sodium and fluid excretion. 2. Diuretics exert their effect by directly or indirectly inhibiting sodium transport. 3. Diuretics usually have a single predominant site of action within the nephron (Note: but events in one region will affect more distant sites) 4. Alterations in body fluid composition induced by diuretics can result in compensatory changes (i.e. neural endocrine) which may oppose action of drug.

B. Major Sites of Diuretic Action (relation to NaCl and water reabsorption) 1. Mechanisms of sodium transport across tubular epithelium – most is transcellular (see Figure 2) a) Electrogenic entry of sodium b) Co‐transport of sodium with organic solute or phosphate c) Entry of sodium in exchange for a proton moving in the opposite direction (antiport) d) Simultaneous entry of sodium, one potassium and two chloride ions. e) Entry of sodium coupled to single chloride ion. f) Paracellular pathway – intercellular spaces 2. Organization of nephron and type of sodium entry (NOTE: Letters refer to mechanisms described above and displayed in Figure 1.) a) Proximal convoluted tubule (PCT) – a, b, c. b) Late proximal tubule (LPT) – a, f. c) Thick ascending part of Henle’s loop – d. d) Distal convoluted tubule (DCT) – e. e) Late distal tubule and collecting duct – a, c.

C. Factors that Determine the Effectiveness of Diuretics 1. Site of action of the drug a) If drug works where only a small amount of sodium is reabsorbed, then drug will be a weak diuretic b) Proximal tubule diuretics are weak because more distal sites reabsorb the bulk. c) Combination of drugs each with different site of action can be effective. 2. Physiological state of animal – particularly volume status. 3. Access to site of action – most diuretics act on receptors in the apical membrane of the lumen and must approach from the tubular fluid (see Table 1)

2. The Diuretics

B. Osmotic Diuretics 1. General Properties a) Compounds that are freely filtered by the glomerulus and that remain in the tubular lumen in high concentrations and that are not pharmacologically active. b) May be exogenous compound (e.g. mannitol – most effective) or endogenous compound (e.g. glucose and urea). 2. Mechanism of Action a) Proximal tubule – non‐reabsorbable solute which exerts an osmotic effect and indirectly inhibits the movement of sodium.

196 VM522P / VM7522 Pharmacology WIMU Fall b) Henle’s loop – these osmotic agents inner medullary flow, reducing hypertonicity and favorable gradient for water and sodium reabsorption. c) Greatest effect is on urine flow (i.e. lose more water than sodium) 3. Therapeutic uses – maintain dilute urine flow a) Early treatment of acute renal failure under diverse conditions b) Reduce cerebral edema c) Reduce intraocular pressure 4. Toxicity and Contraindications a) Volume depletion, dehydration and hypernatremia b) Anuria secondary to renal disease; can prevent e.g. mannitol clearance.

B. Carbonic Anhydrase Inhibitors 4. General Properties e) Historically, one of the most popular but more recently limited usefulness, more common in glaucoma and motion sickness. f) Main site of action is proximal tubule (PCT). g) Representative drug – Acetazolamide 5. Mechanism of Action a) Potent, reversible inhibitors of carbonic anhydrase (require 99% inhibition) ‐ less destruction of bicarbonate in lumen and CO2 diffusion into cell, reduce bicarbonate by 80% and sodium as well. b) Loop of Henle and distal sites can reabsorb sodium, so only weakly diuretic. c) Because of increased urine flow to distal sites more potassium excretion. 6. Therapeutic Uses a) Glaucoma b) Correct severe metabolic alkalosis c) Can be used with Loop Diuretics 7. Toxicity and Contraindications a) Can create metabolic acidosis (by reducing serum bicarbonate levels). b) Hypokalemia – increased urine flow results in potassium excretion.

C. Loop of Henle Diuretics (High Ceiling Diuretics) 6. General Properties a) Potent, rapidly acting and short‐lived (3‐8 hours) b) Site of action ‐ thick ascending Loop of Henle. c) Action is limited by amount of sodium delivered to the Loop. d) Representative drugs – Furosemide, ethacrynic acid, bumetanide 7. Mechanism of Action a) Inhibit electrolyte reabsorption in thick ascending limb of Loop by inhibiting chloride cation cotransporter b) Temporary increase in RBF reduces reabsorption in proximal tubule: although long term use and progressive volume depletion results in enhanced proximal reabsorption (“braking phenomena”) c) May be more distal sites – carbonic anhydrase? d) Most potent class of diuretics – increase sodium excretion by 15‐20x. 8. Therapeutic Uses a) Management of edematous states – congestive heart failure, nephrotic syndrome, liver disease, kidney failure. b) Antihypertensive therapy 9. Toxicity and Contraindications b) Abnormalities of fluid composition – potassium depletion, hyponatremia, metabolic alkalosis. c) Ototoxicity – sudden hearing loss by hair cell damage. 197 VM522P / VM7522 Pharmacology WIMU Fall

D. Thiazide Diuretics and Derivatives 1. General Properties a) Less potent than Loop diuretics (increase sodium excretion by 3‐5x) but longer half‐life b) Site of action: distal convoluted tubule (DCT) c) Some compounds may have proximal tubular action as well. d) Representative drugs: Hydrochlorothiazide, metolazone. 2. Mechanism of Action a) Reduction of sodium transport in the distal convoluted tubule (DCT), probably by blockade of electroneural NaCl transport. b) Less potent because 90% of sodium already reabsorbed prior to site of drug action. c) Cause potassium excretion because of increased tubular flow. 3. Therapeutic Uses a) Edematous states b) Hypertension 4. Toxicity and Contraindications a) Hypokalemia – sometimes combined with potassium sparing diuretic to avoid this condition b) Hyponatremia

E. Potassium Sparing Diuretics– Collecting Tubules 1. General Properties a) Two main types – aldosterone antagonists and sodium channel blockers. b) Site of action: distal convoluted tubule (DCT) – cortical collecting duct c) Natriuretic activity limited because only 2‐3% of filtered load is reabsorbed in this region. d) Primarily used for anti‐kaliuretic properties (usually in combination with other diuretic). e) Representative drugs: Spironolactone (aldosterone antagonist), amiloride (sodium channel blocker) 2. Mechanism of Action a) Alsosterone antagonist – competitive antagonist at cytosolic aldosterone receptors b) Sodium channel blocker – inhibit electrogenic entry of sodium 3. Therapeutic Uses a) Edematous states. b) Hypertension 4. Toxicity and Contraindications –their uses can be dangerous when elevated potassium levels exist (hyperkalemia) a) Renal failure b) Diabetes c) In combination with other drugs that inhibit aldosterone such as angiotensin converting enzyme inhibitors (ACE inhibitors).

F. Other Diuretic Agents 1. Glomerular vasodilator agents – methylxanthines 2. Aquaretic agents – vasopressin antagonists

3. Summary – see Table 2

198 VM522P / VM7522 Pharmacology WIMU Fall TABLE 2

SITE AND MECHANISM OF ACTIONS OF DIURETICS

RESPONSE (% FRACTIO- GROUP SITE MECHANISM NAL Na+ EXAMPLE EXCRETION A. Osmotic Proximal tubule Osmotic 20-30% Mannitol, urea (PCT) and loop

B. Carbonic Proximal tubule Inhibit carbonic 2-3% Acetazolamide anhydrase anhydrase enzyme inhibitors

C. Loop Thick ascending Inhibit Na+2Cl-K+ 25-30% Furosemide, limb of Loop cotransport system ethacrynic acid, bumetanide, piretanide, muzolimine

D. Thiazide Early distal tubule Inhibit distal neutral 5-8% Hydrochloro- and proximal NaCl transport thiazide, tubule chlorthalidone, metolazone

E. Natriuretic Late distal tubule Inhibit luminal Na 2-3% Amiloride, K-sparing and collecting permeability and triamterene, tubule aldosterone action spironolactone

F. Others: a) Aquaretic Collecting tubule Antagonize ADH 1-2% Lithium, effect on water demechlortetra- permeability and cycline, ADH inhibit Na+ transport antagonist, poss. atrial natriuretic peptide (?), poss. ouabain-like natriuretic peptide

b) Glomerular Glomerulus Vasodilation 0-1% Theophylline, atrial natriuretic peptide

199 VM522P / VM7522 Pharmacology WIMU Fall

4. Overview

200 VM522P / VM7522 Pharmacology WIMU Fall

Part 6 – Calculations (M. Meyer‐Ficca) L29‐34: Calculation Issues Objectives 1. Review and reinforce quantitative skills to improve overall accuracy in both solving numerical problems and communicating numerical results. 2. Develop additional skill in solving the types of quantitative problems that frequently arise in the clinics. 3. Solve problems that use quantitative relationships between pharmacokinetic variables to increase the conceptual understanding of the relationship between these variables. 4. Use numerical approaches to understand how dosing regimens are designed and can be manipulated for therapeutic benefit.

