THE CARDIORESPIRATORY AND ANESTHETIC EFFECTS OF CLINICAL AND SUPRA CLINICAL DOSES OF ALF AXALONE IN CYCLODEXTRAN IN CATS AND DOGS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Master of Science in the Graduate

School of The Ohio State University

By

Laura L. Nelson, B.S., D.V.M. * * * * *

The Ohio State University 2007

Dissertation Committee:

Professor Jonathan Dyce, Adviser

Professor William W. Muir III

Professor Shane Bateman If I have seen further, it is by standing on the shoulders of giants. lmac Ne1vton (1642-1727) Copyright by

Laura L. Nelson

2007

11 ABSTRACT

The anesthetic properties of steroid hormones were first identified in 1941, leading to the development of neurosteroids as clinical anesthetics. CT-1341 was developed in the early 1970’s, featuring a combination of two neurosteroids (alfaxalone and alphadolone) solubilized in Cremophor EL®, a polyethylated castor oil derivative that allows hydrophobic compounds to be carried in aqueous solution as micelles. Though also possessing anesthetic properties, alphadolone was included principally to improve the solubility of alfaxalone.

CT-1341, marketed as Althesin® and Saffan®, was characterized by smooth anesthetic induction and recovery in many species, a wide therapeutic range, and no cumulative effects with repeated administration. Its cardiorespiratory effects in humans and cats were generally mild. However, it induced severe hypersensitivity reactions in dogs, with similar reactions occasionally occurring in cats and humans. The hypersensitivity reactions associated with this formulation were linked to Cremophor

EL®, leading to the discontinuation of Althesin® and some other Cremophor®-containing anesthetics.

More recently, alternate vehicles for hydrophobic drugs have been developed, including cyclodextrins. Cyclodextrins are rings of α-1,4-linked glucose subunits that feature a hydrophilic exterior and hydrophobic interior that allows them to complex with iii appropriately sized hydrophobic drugs. One such molecule, hydroxy-β-cyclodextrin, has a very wide therapeutic index, increases the solubility of alfaxalone by over 8,000 times, and has been combined with alfaxalone as Alfaxan-CD RTU®.

The objectives of the described studies were to administer clinical and supraclinical doses of Alfaxan-CD RTU®, to assess the resultant quality of anesthesia, and to assess anesthetic effects on hemodynamic, respiratory, pH, and blood gas variables. We hypothesized that Alfaxan-CD RTU® would produce general anesthesia of rapid onset and brief duration in healthy dogs and cats, that minimal cardiorespiratory depression would result from clinical doses, and that no histaminoid reactions would occur.

Eight healthy adult cats and dogs were included in the study. Each cat was sequentially administered placebo, 5 mg/kg, 15 mg/kg, and 50 mg/kg of Alfaxan-CD

RTU®, with washout periods of 1, 3, and 24 hours between treatments, respectively.

Monitored parameters included respiratory rate, tidal volume, minute volume, lead II electrocardiography, heart rate, arterial blood gas (pH, PaO2, PaCO2, and HCO3), systemic (systolic, mean, and diastolic) and pulmonary arterial pressures, and body temperature. Rate pressure product and systemic vascular resistance were calculated.

The qualities of induction, anesthetic maintenance, and recovery and the response to a noxious stimulus were assessed as categorical variables.

Alfaxan-CD RTU® doses of 2 mg/kg, 6 mg/kg, and 20 mg/kg were administered to each dog as blinded treatments in random order. Each treatment was followed by a 3 hour washout period. Monitored and calculated parameters were the same as for cats, with the addition of left ventricular end-diastolic pressure. iv In cats, dose-dependent decreases in heart rate, blood pressure, systemic vascular resistance, and rate pressure product were noted. These effects were most pronounced at the higher doses, with significant decreases in systolic arterial blood pressure and pulmonary arterial pressure after the 50 mg/kg doses. Hemodynamic changes were mild

at the clinically recommended dose (5 mg/kg). Dose-dependent decreases in PaO2

occurred at all doses, but lasted for 30-60 minutes after the higher doses. The majority of

cats at all doses required supplementation with 100% oxygen and mechanical ventilation

after PaO2 dropped below 60 mmHg, including all cats at the highest dose. Apnea

occurred in 1 and 2 cats at the 5 and 15 mg/kg doses, respectively, but occurred in all cats

at the 50 mg/kg dose. Decreased tidal volume, pH, and HCO3, and increased PaCO2 also

occurred at the 50 mg/kg dose. The qualities of induction and maintenance were

excellent at all doses. The quality of recovery was excellent at lower doses, but poor in

5/7 cats at the 50 mg/kg dose due to prolonged recovery periods.