Overview Doing calculations correctly is critical to the practice of veterinary medicine. After you have determined what is wrong with the patient and what you plan to do, the next question you will ask yourself is how much and for how long. It is critical that you not only make these determinations accurately, but that you also communicate the results of your calculations accurately. Unfortunately, when calculations are not done accurately or communicated accurately, patients can be seriously harmed or even die. This is a documented fact. Taking the time to develop good numeric skills will be time well spent that will last you for the life of your career.

Numeric Conventions I have some very strict rules when it comes to numeric conventions. While these may seem arbitrary or capricious, they have been developed not because I am anal retentive, but because there have been studies that document these conventions avoid errors that can lead to potentially catastrophic results (note the infamous polo pony die off in the Fall of 2009 was likely due to a 10‐fold error in the amount of selenium given to the horses; whether the failure was in the calculation or communicating the result of a calculation has yet to be reported). Once you get used to these conventions I think you will find that they are not unduly burdensome, and that as you advance in your careers and become a consumer of numbers generated by others, you will find that you wished they also followed the conventions.

 Two important points: o Some of these conventions will prevent an error maybe one in a hundred or one in a thousand times – are they really worth the effort? Yes they are! When you think that in a year tens of millions of animals are seen by veterinarians, and with each visit there can be multiple calculated values. Even if the error rate is 1 in a 1,000, with 10 million calculations that is still 10,000 errors. If only 1% of the errors are fatal, they still result in about 100 deaths (many more are not fatal but nevertheless compromise the health of the patient). We all must make efforts to reduce the chances for errors when we can, especially when many of these errors can be prevented by just developing good habits that can become so ingrained in your daily practice that you do not even notice them. o Everyone has to recognize that mistakes happen – we are not inerrant calculating machines; we get distracted, we do things in haste, we use sloppy handwriting, etc. The single most important advanced made in this area in the last 10 years in human medicine has been to recognize that mistakes are part of the human experience, and because of this there needs to be a systematic approach to avoiding errors. This has resulted in changes such as color coding labels, special emphasis in lettering on labels, required double checks on procedures, among others. Part of this effort has also focused on methods that reduce numerical mistakes, and the rules outlined below come from these studies. 201 VM522P / VM7522 Pharmacology WIMU Fall Writing Numbers  Significant digits – final numbers should be written with 2 significant digits (one exception is noted below). There are several reasons for this: o Most measuring devices used in the clinic do not have the ability to give results more accurate than 2 digits. o Thus most numbers you use in your calculations are not determined with any more accuracy than 2 digits. You should recall that in a calculation, the level of accuracy in a final value is no greater than the least accurate value you started with. o When you write a numerical string (e.g., 3.3837) it is not uncommon that two digits will get trans‐ posed when the number is rewritten. With only two digits, such a transposition is very unlikely. Thus the 2 digit rule is also a safety issue! o In practice writing 3 digits is not a major blunder, but more than 3 should definitely be avoided. However, to get you into the habit of only using 2 significant digits, in this course you will lose partial credit on an answer if you violate this rule. o However, when you calculate intermediate values, you should always carry at least 3 digits. This helps to reduce errors due to rounding intermediate values. o In case you have forgotten: . What is a significant digit? It is an indicator of the accuracy with which a value is known. When a value is reported, it should be recognized that the true value is not really known, but that the number represents an approximation of the true value. How close is the reported value to the true value? That is given by the number of significant digits. For example, a value of 1.6 indicates that the true value is between 1.55 and 1.649. When you write more digits than the known accuracy, they are not meaningful, and actually misleading, because they imply the true value is known more accurately than it actually is. . Are leading zeros (as in 0.042) significant? No. The only significant digits in 0.042 are the 4 and the 2; the 0’s are just place holders to get the decimal point in the correct place. Another way to think about this is that significant digits are the ones you would use if you wrote the number in scientific notations. Thus 0.042 is written as 4.2 x 10‐2 . Are trailing zeros (as in 3.0) significant? The answer is yes. The inclusion of the trailing zero indicates that the real value is somewhere between 2.95 and 3.049. Note in this case the trailing zero is not being used as a place holder to get the decimal point in the right place. . What about a number such as 30? This one is trickier. The trailing zero could be significant, or it might be just a place holder. You would know if it was written in scientific notation: 3.0 x 101 or 3 x 101 (the original 0 was significant in the former but not the latter), but when the number is written 30, you just don’t know.

 Leading and trailing zero rules – you were just informed of the 2 digit rule, but now we are going to make some exceptions. These exceptions are made because of safety, not because of any abstract reason about numerical accuracy. o When writing a number with a decimal, always include a leading zero. This draws attention to the decimal so it will not be overlooked: Thus .73 should be written as 0.73

o When writing a number with a decimal, always drop a trailing zero, even if it is significant. This ensures that an error won’t be made if the decimal is overlooked: Thus 5.0 should be written as 5

o Like the 2 significant digit rule, violation of this rule will result in loss of partial credit.

202 VM522P / VM7522 Pharmacology WIMU Fall  Commas – o When more than four digits are present use a comma. Especially if more than 5. 1000 or 1,000 can go either way (but the latter is preferred) 570000 lb should be written: 570,000 lb o Alternatively, especially with large numbers, write the unit term – it is less likely to be confused: 67,000,000 units should be written: 67 million units $14300000000000 is very confusing (national debt in 2011); $14,300,000,000,000 is better; but $14.3 trillion says it all (although the full number with all its digits is quite impressive and pretty scary! Makes your student loans look puny!).

 Spacing numbers and labels. This is less a safety rule, although at times safety issues could arise (as the l in lb might be mistaken for a 1). The conventions are: o If you write number with an abbreviation for a unit, such as: g, or lb, or m, or mM, or mcg, etc. you should leave a space between the number and the unit. (Also, abbreviations for a unit should not be followed by a period.) Thus: 3.4 g (not 3.4g) 21 lb (not 21lb) I think we’d all recognize that 21pounds is incorrect. Thus lettered abbreviations should be spaced like a word. o If you write a number with a symbol, such as $, or %, or ° (for degree) you do not leave a space between the number and the symbol: $35.00 not $ 35.00 14% not 14 % 72° F not 72 ° F and not 72°F

 Rounding o As previously mentioned, it is a good habit to carry three or more digits for intermediate values, and then at the last step reduce to two digits, and then apply the leading/trailing zero rule. This approach reduces rounding errors. o Anything 5 or greater rounds up and anything less than 5 rounds down o Do not sequentially round, as in:

4.3448 rounds to 4.345 rounds to 4.35 rounds to 4.4 This is wrong and will be marked wrong!

o To properly round note the final significant digit in your answer and round from that point. Thus in the example above (final significant digit underlined):

4.3448 would round to 4.3 because the digit just past the 3 is less than 5

203 VM522P / VM7522 Pharmacology WIMU Fall Banned Units! It has been determined by the Joint Commission (a group of health care specialists from a variety of professions) that using certain abbreviations often lead to mistakes. There has been an effort to stop health care providers from using these abbreviations to reduce these errors.