In dogs, hemodynamic changes included dose-dependent decreases in systemic

and pulmonary arterial pressures, rate pressure product, and systemic vascular resistance.

Changes were generally mild and were most pronounced at the 6 and 20 mg/kg doses.

Dose-dependent decreases in respiratory rate, minute volume, and PaO2 and decreases in

PaCO2 and pH were also noted, but lasted for only 15 minutes and were most pronounced

at the 20 mg/kg dose. Apnea was more frequent at higher doses. The quality of

induction, maintenance, and recovery were good to excellent at all doses. The duration

of anesthesia was dose-dependent.

In conclusion, our studies indicate that Alfaxan-CD RTU® produced good to

excellent anesthesia in cats, characterized by rapid induction to anesthesia, excellent v muscle relaxation, unresponsiveness to noxious stimuli, and smooth, uneventful recovery from anesthesia. Hypoventilation and apnea were uncommon at clinically relevant doses, but became the most important adverse effects when larger doses were administered rapidly IV. Arterial blood pressure remained stable following the IV administration of clinically relevant doses (<5 mg/kg) of Alfaxan® CD-RTU, but caution is advised with larger doses or with administration to animals with cardiovascular compromise.

In dogs, Alfaxan®-CD RTU produced safe and effective anesthesia. Induction to anesthesia was rapid and uneventful. The maintenance and recovery periods were characterized by good to excellent muscle relaxation and analgesia. Cardiovascular status was well maintained when dosages of up to 6 mg/kg IV were administered.

Respiratory depression and apnea were the only notable disadvantages, and then only when larger dosages (>6 mg/kg IV) are administered. Alfaxan®-CD RTU should be an excellent alternative to currently available injectable anesthetics when administered for short surgical procedures or as induction to inhalant anesthesia.

vi Dedicated to my mother and father

vu ACKNOWLEDGEMENTS

I wish to thank Dr. William Muir for allowing me to participate in this endeavor and for his patience and assistance in its completion. This thesis would not exist without his help. I thank him for saving yet another lost graduate student.

I thank my advisor, Dr. Jon Dyce, for his steadfast encouragement, advice, and assistance in this and other scholarly endeavors.

I wish to thank Dr. Philip Lerche, whose work in securing funding for and completing these experiments was invaluable. I hope that my assistance in the completion of this project is of some help.

Finally, the skilled assistance of Barbara Lang and Dr. Ashley Wiese in the performance of these experiments was deeply appreciated. I am truly indebted to all of the above individuals for their roles in every facet of this project.

This research was funded by a grant from Jurox Pty. Ltd.

Vlll VITA

June 3, 1978 ...... Born - Canton, Ohio

1996-2003 ...... B.S. Zoology, The Ohio State University

1999-2003 ...... Doctor of Veterinary Medicine, The Ohio State University

2003-2004 ...... Small Animal Intern, Michigan State University College of Veterinary Medicine, East Lansing, Michigan

2004-present...... Resident in Small Animal Surgery, The Ohio State University

PUBLICATIONS

Research Publication

1. Kolin A, Gaumer J, Ravula V, Urban R, Gilbertson L, Bos G, Dey T, Nelson L, Dyce J, Lannutti J: Three-dimensional laser micrometry characterization of surface wear in total hip arthroplasty. J Biomed Mater Res B Appl Biomater 2007.

FIELDS OF STUDY

Major Field: Veterinary Anesthesia

IX LIST OF TABLES

Table Page

1.1 Main pharmacokinetic variables after bolus intravenous administration of alfaxalone as Alfaxan-CT RTU® in eight healthy beagle dogs ...... 14

2.1 Description of anesthetic induction, recovery, and maintenance scores ...... 28

2.2 Hemodynamic average and standard deviation values for cats ...... 29

2.3 Respiratory and blood gas average and standard deviation values for cats ...... 30

2.4 Summary of time-related and categorical, descriptive variables for Alfaxan-CD RTU® anesthesia in cats ...... 31

3.1 Hemodynamic average and standard deviation values for dogs ...... 40

3 .2 Respiratory and blood gas average and standard deviation values for dogs ...... 41