 Some examples that might get used in this course that you should not use (these will be marked wrong): cc for ml (cc can be confused for two zeros) g for microgram (the sometimes confused for m, use mcg) (This is especially an issue when blocks of text are converted from one font to another – Greek letters are often converted back to an Arabic letter. Thus g becomes mg, and spell checkers will not catch the error.) U or u for unit (U can be confused for a zero, write out ‘unit’) IU for international unit (IU can be confused for 10, write out ‘international unit’)

Some other commonly used abbreviations that are considered dangerous (bolded ones are banned by the Joint Commission): Bad abbreviation Instead use qd daily qod every other day

MS or MSO4 morphine sulfate

MgSO4 magnesium sulfate qhs nightly qn nightly X 3d for 3 days SC, SQ, or sub q subcut (or subcutaneously) ss one half SSI sliding scale (insulin)

As a general rule there has been a movement away from using Latin abbreviations. Often non‐medical personnel do not know what these mean, and yet they are required to read directions given by physicians, veterinarians, nurses, pharmacists, etc. If any of you use or pick‐up prescription medications in the last few years (and had done so more than 10 years ago) it is very noticeable how labels have changed. Now instructions for use have become much more explicit with virtually no abbreviations. This has happened as a result of a concerted push by safety conscious people (and a few lawsuits). You should try to break the habit of using abbreviations particularly in regards to use instructions, and especially if communicating with non‐professionals. Spell out in English what you want.

204 VM522P / VM7522 Pharmacology WIMU Fall Calculation Issues for Unit 1

o Conversions, abbreviations, and prefixes o A conversion table is given on the next page. Use this for the problem sets and homework assignments. Conversions needed on quizzes and tests will be provided except as noted below.

o You should know the bolded items below by heart – they will not be given on quiz/test papers. More rarely used conversions, especially metric to English units, will be given.

o I expect that you are familiar with the major metric units for volume, distance, and weight (respectively): liter: L meter: m gram: g

o I also expect that you are familiar with the associated prefixes used in international units. These include: giga: G (109) mega: M (106) kilo: k (103)

deci: d (10‐1) centi: c (10‐2) milli: m (10‐3)

micro: mc (10‐6) nano: n (10‐9)

(Particularly noteworthy is deci d, of which many of you may be unfamiliar – it is often used as a unit when plasma levels of drugs or biologically important molecules are given; as in mcg/dL. 1 dL is 100 ml or 0.1 L)

o You should also know that: ppm is mg/kg (for solids) or mg/L (for solutions) ppb is mcg/kg (for solids) or mcg/L (for solutions)

o You should know the following standard U.S. conversions (hopefully you remember this from 3rd grade): 8 liquid oz = 1 cup 2 cup = 1 pint 2 pint = 1 qt 4 qt = 1 gal 16 dry oz = 1 lb 2000 lb = 1 ton 12 in = 1 ft 3 ft = 1 yd

o Note that a liquid oz ≠ dry oz (unlike the metric system where 1 ml ~ 1 g). Thus when converting from metric to avoirdupois (the official name of the American system of weights and measures) you need to select the correct conversion (solid or liquid).

o Note that when the term ’ton’ is used alone, it means a U.S. ton (2000 lb). When a ‘metric ton’ is the unit of measure, the modifier ‘metric’ must be present. Also know: 1 metric ton = 1000 kg

o Just to be explicit ‐ you should know time conversions and have the ability to convert from a 12 h clock to a 24 h clock (and vice versa): 1 min = 60 sec 60 min = 1 h 24 h = 1 day 7 day = 1 week

o One U.S. to metric conversion that you should know by heart: 2.2 lb = 1 kg

205 VM522P / VM7522 Pharmacology WIMU Fall Ratios: o Express ratios in their simplest form: 1:1,000 or 1:33 or 2:3 but not 25:100 (this would be 1:4) or 14:28 (this would be 1:2)

o When the numerator is small compared to the denominator they can be used as a fraction: 1:2,000 is virtually the same as 1/2,000 [but technically it is 1 / (2000 + 1)]

o Be careful when the ratio has numbers of similar magnitude, then use the technical conversion. For example: When items are present in a ratio of 1:2 the first component is 1/3 of the total, not 1/2. [1 / (1 + 2) ]

o Some important ratios to keep in mind: 1:1,000 solution is 1 mg in 1 ml 1:100 is ~1% (actually 0.99%) 1 ppm is 1:1,000,000 1 ppm is also 1 mg/kg (wt/wt) or 1 mg/L (wt/vol)

o When expressing things as ratios, make sure they are the same or equivalent units.

For example, both terms are g, or both are mg.

Where this gets tricky is when things are expressed as ratios, but they involve a weight unit and a volume unit. This happens when solutions are made and then the concentration is expressed as a ratio. In this case remember that 1 ml of water ~1 g. If 1 mg is mixed into 3 ml, the concentration, expressed as a ratio, is 1:3,000

What is deceptive is that although both units are modified by milli, they are by weight 1,000 fold different. This is a frequent mistake and often causes people to have to retake the calculation exam! You may believe this is minor issue, but misreporting a value in this manner can cause a catastrophic error because of the size of the error (1,000‐fold).

Molarity (or molar concentration): o Molarity is expressed as moles / L. o To convert a mass of a substance to moles you need to know its molecular weight (MW) (e.g., the MW of NaCl is 58.4 which means there are 58.4 g of NaCl / mole) o Question: How many g of NaCl are needed to make 2 L of a 3 mM solution? 3 x 10‐3 mole / L * 2 L * 58.4 g / mole = 0.35 g

206 VM522P / VM7522 Pharmacology WIMU Fall WEIGHTS, MEASUES, EQUIVALENTS AND CONVERSION FACTORS METRIC PREFIXES:

name ABBREVIATION VALUE EXAMPLE METRIC RELATIONSHIPS IN PARTS giga G 109 Gbyte parts per million (ppm) = mg/kg or mg/L mega M 106 Mbyte parts per billion (ppb) = g/kg or g/L kilo k 103 kg parts per trillion (ppt) = ng/kg or ng/L hecto h 102 deca da 101 100 liter (L), meter (m), or gram (g) deci d 10‐1 dL centi c 10‐2 cm milli m 10‐3 ml micro mc () 10‐6 mcg (g) nano n 10‐9 nm (equivalent to 10 Angstroms) pico p 10‐12 pg femto f 10‐15 fL

WEIGHTS:

Metric KILOGRAM (kg) GRAM (g) MILLIGRAM (mg) MICROGRAMS (g) NANOGRAMS (ng)

1 = 1000 = 106 = 109 = 1012 1 = 1000 = 106 = 109 1 = 1000 = 106 1 = 1000

Avoirdupois POUNDS (lb) OUNCES (oz) DRAMS (dr) GRAINS (gr)

1 = 16 = 256 = 7000 1 = 16 = 437.5 1 = 27.34375

Apothecary/Troy POUNDS (lb) OUNCES (oz) DRAMS (dr) SCRUPLES (s) GRAINS (gr)

1 = 12 = 96 = 288 = 5760 1 = 8 = 24 = 480 1 = 3 = 60 1 = 20

An avoirdupois ton is 2000 lb whereas a metric ton is 1000 kg or 2200 lb. If not specified (i.e., if only the word ‘ton’ appears) assume it is an avoirdupois ton. Only assume a metric ton if it is specifically stated ‘metric ton’.

metric to AVOIRDUPOIS TO METRIC METRIC TO APOTHECARY APOTHECARY TO METRIC avoirdupois kg = 2.2 lb lb = 0.4545 kg kg = 2.679 lb lb = 0.373 g = 0.03527 oz oz = 28.35 g g = 0.03215 oz oz = 31.104 g g = 15.434 gr gr = 64.79 mg g = 15.434 gr gr = 64.79 mg

NOTE: A grain is the same in Avoirdupois and Apothecary. All others scale accordingly. For example: the conversion between an Apothecary “lb” to an Avoirdupois “lb” is 5760/7000 or 0.823.