3.3 Summary of time-related and categorical, descriptive variables for Alfaxan-CD RTU® in dogs ...... 42

x LIST OF FIGURES

Figure Page

1.1 a. Testosterone b. Progesterone ...... 1

1.2 Hydroxydione ...... 2

1.3 Cholesterol molecule with numbered carbon atoms ...... 3

14. a. Alfaxalone b. Alphadolone ...... 3

Xl TABLE OF CONTENTS

Page Abstract ...... iii Dedication ...... vii Acknowledgements ...... viii Vita ...... ix List of Tables ...... x List of Figures ...... xi

Chapters

1. Introduction 1.1 Origins of neurosteroid anesthetics ...... 1 1.2 Pharmacologic and clinical properties of Saffan®I Althesin® ...... 4 1.3 Alternative vehicles for hydrophobic drugs ...... 9 1.4 Pharmacokinetics and pharmacodynamics ...... 13 1.5 Alfaxalone in cyclodextrin-hypothesis ...... 17

2. Cardiorespiratory and anesthetic effects of alfaxalone in ~-cyclodextrin in the feline 2.1 Materials and methods ...... 19 2.2 Results ...... 24 2.3 Tables ...... 28

3 Cardiorespiratory and anesthetic effects of alfaxalone in ~-cyclodextrin in the canine 3.1 Materials and methods ...... 32 3.2 Results ...... 37 3.3 Tables ...... 40

4. Discussion and conclusions 4.1 Overview ...... 43 4.2 Cardiovascular effects of Alfaxan-CD RTU® ...... 44 4.3 Respiratory effects of Alfaxan-CD RTU® ...... 49 4.4 Anesthetic properties of Alfaxan-CD RTU® ...... 53 4.5 Conclusions ...... 57

List of references ...... 58

xii CHAPTER 1

INTRODUCTION

1.1

ORIGINS OF NEUROSTEROID ANESTHETICS

In 1941, a landmark paper was published by Hans Selye regarding the anesthetic actions of steroid hormones.[1] Dr. Selye, best known for his work in the description and characterization of the stress response, noted that steroid hormones such as progesterone, desoxycorticosterone acetate (DCA), androgens, and estrogens resulted in general anesthesia when injected intraperitoneally into mammals.[1] In particular, progesterone, pregnanedione, and DCA showed potent anesthetic activity and were readily absorbed after intraperitoneal and enteral administration.[!, 2] Though anesthetic effects were observed after administration of a number of hormonally significant compounds, others

(e.g. pregnanedione) produced anesthesia without significant endocrine effects.[1, 3]

Figure 1.1: a. Testosterone; b. Progesterone

1 Progress in the further development of anesthetic steroids (also called neurosteroids or neuroactive steroids) was hindered by their poor solubility.

Hydroxydione (Fig. 1.2), a 21-hydroxy derivative ofpregnanedione, was made water soluble through esterification at C21 and developed for use as an anesthetic.[3] Though it produced anesthesia with a high therapeutic index and a smooth recovery, hydroxydione did not find long-term clinical use due to its relatively large dose, prolonged induction period, alkalinity, and high incidence of thrombophlebitis. [4] No a I The search for steroid anesthetics )-0 with similar safety and efficacy but greater 0-( 0 ease of administration led to the investigation of structure-activity relationships for steroid molecules.[3] The more important of these are listed below:

Figure 1.2: Hydroxydione

• Anesthetic activity necessitated the presence of an oxygen function

(hydroxy or ketone) at the C3 and C20 positions of the pregnane molecule

(C 17 of the androstane molecule).

• Substitutions into the steroid structure reduced anesthetic activity and

occasionally introduced convulsant properties.

• Both Sa and SB compounds were highly active

• The C3 hydroxyl group could be in the a or Bconformation, with the 3a-

hydroxy-Sa and 3a-hydroxy-SB having the greatest anesthetic activity.

2 • Esters of hydroxy compounds were less active and rapid-acting than the

parent alcohols.[3]

Figure 1.3: Cholesterol molecule with numbered carbon atoms

In 1971, Child et al. described the properties of a new neurosteroid formulation consisting of one part 21-acetoxy-3a-hydroxy-5a-pregnane-11,20-dione (alphadolone) to three parts of 3a-hydroxy-5a-pregnane-11,20 dione (alphaxolone or alfaxalone) solubilized in Cremophor EL, a polyoxyethylated castor oil diluent.[5] This formulation

(CT-1341) was ultimately marketed in veterinary and human markets as Saffan® and

Althesin®, respectively. Alphadolone, though possessing anesthetic properties, was included in the formulation largely because it greatly increased the solubility of alfaxalone in the diluent.[5]

a\. OH

a. H b.

Figure 1.4: a. Alfaxalone; b. Alphadolone 3 1.2

PHARMACOLOGIC AND CLINICAL PROPERTIES OF SAFFAN®/ALTHESIN®

The initial pharmacologic properties of CT-1341 were described for the mouse, rat, rabbit, cat, monkey, and dog.[5] The formulation was most effective in the non­ rodent species. Overall, CT-1341 was active at a lower dose than other induction agents and resulted in immediate unconsciousness in all species at the lowest active dose.[5]

Compared to thiopentone and methohexitone, CT-1341 did not result in a cumulative effect when administered repeatedly to mice or when infused intravenously into cats.[5]

The formulation was not found to induce thrombophlebitis in any species irrespective of dosage or intra-arterial administration. [5]

In the cat, CT-1341 at a dose of 1.2 mg/kg produced transient hypotension and

loss of consciousness within 10-30 seconds of administration.[5] Transient decreases in blood pressure and tachycardia occurred immediately after injection, followed by a mild pressor effect on emergence from anesthesia. Cats regained consciousness 6-9 minutes

after injection, rapidly returning to normal.[5] These responses were similar to those produced by low doses ofthiopentone (3 mg/kg) and propanidid (8 mg/kg). At higher

doses, (19.2 mg/kg of CT-1341, 24 mg/kg ofthiopentone, and 32 mg/kg ofpropanidid),

CT-1341 was the only agent not to produce apnea, but it did result in a prolonged

hypotensive response similar to that observed with thiopentone.[5] Doses of 32 mg/kg

resulted in apnea, cardiovascular collapse, and death. CT-1341 was not noted to interact

4 adversely with a variety of other medications, including neuromuscular blocking drugs, halothane, chloroform, atropine, and chlorpromazine.[5]

Subsequent clinical trials of CT-1341 in cats yielded similar results. Child et al. compared CT-1341 to thiopentone, methohexitone, pentobarbitone, propanidid, and ketamine at doses ranging from the minimal necessary to produce loss of the righting reflex to a maximum dose determined from previous experiments (twice the maximum dose was fatal in all cats).[6] Cardiovascular effects were characterized by transient decreases in blood pressure and tachycardia, followed by a second decrease in blood pressure 2.5-5 minutes after injection.[6] At the two highest doses, recovery from this decrease did not occur for 30-60 minutes. Respiratory effects of CT-1341 were mild in comparison to other agents, with only 3/7 cats experiencing slow, shallow breathing after the maximum dose.[6] Cats receiving barbiturates experienced apnea and respiratory depression at high doses. Overall, the therapeutic index of CT-1341 was higher than that of barbiturates and propanidid and similar to that ofketamine.[6] CT-1341 induced less respiratory depression than the barbiturates, and was not accompanied by the circulatory stimulation, catatonia, and bizarre behavior noted with ketamine.[6]

Haskins et al. compared CT-1341 to ketamine and xylazine in the cat after intramuscular (IM) administration. [7] CT-1341 resulted in a sustained decrease in blood pressure compared to baseline values and those noted after ketamine or xylazine administration. Central venous pressures and blood gas values were not substantially changed. CT-1341 resulted in faster recovery times, less spontaneous movement, and eyelid closure.

5 Dyson et al. evaluated the effects of Saffan® on cardiopulmonary function in healthy cats.[8] Respiration was significantly diminished during the first 15 minutes after induction, but blood gas changes were not statistically significant. Cardiac output, stroke volume, and systolic arterial blood pressures all fell significantly below baseline values at various points after anesthetic induction, similar to that noted in other studies.[8]

Middleton et al. compared the physiological effects of thiopentone, ketamine, and

CT-1341 at recommended clinical dosages in the cat, evaluating their effects on blood gas values, mean arterial blood pressure, and heart rate.[9] Compared to other agents tested, CT-1341 had the least effect on blood gas parameters, with pH not statistically different in any cat compared to baseline and decreases in carbon dioxide (pC02) only at

30 minutes post-administration.[9] Hemodynamic parameters showed a statistically significant drop in arterial blood pressure at all time points, with a similar initial drop in pressure, partial recovery, then a sustained hypotensive episode. These hemodynamic changes were similar to but more consistent and profound than those observed after thiopentone administration. Tachycardia was present only during the initial phase (1-5 minutes post-injection). [9]