207 VM522P / VM7522 Pharmacology WIMU Fall WEIGHTS, MEASUES, EQUIVALENTS AND CONVERSION FACTORS

VOLUME:

Metric LITER (L) MILLILITER (ml) MICROLITER (L)

1 = 1000 = 106 1 = 1000

Apothecary (U.S.) GALLON (gal) QUARTS (qt) PINTS (pt) CUPS (c) OUNCES (oz) DRAMS (dr)

1 = 4 = 8 = 16 = 128 = 1024 1 = 2 = 4 = 32 = 256 1 2 = 16 = 128 1 = 8 = 64

metric to APOTHECARY TO METRIC TEASPONS (tsp) AND TABLESPOONS (tbs) apothecary L = 0.264 gal gal = 3.786 L U.S.: tsp ~ 1 dr tbs ~ 3 tsp L = 1.0565 qt qt = 0.9465 L tsp ~ 5 ml tbs ~ 15 ml ~ 0.5 oz ml = 0.0338 oz oz = 29.577 ml

VOLUME TO WEIGHT (FOR WATER AT 4C) 1 cm3 = 1 cc = 1 ml = 1 g

NOTE: a wet oz (29.6 ml)  a dry oz (28.35 g)

DISTANCE:

Metric KILOMETER (km) METER (m) CENTIMETER (cm) MILLIMETER (mm) MICROMETER/MICRON (m)

1 = 1000 = 105 = 106 = 109 1 = 100 = 1000 = 106 1 = 10 = 10,000 1 = 1000

British and American MILE (mi) YARD (yd) FOOT (ft) INCH (in) AMERICAN TO METRIC metric to 1 = 1760 = 5280 = 63,360 1 = 3 = 36 american 1 = 12 km = 0.621 mi mi = 1.609 km

m = 1.094 yd yd = 0.9144 m m = 39.4 in in = 0.0254 m cm = 0.394 in in = 2.54 cm MOLECULAR WEIGHT (MW) & MILLIEQUIVALENTS (meq) OF COMMON BIOLOGICAL IONS

ION MW VALENCE mg/meq ION MW VALENCE mg/meq H+ 1 1 1 K+ 39 1 39 Na+ 23 1 23 Mg2+ 24 2 12 Cl‐ 35.5 1 35.5 Ca2+ 40 2 20 208 VM522P / VM7522 Pharmacology WIMU Fall

Unit 1 – Problem Set –conversions, ratios, molarity, ppm/ppb

1. How many g of CaCl2 (MW 111 g/mol) are needed to make 6 L of a solution that is 7 mM?

2. An antibiotic is added to feed. How many mg of the antibiotic is needed to make 2 metric ton of feed at 48 ppb?

3. A protocol calls for 6 ml of a solution. How many teaspoons (tsp) is this?

4. You mix 65 mg of an antibiotic into 25 kg of feed. What is the final concentration of antibiotic in the feed (in ppm or ppb as is appropriate, i.e., avoid values <1 or >1000)?

5. A mixture calls for 135 lb of material. How many kg is this?

6. You have 6 ml of a solution of antibiotic that is 5 ppm. How much antibiotic (in mcg) is in the solution?

209 VM522P / VM7522 Pharmacology WIMU Fall 7. You hear back from the lab that the concentration of a toxin in the plasma is 3.9 mcg/ml. Express this value in ppm.

8. You add 5 g of glucose (MW 180 g/mole) to 1 L of water. What is the molarity of the solution in mM?

9. You mix 9 g with 2 kg. Express this as a ratio in simplest form.

10. You mix 6 mg into 10 ml. Express this as a ratio in simplest form.

210 VM522P / VM7522 Pharmacology WIMU Fall Calculation Issues for Unit 2

 Percentages: o A percentage is the same as saying parts per 100. It is simply a fractional value expressed with 100 as the denominator. Thus any fraction can be converted to a percentage by setting up a proportionality. For example, what percent is 4/7? 4 X 4 / 7 = / 100 Thus X = ( / 7) * 100 = 57%

o Calculating percentages also depends on the nature of the compounds combined: a) For two solids use wt/wt (denominator is total wt) A paste has 4% drug. This means it has 4 g of drug for every 100 g of total product. A 100 g of product would contain 4 g drug plus 96 g of inert ingredient.

Question: You mix 3 g of drug with 15 g of binding agent. What is the percent active ingredient in the final mix?

3 g [ / (3 g + 15 g) ] * 100 = 16.66% write as two significant digits: 17%

b) For a solid dissolved in a liquid use wt/vol (denominator is final total vol) A 10% glucose solution has 10 g of glucose dissolved in water brought to 100 ml final vol (not 10 g of glucose plus 90 ml of water; that would be wt/wt).

Question: If you want 1.5 g of glucose, how much 10% solution do you take? (Note: express a 10% solution as 10 g / 100 ml)

100 ml [ / 10 g ] * 1.5 g = 15 ml

c) For two liquids use vol/vol (denominator is final total volume) A 70% ethanol solution (vol/vol) contains 70 ml of ethanol diluted to 100 ml (not 70 ml of ethanol with 30 ml of water added)

Question: If ethanol has a density of 0.8 g/ml, what is the % of a vol/vol solution in wt/vol? 70 ml * 0.8 g/ml = 56 g ethanol thus: [ 56 g / 100 ml ] * 100 = 56%

211 VM522P / VM7522 Pharmacology WIMU Fall  Some additional important issues with percentages: o Note that 1 ml = 1 g. Thus, when given a solution that is a percentage, it can quickly be converted to metric units, but be aware that the units are g per 100 ml (remember parts per 100). You can use this to answer the following question: What is the concentration of a 0.05% solution in mg/L?

0.05 g First express 0.05% as / 100 ml now convert this to mg/L:

0.05 g 1000 mg 1000 ml / 100 ml * /g * / L = 500 mg / L

o It also follows from 1 ml = 1 g that 1 mg in 1 ml is not 1%. It is 0.1%

1 mg 1 ml 1 g [ / 1 ml * / 1 g * / 1000 mg ] * 100 = 0.1%

o Pay attention to units!! Many people make this mistake on the exam and it makes them to have to repeat the test!

You add 3 g to a final volume of 10 ml. What is the % solution?

3 g [ / 10 ml] * 100 = 30% ( this works because ml ~ g and units cancel)

Versus: You add 3 mg to a final volume of 10 ml. What is the % solution? (you need to get the mg into an equivalent unit , i.e. , g)

3 mg 1 g [ / 10 ml * / 1000 mg ] * 100 = 0.03%

212 VM522P / VM7522 Pharmacology WIMU Fall Unit 2 – Problem Set – percentages

1. You add 14 g of boric acid to water brought up to a final volume of 2 L. What is the percent concentration (wt/vol)?

2. Express the ratio 2:9 as a percentage.

3. You mix 16 mcg of drug into 38 mg of inert paste. Express the final concentration of the preparation as a percent (wt/wt).

4. You have a mix that is 8 parts of active ingredient to 390 parts inactive ingredient. Express this as a percentage.

5. You have a 1.2 ml ampule that contains 68 mg of methscopalamine. What is the percent concentration of the drug solution?

6. How many g of mannitol are in 145 ml of a 3.6 percent solution?

213 VM522P / VM7522 Pharmacology WIMU Fall 7. A sample of feed comes back from the lab with the report that it contains 900 ppm of a toxin. What is the percentage concentration of the toxin in the feed?

8. Each kg of a medicated soap contains 16 g active ingredient. Express this as a percentage (wt/wt).

9. A stock drug solution comes in a 1‐ml ampule at a concentration of 1:500. How much drug (in mg) is in the ampule?

10. A premedicated preparation has 3.2% active ingredient. How many mg of active ingredient would be in 15 g of the preparation?

214 VM522P / VM7522 Pharmacology WIMU Fall Calculation Issues for Unit 3

 Setting up and solving problems: o A frequent concern of students, especially when they first start with these calculations, is they do not recognize how to even start to approach a problem. I find a useful way to approach these calculations is to calculate intermediate values (in other words, take single steps). For example, if the problem involves some seemingly complex calculation involving a dose or exposure for a particular animal, first calculate the amount needed for the dose or the amount the animal has been exposed. Then relate this amount to the preparation that the drug or toxin is in. By taking these single steps you can often walk your way through what appears to be a complex problem.

o If you solve a problem in parts, remember to carry three or more digits when calculating intermediate values. This will avoid accumulating rounding errors. These can become quite significant if you do two or more intermediate values and the rounding all lines up in the same direction.

o Another useful strategy is to carry these excess digits all the way to the final answer. Then at the end, make the appropriate changes (two significant digits with the leading/trailing zero rules).

o And finally, double check that you have the answer in the units requested! If a colleague asks for how many g, and you give them a number that happens to be in mg but didn’t include the unit, a 1000‐fold error might occur.

 Dilutions: o Remember conservation of mass from basic chemistry: V1 * C1 = V2 * C2

V1 is the volume of solution 1 and C1 is the concentration of solution 1.

V2 is the volume of solution 2 and C2 is the concentration of solution 2.

. This is called the conservation of mass because the mass of the solute is the same on both sides of the equation. All that has happened is the volume this mass of substance has been dissolved into has changed (thus the change in concentration).

. It does not matter which solution is called solution 1 and solution 2 – either one can be the stock solution or the working solution.

. You can solve this problem in any direction. For example, how much working solution can be made from a particular amount of stock, or how much stock solution is needed to make a particular volume of working solution – just algebraically solve for the unknown.

. Finally, any concentration unit can be used (e.g., mg/ml; g/L; molarity; percentage; ratios). Just make sure the concentration units are the same for both solutions.

o A second related dilution topic that we will cover involves calculating a desired concentration for a working solution, and then making the dilution from a known amount of stock. Don’t let this problem throw you; it is simply a two part calculation of two things you know how to do.

215 VM522P / VM7522 Pharmacology WIMU Fall  Conversions (some additional hints and advise): o Convert initial values to metric units as soon as possible to minimize potential conversion errors. In other words, if possible, avoid generating long strings that need to be calculated at the end of a calculation. It is very easy to accidentally overlook a variable or put in a conversion that is inverted when it shouldn’t be. This especially applies to the weight of the animal (2.2 lb = 1 kg).

o Note that a dry oz ≠ a wet oz. When converting metric to avoirdupois make sure you use the correct conversion.

If a weight, then 1 oz = 28.4 g.

If a volume, then 1 oz = 29.6 ml

o Become familiar with common conversions that involve 1000‐fold units. Becoming very familiar with the metric prefixes makes this task much easier. For example:

. mg/L is the same as a mcg/ml a mcg is 1/1000 of a mg, and a ml is 1/1000 of a L, so the 1000’s just cancel

12 mcg 1000 ml 1 mg 12 mg /ml * / L * / 1000 mcg = /L

. Another common step is to convert between units without writing out all the cancelations. Just remember to move the decimal place three spaces. For example: Write: 32 mg as 0.032 g write: 570 ml as 0.57 L write: 0.05 kg as 50 g

216 VM522P / VM7522 Pharmacology WIMU Fall Unit 3 – Problem Set – conversions, dilutions, simple doses, exposures

1. A sample from a cow carcass is found to have 82 ppm of a toxin. The carcass weighed 1,450 lb. Assuming equal distribution of the toxin throughout the carcass, how many g of toxin is in the total carcass?

2. You have a stock solution of NaCl that is 1:70. How many ml of 1:800 NaCl can be made from 36 ml of the stock solution?

3. A medication given in drinking water meant for pigs contains 400 ppm antibiotic. However, this antibiotic is toxic to goats when a goat is exposed to more than 10 mg/kg. How many oz of medicated drinking water would a 110 lb goat have to consume to obtain a toxic exposure?

4. You have 9 qt of a 15.5% solution. You need to make 8 L of a solution that is 300 ppm. How much of the stock, in tsp, do you need?

5. You add 3 cup of a solution that is 2% amtriple to 32 gal (final volume). Express the final concentration of amtriple in mg/L.

6. You have 12 oz of a powder that is 167 ppm phosphate. How many mg of phosphate are present?

217 VM522P / VM7522 Pharmacology WIMU Fall 7. You have an ampule that contains 3 ml of a 1:3,000 of dibucaine. You want to treat a 10 lb cat with 0.01 mg/kg, however, you want your injection volume to be 0.5 ml. To what total volume (in ml) would you dilute the contents of the ampule so that 0.5 ml contains the appropriate dose?

8. How many ml of a 70% alcohol solution is needed to make 2 L of a solution that is 30%?

9. You plan to treat a 66 lb dog with a drug at a dose of 9 mg/kg. The drug comes in a vial that is 30 mg/ml. How many ml do you administer?

10. You need to make a 9.5% solution of glucose. You have a container that will hold 2.5 gal. How many g of glucose do you add to the container before bringing it to final volume?

218 VM522P / VM7522 Pharmacology WIMU Fall Calculation Issues for Unit 4

 Volume of Distribution o Use the equation that relates Vd, Cp, and amount given (dose) and algebraically solve for the unknown: Cp = dose / Vd o For some of the problems you may need to first calculate one of the terms before you use the equation.

 Clearance o Use the relationship we derived in class for renal clearance: Amount in urine (mg/min) Clrenal = / Cp (mcg/ml)

o Remember to correct final value to the weight of the animal.

 Henderson‐Hasselbach Equation and Percent Ionized. o Use the appropriate HH equation (barbiturates are weak acids). o Once you know the ratio of ionized to un‐ionized express as a percentage. Remember in calculating the percent ionized the numerator is the amount ionized and the denominator is the total amount of drug present (ionized + unionized). This fraction is then multiplied by 100 to get the percentages. o This problem was generated to help understand the recommendations in Plumb’s compendium that alkalinizing the urine can be useful in a phenobarbital overdose but is not much use in a pentobarbital overdose. Why should this be recommended for phenobarbital and not pentobarbital? One item you should see from the calculation is the magnitude of the effect on changing the ionization state of the two drugs. In the calculation section we will discuss this and other aspects of the pharmacokinetics of these compounds to understand these recommendations.

 Making an Infusate o The infusion rate (mg/kg/min) / flow rate (ml/kg/min) will equal the needed concentration for the infusate (mg/ml). o Note that when three units are listed as follows: ml/kg/min it is the same as: ml / (kg∙min)

219 VM522P / VM7522 Pharmacology WIMU Fall  Diluting into a Fixed Volume o Note that in the dilution described that you do not know the final volume of the working solution (C2) because it will be the sum of the fixed volume given (V2) plus how much stock solution you add (V1). Thus we must modify the dilution equation to account for this (note the stock concentration is C1 and the final concentration is C2): V1 * C1 = (V1 + V2) * C2 V1 * C1 = (V1 + V2) * C2 Multiply out right side: V1 * C1 = V1 * C2 + V2 * C2 Isolate terms with V1 on left side:

V1 * C1 - V1 * C2 = V2 * C2 Factor out V1 on the left side: V1 (C1 – C2) = (V2 * C2) Isolate V1( divide both sides by (C1 – C2)) V1 = (V2 * C2) / (C1 – C2)

o Using algebra to solve for V1 reveals:

(V2 * C2) V1 = / (C1 – C2)

o Do not worry about memorizing equations or derivations – on any quiz/test you will be given a section that has a list of equations.

220 VM522P / VM7522 Pharmacology WIMU Fall Unit 4 – Problem Set – Vd, more dilutions, Clr, HH, preparation of infusates

1. A drug has a volume of distribution in sheep of 1.1 L/kg. You give 95 mg to a 110 lb sheep. What plasma concentration (Cp) in mcg/ml do you expect this to produce?

2. You give 60 mg of a drug IV to a 65 lb dog. Shortly thereafter you obtain a plasma sample and the concentration of the drug is 5 mcg/ml. What is the Vd for this drug (in L/kg)?

3. You want to give erythromycin to a 270 kg cow. The volume of distribution for erythromycin in the cow is 0.8 L/kg. The lab report indicates you will need a plasma concentration of 5 mcg/ml to achieve a minimal effective concentration (MEC). You target twice the MEC. Erythromycin comes in an injectable solution at 200 mg/ml. How much erythromycin (in ml) do you give?

4. Rather than waiting for drug levels to build up during an infusion you may instead give a bolus of drug that rapidly achieves the desired plasma concentration. How much drug do you need to give (in mg) to a 42 lb dog if the drug has a Vd of 0.3 L/kg and you are targeting a plasma concentration (Cp) of 10 mcg/ml?

5. You are working for a biotech start‐up company developing a new antibiotic drug for bovine respiratory disease. As part of a new drug application to the FDA you want to know the rate of renal clearance of the drug in cows. In a 1200 lb cow you measure a plasma concentration of the drug of 14 mcg/ml. After 2.5 hours (h) you collect 170 ml of urine and find the concentration of drug in the urine to be 0.7 mg/ml. What is the renal clearance (in ml/min/kg)?

6. You want to estimate the shift in percent of phenobarbital (organic acid, pKa = 7.4) and pentobarbital (organic acid, pKa = 8.0) to an ionized state if you alkalinize the urine by 3.0 pH units. a. At normal urine pH (5.0), what percentage of phenobarbital and pentobarbital is ionized? For each compound indicate whether it is likely to be reabsorbed or trapped in the urine.