Adverse effects of CT-1341 in cats other than hypotension included edema and flushing of the ear skin and face and swelling of the forepaws. In the report by Middleton et al., 6of11 cats experienced side effects similar to those previously reported (swollen muzzle, flushed pinnae, lacrimation, coughing).[9] Another report found histaminoid reactions following 69% of administrations, occasional cyanosis, laryngospasm, coughing, vomiting, opisthotonus, and occasional excitement during recovery.[1 O]

Vomiting and defecation were noted during induction and recovery by Haskins et al., as

6 were sneezing and nose-rubbing in recovery.[7] Many of these signs are suggestive of histamine release, were recognized early in the evaluation of CT-1431, and were attributed to the vehicle (Cremophor EL).[6] Cremophor administered intravenously to cats resulted in the same effects as CT-1431.[6] Moreover, the hemodynamic effects of histamine release parallel the decrease in systolic blood pressure noted after CT-1341 administration in cats.[9] Despite the relatively high incidence of histaminic reactions,

Saffan® became widely used as an induction agent and injectable anesthetic in the United

Kingdom and Australia.

CT-1341 found only limited use in the canine due to consistently severe histaminoid reactions following administration.[3] Reports of severe reactions to use of

CT-1341 use in dogs included death after a period of excessive salivation, diarrhea, extensive edema, and respiratory difficulty.[11] Safe use of Saffan® was achieved by administering the formulation concurrently with chlorpheniramine (an antihistamine) in several reports.[12, 13] Reported benefits of neurosteroid anesthesia in dogs included a wide margin of safety, rapid recovery in all breeds of dog (including sighthounds), no tissue damage with perivascular injection, minimal depression of pups after Caesarean section, and a small volume of injection required.[13]

In humans, Althesin® was found to induce relatively mild hypotension, to produce mild respiratory depression, and to feature relative ease of administration, increased pulmonary compliance, and rapid recovery with pleasant euphoria. [ 14] A clinical trial in healthy patients demonstrated no respiratory depression and an overall increase in respiratory rate.[15] Mean arterial blood pressure consistently decreased following induction, but no significant changes in mean cardiac output were noted.[15] Other

7 studies demonstrated that a period of hyperventilation occurred after induction, followed by apnea, then tachypnea.[3] Althesin® also decreased intracranial pressure due to a reduction in cerebral blood flow and cerebral blood volume.[3] Althesin® did not have an effect on renal or hepatic function and appeared safe in the management of patients susceptible to malignant hyperpyrexia.[3]

Though the occurrence of histaminoid reactions in humans was less frequent than that observed in cats and dogs, allergic reactions to Althesin® received significant attention in the medical literature.[3] Reactions to Althesin® included facial edema, hypotension, bronchospasm, and cyanosis. [ 16, 17] The incidence of such reactions ranged from 1/430 patients to 1111000 patients.[18] Increased risk ofreaction was described in patients that had previously received Althesin® or other drugs using

Cremophor EL as a carrier (e.g. propanidid), women, patients with atopic or allergic disorders, children, pregnant women, patients receiving a large total dose, and patients to whom the dose was administered rapidly.[18-20] Multiple mechanisms are likely involved in the development of hypersensitivity reactions. First exposure reactions may be related to mast cell degranulation or alternative pathway complement activation. [ 19]

Repeated exposure is more likely to involve activation of the classical complement pathway.[19, 21]

Despite the incidence of substantial hypersensitivity reactions in humans,

Althesin® was, for many anesthetists, an ideal drug for use by constant infusion because of its rapid metabolism and for neurological surgery due to its safety in patients with increased intracranial pressure.[22] The drug was withdrawn by its manufacturers after the Italian government banned its use and the use of propanidid, another Cremophor-

8 containing anesthetic, leaving a gap in the available techniques that was upsetting to many anesthetists.[22]

9 1.3

ALTERNATIVE VEHICLES FOR HYDROPHOBIC DRUGS

The purpose of a vehicle in the administration of an injectable drug is to evenly disperse the active ingredient in a volume that allows the clinician to administer the drug in a convenient dose size while minimizing side effects. Highly lipophilic drugs like propofol and alfaxalone require the addition of an emulsifier or surfactant to allow the drug to be dispersed evenly in aqueous solution.[23] The technologies available to accomplish this goal have progressed substantially since the introduction of CT-1341.

Cremophor EL is a nonionic surfactant synthesized by the polyethylation of castor oil by treatment with ethylene oxide. When mixed with water, it aggregates into micelles of <100 nm size. Hydrophobic drugs, such as propofol, propanidid, and CT-1341, reside in the hydrophobic core of the micelle. Unfortunately, allergic reactions related to

Cremophor administration are well-documented, though it continues to be used as the vehicle for several antineoplastic drugs (teniposide, paclitaxel, aplidine, clanfenur, etc.).[24] Its use as a vehicle for antineoplastic drugs has also brought to light its effects on the pharmacokinetics and pharmacodynamics of drugs coadministered with it; interactions that can impact the overall toxicities of antineoplastic agents.[24]

Alternative vehicles for hydrophobic drugs have been developed. After initial micellization in Cremophor EL, propofol was formulated as a lipid emulsion. The emulsion formulation chosen for development had the same components as the parenteral

10 fat formulation, Intralipid® (soybean oil, egg yolk lecithin, and glycerol).[23] The propofol is principally dispersed in the soybean oil, with the lecithin serving to stabilize small propofol-soybean oil droplets and the glycerol present to maintain a formulation isotonic to the blood.[23] Upon administration of the emulsion, propofol must dissolve out of the droplet and into the bloodstream, a process governed by the drug concentration gradient, the partition coefficient, the drug diffusivity in both phases, and the interfacial area of the oil-drug droplets.[23] Emulsions will always slow the availability of free drug compared to drugs administered in solutions in which they are dissolved.[23]

Emulsions, as opposed to micellar solutions, are composed of emulsified oil droplets that can be unstable over time. The size of the oil droplets in an emulsion determine the appearance of the formulation (milky vs. clear) and determine whether it is considered a coarse or fine macroemulsion or a microemulsion.[23] Emulsions with smaller droplets allow for more rapid drug dispersion. Microemulsions and micellar solutions are thermodynamically stable and remain intact for long periods.

Macroemulsions like propofol (as currently available) are relatively unstable due to a tendency for the mixture to separate completely into two phases (upper oil layer and a lower water layer). The emulsifier reduces the interfacial tension of the oil-water interface and allows the oil to form stable dispersed droplets within the water phase.[23]

Even in spite of this, emulsions tend to degrade over time, resulting in increased droplet size and variations in drug concentration within a volume of emulsion.[23]

Alternatives to traditional micellization or emulsification of hydrophobic drugs include the use of cyclodextrin formulations and use of poloxamer to create polymeric micelles.[23] It is the use of cyclodextrin formulations that currently shows the most

11 promise as a safe replacement for Cremophor EL as the vehicle for neurosteroid anesthetics.

Cyclodextrins are cyclic oligomers that contain various numbers of a-1,4-linked glucose units, the number of which determines the nomenclature for each compound (a indicates the glucose hexomer, p the glucose heptomer, y the octomer, etc.).[25] These molecules assume the shape of a truncated cone or torus in which the polar sugar groups are oriented to the exterior and the interior is non-polar and lipophilic. This property allows cyclodextrins to form soluble, reversible inclusion complexes with compounds insoluble in aqueous solution. [25] The cavity within the cyclodextrin must at least partially accommodate the drug molecule in question, a qualification for which the P­ cyclodextrins are often the most appropriate.[25] The highly stable crystal lattice of P­ cyclodextrins makes them poorly soluble in aqueous solution. To improve this, the

structure of the ring has been modified by alkylation and hydroalkylation to produce more soluble cyclodextrins.[25] Though many cyclodextrins result in increased aqueous

solubility of alfaxalone, hydroxypropyl-P-cyclodextrin (HPCD) was selected for further

study based on its low systemic toxicity .[25] The aqueous solubility of alfaxalone is

increased 8056 times when combined with HPCD, almost 10 times greater than that

achieved with Cremophor EL.[25] The combination of alfaxalone and HPCD is also able to be lyophilized into a reconstitutable, stable, white powder and can be autoclaved.[25]

Intravenous toxicity studies of HPCD have also been encouraging. Subacute and

subchronic studies of HPCD in rats and monkeys did not result in treatment-related

abnormalities or deaths.[26] An acute high dose study did not result in subject mortality,

with hematuria occurring in only two animals at the 10 g/kg dose. [26]