221 VM522P / VM7522 Pharmacology WIMU Fall b. At the new urine pH, what percent of phenobarbital and pentobarbital is ionized? For each compound indicate whether under these new conditions whether it is likely to be reabsorbed or trapped in the urine.

7. You want to make a drug solution for infusion into a 45 lb dog. You want the drug infusion to be 300 mcg/kg/min and the flow rate to be 0.05 ml/kg/min. a. What concentration should your drug be in the infusion solution (in mg/ml)?

b. You use a minidrip system (60 drops/ml) to administer the infusion. To what drip rate (drops/10 sec) do you set the minidrip system? (round to the nearest whole number)

c. You need to make the infusion solution into a fixed volume of 400 ml using a drug stock that is 20 mg/ml. How many ml of stock do you add?

222 VM522P / VM7522 Pharmacology WIMU Fall Calculation Issues for Unit 5  Relationship of t1/2 to volume of distribution (Vd) and clearance (Cls): (Vd * 0.693) t1/2 = / Cls (See class notes for more information.)

 Bioavailability (F) o F is the fraction of given drug that is absorbed into the systemic circulation when the drug is given orally. Thus if a drug has 60% oral bioavailability, F = 0.6; and (dose * F) should be substituted for (dose) anytime you need to know the amount of drug that makes it into the general circulation. Some notes: . By definition, F is equal to 1 for an IV dose.

. If a bioavailability term is not given, you can assume that it is 100% and F = 1.

o When moving from one dosing regimen to another when bioavailability changes (e.g., going from IV to oral, or switching oral preparations that have different bioavailabilities) the goal is to give the amount of drug such that the amount absorbed is identical. You are essentially solving the following equation: F1 * dose1 = F2 * dose2

In any question you will have the values for F (again, remember for an IV dose, it is equal to 1) and one of the dose values, so all you have to do is solve for the unknown dose.

 Future plasma levels given an initial concentration and a t1/2 o There is a formulaic approach to doing this as follows: (number of half lives) Cpt = (Cpo) / 2

Where: Cpt is the concentration at the time of interest

Cpo is the initial concentration And the number of half‐lives is the time of interest expressed in half‐lives Although precise, this is unnecessarily complicated; you are not likely to remember this formula in the future and it involves a log calculation, so you are unlikely to want to do the calculation. Thus it doesn’t have much value.

o A more intuitive approach is to just serially divide the Cp by 2 (that essentially is all the equation above is doing): . If you need to know the plasma level at a certain time in the future, just serially divide the initial plasma concentration by 2 adding one t1/2 each time you divide. When the sum of the half‐ lives equals your time of interest, you have an estimate of plasma levels at that point in time.

. If you need to know the time until a certain level is achieved, again, serially divide the initial concentration by two until you the level of interest is found, then count the number of times you divided and multiply by the t1/2.

223 VM522P / VM7522 Pharmacology WIMU Fall  Relating to a clock. o Using a 24 hour clock (e.g., 0800 or 1800 or 2300) is less ambiguous because no one can confuse the time of day, and it makes calculating easy (simply add the number of hours to the present time and then serially subtract 24 until the number is less than 24 to get the number of days, and the remainder will be the time on the day of interest; with a 12 hour clock you have to keep track of whether you are AM or PM). However, it can be confusing to some clients who are not comfortable with such terminology (you would be surprised). Because the 24 hour clock is less error prone, you should use it for your calculations, but you may want to convert the final answer to a 12 hour clock with AM and PM.

 Complex dilutions – increasing the concentration of a drug in a volume that already contains a drug: For example, you have 100 ml of a solution that already has 2 mg/ml. You want to increase the concentration in the solution to 5 mg/ml, and you have a stock solution that is 10 mg/ml. How many ml do you add to the 100 ml? You can’t do this by methods we have previously developed because you need to account for the drug currently in the solution. However, as before, if one keeps in mind the conservation of mass, a formula can be determined: (V1 * C1) + (V2 * C2) = (V1 + V2) * C3 Where: C1 is the concentration in the stock solution (in the example: 10 mg/ml) V1 is the amount of stock you add (the unknown you are trying to solve for) C2 is the concentration already in the working solution (2 mg/ml) V2 is the current volume of the working solution (100 ml) C3 is the final desired concentration (5 mg/ml) Note: V1 * C1 gives the mass of drug you are adding V2 * C2 gives the mass of drug you have (V1 + V2) gives the final volume (V1 + V2) * C3 gives the mass of drug present in the final preparation Solving for V1 gives: (V1 * C1) + (V2 * C2) = (V1 + V2) * C3 multiply out right side V2 * (C3 ‐ C2) (V1 * C1) + (V2 * C2) = (V1 * C3) + (V2 * C3) isolate V1 on left, V2 on right V1 = / (C1 – C3) (V1 * C1) – (V1 * C3) = (V2 * C3) – (V2 * C2) factor out V1 and V2

V1 * (C1 – C3) = V2 * (C3 – C2) isolate V1 on the left V1 = V2 * (C3 – C2) / (C1 – C3) For the example: (5 mg/ml – 2 mg/ml) V1 = (100 ml * / (10 mg/ml – 5 mg/ml) = 60 ml

 Complex dose calculations ‐ reminders: o Change any doses given into metric units (mg/kg) o Calculate out the dose you want to administer, and then put it into a volume you want to administer. Now you have a concentration. o Make the appropriate working solution from the stock solution.

224 VM522P / VM7522 Pharmacology WIMU Fall Unit 5 – Problem Set – t1/2, bioavailability, Clr, complex dilutions (to be discussed Fri, Sep 21)

1. A drug has a volume of distribution (Vd) of 3 L/kg and a total body clearance of 7.4 ml/min/kg. What is the t½ (half‐life) in hours (h) for this drug?

2. A drug has a t½ (half‐life) of 2 h. If at 6 AM the plasma concentration is 1 mcg/ml, and no further drug is given, what will the plasma concentration be 8 h later?

3. The half‐life of a toxicant is 11 h. Symptoms of toxic exposure to this compound appear when plasma levels exceed 0.1 mcg/dl. If circulating levels are found to be 12.8 mcg/dl, how long will it take (in h) before the plasma concentration falls below the level that produces symptoms?

4. A drug is given orally to dogs at 1 mg/kg. The drug has an oral bioavailability of 75 percent. However, Leo has a broken jaw and you want to give the drug IV. What is the amount of drug (in mg) that should be given by injection if Leo weighs 4 kg?

5. You have a patient with congestive heart failure that is on a preparation of digoxin that has an oral bioavailability of 50 percent. Your client wants to switch to a different preparation of digoxin that is less expensive, but the new preparation has an oral bioavailability of 30 percent. If the patient is currently taking 0.8 mg of digoxin once a day, how much of the new preparation should the patient take at each dose?

6. A drug has a t½ of 10 h. If the total body clearance for the drug is reduced to 65% of normal, what will be the new expected value for the t½?

225 VM522P / VM7522 Pharmacology WIMU Fall 7. A drug has an oral bioavailability of 45 percent. If 9 mg of drug given IV to a 45 lb dog produces a plasma concentration of 6.3 mcg/ml, how much of the drug (in mg) needs to be given orally to produce the same plasma concentration?

8. A plasma sample is taken at 5 AM and at 8 AM you received the lab report that the concentration of drug (t½ is 6 h) is 9.6 mcg/dl. You do not want the drug concentration to fall below 0.3 mcg/dl. What is the longest you can wait before giving the next dose of drug? (Give answer in terms of a 12 hour clock and whether the time is the same day or next day. Note: noon is 12 PM, midnight is 12 AM.)

9. You have 90 mls of a solution that contains 0.6 mg/ml of a drug. You want to increase the concentration of drug in the solution to 2.4 mg/ml. How much stock solution (7.2 mg/ml) do you add (in ml)?

10. An antiparasitic compound comes in a preparation that is 0.5% active ingredient. The label says to administer the drug subcut to dogs at 1 ml per 100 lbs body weight. You want to test the drug in rats, and while you want to give the dose recommended for dogs, you also want to modify the concentration so that you inject 0.1 ml of preparation for every 100 g of weight. What final volume (in ml) do you make if you start with 0.5 ml of the preparation made for dogs?

226 VM522P / VM7522 Pharmacology WIMU Fall Calculation Issues for Unit 6  Calculating infusion rate to produce a desired Cp or an expected Cp from a specific infusion rate.

Given one of the infusion equations, you should be able to algebraically solve for any of the other parameters.

I = CLs * Cplat can be rearranged to: Cplat = I / CLs

I = (Cplat * 0.693 * Vd) / t1/2 can be rearranged to: Cplat = (I * t1/2) / (0.693 * Vd)

 More practice with complicated dilutions.

 Be able to determine a specific time when Cp is at an estimated level. o The critical element in these calculations is to apply the gap rule. The gap rule: the gap from where Cp is currently at to where Cp is going closes by ½ in a half‐life. This happens whether Cp is increasing or decreasing, or starting from 0 or going to 0. o What follows from the gap rule is you need to note where Cp is currently at, and where Cp is going. o Finally, remember to apply the gap rule sequentially until you get the desired Cp. Then count the number of half‐lives it took to achieve the target Cp. o A shortcut worth remembering: once you have the relationship between a particular infusion rate (I) and the plasma concentration (Cp) this will produce, this calculation is quite simple (because they are directly proportional to one another). Thus, if you double the infusion rate, then Cp will double. If you increase the infusion rate by a factor of 4, the Cp will increase by a factor of 4. If you half the infusion rate, Cp will half. You don’t have to recalculate the entire equation.

 Bioavailability o Remember to examine bioavailability when changing between oral dosing and IV dosing. Use the equation previously given: (F1 * dose1) = (F2 * dose2) o For an IV dose F is = to 1.0

227 VM522P / VM7522 Pharmacology WIMU Fall  Establishing a range of Cp for intermittent dosing o Remember to examine toxic levels and minimal effective therapeutic levels. o You need to stay below toxic levels and above minimal therapeutic levels. o These values define the maximum range. You can choose other values as long as they keep Cp between the toxic levels and within the therapeutic level. o It is often convenient to choose values that are related by a factor of 2 (2‐fold, 4‐fold, 8‐fold, etc) because these are easy to think about in terms of half‐lives. o Remember that because absorption is not instantaneous, in these theoretical calculations it is okay to target the specific toxic level given because it is not likely that they would be achieved exactly (there is likely to be rounding off because of some delay in the absorption of the drug). o One thing to remember about values from a text – these are best estimates – not every animal will show toxicity at the reported toxic level, and some animals might show signs of toxicity before the levels are achieved. (Same thing goes for therapeutic levels). o Also remember that in real life the pharmacokinetic parameters for a given drug are not absolutely identical in every animal of a given species. o Until you measure plasma levels, you do not know if the reason an animal is resistant (or extra‐ sensitive) to a drug is because of a pharmacokinetic reason (CLs, Vd, or bioavailability) or pharmacodynamic reason (target site).

 Dosing with tablets: o Although you can calculate doses exactly, tablet sizes will restrict your ability to get exact doses. A few things to keep in mind: . Client/patient compliance is your number one problem. Try to keep regimens as simple as possible if you want good compliance. The fewer sizes and number of pills the better. They are less likely to get confusing. . For example, a protocol with 2 small and one large table might get turned around by the client into one small and two large. One tablet of each size cannot get confused. . Changing doses from day to day can also get confusing and overlooked. As a rule it is best to keep a consistent amount. Remember your client may not be as motivated as you or one of your staff to keep the patient on a schedule. . Half‐tablets can be used but in general they also can cause problems (broken crumbly tablets, imperfect divisions, etc.). . Thus anything you can do to make it simple is to be desired.

228 VM522P / VM7522 Pharmacology WIMU Fall Unit 6 – Problem Set –Infusions and Intermittent Dosing

Betsy, a 5 year old 44 lb mixed‐breed dog, enters your clinic with a cardiac arrhythmia but in otherwise normal health. You decide to treat with procainamide and find the following pharmacokinetic parameters for procainamide in dog: ∙ t1/2 = 2 h ∙ Vd = 2 L/kg ∙ 50% excreted unchanged in the urine ∙ oral bioavailability on an empty stomach 85% ∙ therapeutic plasma levels 3 ‐ 8 mcg/ml ∙ toxic levels >21 mcg/ml

‐ Procainamide comes in 10 ml vials (50 mg/ml) and tablets of 250, 375, and 500 mg. ‐ You target a plasma level of 4 mcg/ml. ‐ You plan to administer the drug in a 100 ml IV bag with a flow rate of 0.2 ml/min.

======

1. What should the infusion rate of the drug be (in mg/h)?

2. How many ml of the injectable preparation should be added to the IV bag to get this desired infusion rate (in ml; remember to correct for volume added to bag)?

3. How much should be given as an IV loading dose to reach the desired Cp (in ml of injectable preparation?)

4. After 4 hours it is decided that more drug is needed. The plan is to increase plasma levels to a target of 8 mcg/ml (double the original level) without increasing the flow rate (that is, the concentration of drug in the infusate needs to be doubled). How many ml of the injectable preparation of drug would you add to the remaining solution in the IV bag (in ml; remember to correct for the amount of solution already used, to correct for the drug already in the infusion solution, and to correct for the volume of stock to be added to the infusion solution)?

5. How long would it take (in h) before the Cp reached 7.5 mcg/ml?

229 VM522P / VM7522 Pharmacology WIMU Fall 6. If you were impatient and also doubled the flow rate (in addition to doubling the drug concentration in the infusion solution), what would the Cp be 2 h after this manipulation (in mcg/ml)?

7. It is now time to put Betsy on oral doses so she can go home (assume she has reached a Cp steady‐ state of 8 mcg/ml). Based on your observations during the initial therapy you do not want plasma levels to fall below 5 mcg/ml and yet you want to avoid any time at potentially toxic concentrations (20 mcg/ml). At the same time you want to maximize the dosing interval. What would you use for a dosing interval (in h)?

8. How much would you give at each dose (in mg)? Note: because you are switching from an established IV dose to oral dosing, you also need to correct for limited oral bioavailability.

9. Choosing from the list below, how many of what size tablets (small 250 mg; medium 375 mg; large 500 mg) would you dispense for each dose? Use the following rules:  You can use only up to 2 different size tablets.  You can break a tablet in half, but the half tablet counts as one of your two sizes of tablets.  You can exceed the calculated dose, but by no more than 5%, otherwise you should under dose.  If you have a choice (slight under dose or slight over dose), you should use the combination that gets you closest to the actual calculated dose. A) 1 small tablet G) 1 large and 1 medium tablet B) 1 medium tablet H) 2 large tablets C) 1 large tablet I) 2 large and 1 small tablet D) 1 large and ½ small tablet J) 2 large and ½ medium tablet E) 1 large and 1 small tablet K) 2 large and 1 medium tablet F) 1 large and ½ medium tablet L) 3 large tablets

10. How long after you stop the infusion should you wait before you give the first oral dose (in h; whole number only)?

230 VM522P / VM7522 Pharmacology WIMU Fall Calculation Issues for Unit 7  Calculating an impact factor (If).

o You need to estimate both KF (can be obtained from estimating renal clearance from creatinine or B.U.N. – see class notes) and find the fraction excreted unchanged in the urine (FUR)

o The use the following relationship:

If = 1 ‐ Fur * (1 – KF)

o The number one reason students miss this problem is due to errors in order of operation. Remember to evaluate the expression within parenthesis first, then all multiplications/divisions, and then additions/subtractions. Thus, determine (1 – KF), then multiply the parenthetical value by FUR before you subtract FUR from 1 (FUR * the parenthetical value should be subtracted from 1).

o The second most common reason students miss this problem is they don’t remember if the dose is multiplied or divided by If (same with interval). The If is a fraction that tells you how much to reduce overall drug administration (for example, if it is 0.5, then drug needs to be cut in half by 50%). Remember, in all cases the goal is to reduce total dose. Thus either the dose needs to be made smaller (multiple dose by If) or the interval longer (divide interval by If). Always, as a final check, examine if the new value(s) you calculate accomplish this goal – i.e., total drug exposure has been reduced.

o Making an adjustment using total daily dose.