12 1.4

PHARMACOKINETICS AND PHARMACODYNAMICS

Alfaxalone in HPCD has been developed for clinical use in small animals under the trade name Alfaxan-CD RTU® (Jurox Pty. Ltd, Rutherford, Australia). The pharmacokinetic parameters of this new formulation in the dog have been described by

Ferre et al. [27]. Dosages of 2 and 10 mg/kg were administered separately to healthy

Beagle dogs as intravenous boluses over 60 seconds, the first dose representing the recommended clinical dose and the second a supraclinical dose.[27] Induction of anesthesia was rapid in all dogs ( <1 minute). The duration of anesthesia ( endotracheal intubation to extubation) was 6.4 ± 2.9 and 26.2 ± 7.5 minutes for the 2 and 10 mg/kg doses, respectively.[27] No adverse events were noted in the study apart from mild agitation and noise hypersensitivity during recovery.[27]

Alfaxalone was quantifiable in blood in all dogs 2 hours after the 2 mg/kg dose and up to 4 hours in four dogs after the 10 mg/kg dose. The pattern of alfaxalone concentration after administration was characterized by a minor "plateau" phase after the initial slope, followed by a return to the slope within 60 minutes after drug administration.[27] Major pharmacokinetic parameters are summarized in Table 1.1 below. Statistically significant differences were noted between average plasma clearance

(Clp) between the 2 and 10 mg/kg doses, as was the average mean residence time

(MRT).[27]

13 Table 1.1: Main pharmacokinetic variables after bolus intravenous administration of alfaxalone as Alfaxan-CT RTU® in eight healthy beagle dogs.[27] Dose 2 mg/kg 10 mg/kg Cmax (mg/kg) 2.3 ± 1.5 9.4± 2.3 AU Co~ (minute·mg/L) 35.5 ± 8.7 198.0±53.2 Clp (mL/minute·kg) 59.4 ± 12.9 52.9± 12.8 MRT (minute) 29.7 ± 9.0 38.5 ± 9.1 Yd (L/kg) 2.4 ± 0.9 2.9 ± 0.4

T112 (minute) 24.0 ± 1.9 37.4 ± 1.6

Cmas, maximum plasma concentration; AUC, area under the curve; Yd, volume of distribution; T 112 , plasma half-life

The cause of the "plateau" effect is not well understood, but may relate to blood flow and cardiac output changes resulting from skeletal muscle movement during recovery.[27] Alternatively, the lung may act as an alfaxalone reservoir, releasing drug into the pulmonary vein after a blood flow-dependent retention time.[27]

Alfaxalone is 30-50% protein-bound in rats, cats, horses, and humans. It is metabolized rapidly in the liver, with glucuronide synthesis playing a major role in its metabolism to 2-a-hydroxyalfaxalone and other compounds and its ultimate excretion.[6]

No appreciable hepatic or renal storage of the drug is present.[28] The majority of a dose of alfaxalone is eliminated as metabolic products in the feces within 5 days after intravenous injection, with the remaining 20-30% eliminated in the urine during the same period. [29]

Alfaxalone, like most injectable sedative and anesthetic drugs including the benzodiazepines, barbiturates (thiopental), propofol, and etomidate, produces dose- dependent unconsciousness and cardiorespiratory depression by potentiating the activity of inhibitory synaptic receptors in the central nervous system. The most important site of action of alfaxalone and other general anesthetics is on y-aminobutyric acid (GABA) receptors, particularly GABAA receptors.[30] The GABAA receptor is a pentameric 14 ligand-gated chloride ion channel with binding sites for GABA and separate binding sites for modulatory compounds, including barbiturates, benzodiazepines, propofol, and neurosteroids. [31]

The GABAA receptor is a macromolecular complex consisting of five subunits, similar to the nicotinic receptor. Six subunit types (a, p, y, o, p, andµ), each with multiple subtypes, have been described.[32] A drug's individual activity is determined by its subunit selectivity and the composition of GABAA receptors [3 3]. Central nervous system (CNS) neurons express a complex array of GABAA receptor subunits (a, p, y, o,

E) and other neuromodulatory receptors, complicating the prediction of individual drug pharmacology. For example, thiopental and propofol act somewhat differentially at both

GABA and glycine receptors, while etomidate acts almost exclusively at GABAA receptors.[34] These drug-specific differences in GABAA activity are likely responsible for the intensity and spectrum of pharmacologic and anesthetic effects produced by each drug, including sedation, anxiolysis, muscle relaxation, and general anesthesia.