. Students often make this more complicated than need be. When the request is to keep total exposure, total dose, Cpavg, etc. constant, you do not need to calculate Cpmax, Cpmin, and/or Cpavg. Simply sum the total daily dose and then divide that value by the number of times the drug is to be administered.

. If going from two times a day to three times a day dosing (assuming a dose was 6 mg), then the total daily dose is 12 mg (6 * 2), and the new individual dose will be 12 ÷ 3 = 4 mg.

. On the other hand, going from 3x a day to 2x a day dosing (again assuming a dose of 6 mg), then the total daily dose is 18 mg (3 * 6 mg), and the new individual dose will be 18 ÷ 2 = 9 mg.

. This can be done whether you are making adjustments to the overall dose or not (we developed this manipulation in the context of changing total dose, but it applies whether we change total dose or not). But if adjusting doses, you can make the adjustment by multiplying the total daily dose by the If, or the individual doses by If – just remember to do it only one time.

. The next critical element you need to think about is the consequence of any change you impose:

. If you reduce the frequency (but increase individual dose) variation will increase. (Cpmax will be greater and Cpmin will be less than the standard therapy).

. If you increase the frequency (but reduce individual dose) the variation will decrease. (Cpmax will be less and Cpmin will be greater than the standard therapy).

. Cpavg will stay the same.

231 VM522P / VM7522 Pharmacology WIMU Fall  Reminder of some relationships:

o Cpavg = (D x t1/2) / (0.693 x Vd x DI) = D / (CLs x DI)

o Cpmax = (dose / Vd) x FAC (to get the accumulation factor one needs to know the RDI and the chart) RDI o Cpmin = Cpmax / range = CPmax / 2  Therapeutic drug monitoring. o There are several issues that need to be examined. These are not so much a specific calculation issue as it is applying pharmacokinetic understanding to the situation. o However, one item is clear: The best single time to obtain a plasma sample is at the end of a dosing interval. Some reasons:

. Plasma levels are more stable at the end of a dosing interval than at the beginning. At the beginning, since you do not necessarily know the absorption characteristics of the preparation, you can be confused by multiple issues. For example, is the sample you measured on the upstroke or has the peak already occurred and plasma levels are falling? Perhaps absorption is very fast and the high level is due to ‐phase issues? Thus it is just best to avoid these issues by sampling later in the dosing interval.

. An alternative might be to collect at the middle of the regimen. This might seem to represent and ‘average’ of sorts and thus have some inherent value because of that. However, one problem is what level do you expect in the middle of the regimen? It is not a point that is easily calculated. Further, it is not particularly informative. It does not tell you if you are maintaining adequate levels through the entire dosing interval. Thus while better than taking a sample at the beginning of the interval, it is not near as useful as a sample taken at the end of an interval.

. Since we know the ending value is dependent on both the Vd and CLs, taking a sample at the end of the interval is very informative. First, if the level is what is expected, and therapeutically the drug is not performing (or causing undesired side expects), then the issue must be pharmaco‐ dynamic (i.e., the condition is resistant to the drug or the patient extra sensitive to the drug). On the other hand, if the level is higher or lower than expected, it indicates that an assumption about the pharmacokinetics is in error. Based on a single sample the exact identity of an erroneous assumed pharmacokinetic parameter cannot be determined, but if two samples are taken within the same dosing interval, the clinician can begin to gain some insights – are the differences between samples what would be expected based on kinetics (i.e, a simple half‐life consideration)? From this one can begin to determine if an assumption about Vd is in error (is the animal especially emaciated or fat?) or elimination (are there signs of kidney or liver disease, is there some other reason that could cause the difference – such as enzyme induction or inhibition)?

o Exactly which conclusion to draw will depend on a variety of clinical signs and your experience with the drug, but the first thing you need to understand is the dynamics of the system. The dynamics are caused by pharmacokinetic considerations, and the better you understand these factors, the more insightful any conclusions you draw will be.

o A final more trivial issue that sometimes arises in these calculations (i.e., sometimes someone will miss a problem because of this) is to remember that the critical time associated with therapeutic drug monitoring is when the sample is obtained from the patient, in absolute terms or relative to dose administration, and not when you send the sample to the lab or get a report.

232 VM522P / VM7522 Pharmacology WIMU Fall Unit 7 – Problem Set –Adjustments and Drug Monitoring

1. You plan to give an antibiotic to a 10 lb cat. The recommended dose of this antibiotic is 2 mg/kg IM every 8 hours. In cats the t1/2 for this antibiotic is 1.5 h and the Vd is 0.35 L/kg. The fraction of the compound eliminated unchanged in the urine is 85%. In this cat you found plasma creatinine to be 2 times normal. a) Based on the altered kidney function, how much should the dosing interval or dosing amount be altered?

Either the interval should be changed to ______h (nearest whole number),

or the amount should be changed to ______mg (two significant digits).

b) Now make the adjustment but also change to twice a day dosing. Answer in amount of drug needed (in mg) twice a day.

2. You have decided to treat Spike, a 40 lb mixed‐breed dog, for an arrhythmia with oral quinidine therapy. The pharmacokinetic parameters reported for quinidine in the dog are: ∙ t1/2 = 6 h ∙ Vd ~ 2.9 L/kg ∙ oral bioavailability ~ 100% ∙ 20% excreted unchanged in the urine, the rest is metabolized in the liver ‐ Quinidine comes as 50, 100, 200 and 300 mg tabs and an inj prep of 50 mg/ml in 10 ml vials. ‐ Therapeutic plasma levels are reported to be 2.5 to 5.0 mcg/ml; toxic effects seen at > 10 mcg/ml ‐ The recommended dosing schedule is 6 ‐ 16 mg/kg PO every 6 h. ======a) Choosing from the list below and using the recommended dosing protocol, what combination of tablets would make a single dose? Note:  no more than two sizes of tablets  do not break tablets  give as large a dose as possible without exceeding 16 mg/kg A) 1 ‐ 50 mg tablet B) 1 ‐ 100 mg tablet C) 1 ‐ 100 mg tablet and 1 ‐ 50 mg D) 1 ‐ 200 mg tablet E) 1 ‐ 200 mg tablet and 1 ‐ 50 mg F) 1 ‐ 300 mg tablet G) 1 ‐ 300 mg tablet and 1 ‐ 50 mg H) 2 ‐ 200 mg tablet I) 1 ‐ 200 mg and 1 ‐ 300 mg tablet J) 2 ‐ 300 mg tablets

233 VM522P / VM7522 Pharmacology WIMU Fall b) Calculate the theoretical loading dose for Spike (in mg, use the specific dose chosen in the previous question for the calculation)?

c) For the dosing regimen determined above for Spike, calculate Cpavg (answer in mcg/ml):

d) For the dosing regimen determined above for Spike, calculate Cpmax (answer in mcg/ml):

e) For the dosing regimen determined above for Spike, calculate Cpmin (answer in mcg/ml):

f) Given the plasma levels associated with therapeutic effects and toxic effects, how well does the dosing regimen selected stay within these limits? (Note: it is safe to assume that absorption is not instantaneous, so peak levels actually observed are going to be less than the Cpmax you calculated. For our purposes, assume the actual Cpmax will be at least 15% less than the calculated Cpmax.)

After a day the arrhythmia is not controlled during the entire interval between doses. You would like to determine if this is due to an inherent insensitivity of the arrhythmia to quinidine or if the regimen is not producing adequate plasma levels of quinidine. To do this you plan to draw plasma samples and measure the quinidine present. g) Dr. Nothought draws a sample 30 min after a dose and finds a plasma level of 6.9 mcg/ml. What conclusion can Dr. Nothought justify with this information?

h) Dr. Thoughtful draws a sample 20 min before a scheduled dose and finds a plasma level of 1.8 mcg/ml. What conclusion can Dr. Thoughtful justify with this information?

i) Dr. Insightful draws both samples. What conclusions can Dr. Insightful make about the Vd and t1/2 of quinidine in Spike with this information? What would Dr. Insightful’s conclusion be regarding the lack of consistent response in Spike’s therapy?

j) You want Spike’s minimal plasma level to remain at or above 3 mcg/ml. There are two ways of doing this. What are they and which one is safer? Why?

234