Alfaxalone demonstrates stereoselective potentiation of GABA actions by increasing the duration of time that the GABAA receptor channel is open, increasing the duration and intensity of early GABA-mediated inhibition of neuronal depolarization.[30,

35] Other studies suggest that they potentiate GABA-evoked currents at nanomolar concentrations, but can directly activate GABAA receptors in the absence of GABA when at micromolar concentrations. [36]

Neurosteroids may act in a paracrine fashion to locally and selectively influence different neurons and GABAA receptor pools with different receptor isoforms.[33] The effects of neurosteroids vary considerably between different regions of the brain, likely

15 due to regional differences in the distribution of appropriate binding sites.[30, 32]

Additionally, different neurosteroids likely vary in their binding affinities for different subtypes.[32] As neuroactive steroids are evaluated for their use in the management of epilepsy, anxiety, insomnia, migraine headaches, drug dependence, depression, stress, and other psychiatric and neurologic conditions, further elucidation of the effects of these drugs on specific receptors and the development of nonanesthetic neurosteroids is likely to continue.[36]

Additional effects of alfaxalone may include modulation of the autonomic nervous system. Alfaxalone inhibits nicotine-induced [Ca2+]i increases and nicotine­ induced inward currents in cultures of adrenal chromaffin cells. [3 7] This inhibition of nicotinic acetylcholine receptors occurs at anesthetically relevant concentrations of alfaxalone and is independent of its effects on GAB AA receptors. [3 7]

16 1.6

ALF AXALONE IN CYCLODEXTRIN--HYPOTHESIS

The favorable safety and anesthetic properties of neurosteroid anesthetics and the body of experience developed with their use in previous formulations (Althesin®,

Saffan®) make the reformulation of alfaxalone with cyclodextrin an encouraging step toward the reintroduction of neurosteroid anesthetics into clinical practice.

Pharmacokinetic and toxicity studies have been previously performed for Alfaxan-CD

RTU®.[28, 37]

Our goals were to evaluate three categories of responses to the administration of

Alfaxan-CD RTU® in the canine and feline:

• Hemodynamic, respiratory, blood pH, and blood gas (P02, PC02)

• Anesthetic quality, including the quality of induction, maintenance, and

recovery of anesthesia and responses to nociceptive stimuli

• The duration of specific anesthetic events, including time to induction,

time to response to a noxious stimulus, time to extubation, the incidence

and duration of apnea, and the duration of cardiorespiratory alterations.

These parameters were assessed for the clinically recommended dose in each species as well as supraclinical doses three and ten times the recommended dose. The evaluation of multiple doses allowed for assessment of therapeutic index of the drug in each species.

17 We hypothesized that Alfaxan-CD RTU®would produce general anesthesia with rapid onset and brief duration. We predicted that cardiorespiratory depression would be minimal at the clinically relevant dose, similar to that described for previous neurosteroid formulations. Finally, we predicted that flushing, facial edema, bronchospasm, and histamine-related hypotension would not be produced with this Cremophor-free formulation.

18 CHAPTER2

CARDIORESPIRATORY AND ANESTHETIC EFFECTS OF ALFAXALONE IN~­ CYCLODEXTRIN IN THE FELINE

2.1

MATERIALS AND METHODS

The experimental protocol was approved by The Ohio State University

Institutional Laboratory Animal Care and Use Committee. Eight adult purpose-bred domestic shorthair cats (4 male; 4 female) weighing between 3.71and5.91 kg were selected. All cats were judged to be in excellent health based upon a physical examination, electrocardiogram, complete blood count (CBC), and blood chemical profile (total protein, albumin, sodium, potassium, chloride, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, creatine phosphokinase, creatinine, and blood urea nitrogen).

Experimental Preparation and Procedures-All cats were instrumented with vascular catheters the day before (Day -1) the experiment. Vascular catheterization procedures were completed after the administration of propofol (4-6 mg/kg IV;

PropoFlo™; Abbott Laboratories, North Chicago, Illinois, USA) during isoflurane (2-

3%; IsoFlo™; Abbott Laboratories) in oxygen anesthesia. The skin adjacent to the right carotid artery and jugular vein was surgically scrubbed and prepared for placement of catheter introducers. Once positioned, the catheter introducers (Arrow International Inc.,

19 Reading, Pennsylvania, USA) were flushed with heparinized (1000 units/500 mL) saline,

subcutaneously tunneled to exit the skin, and secured with silk suture and bandages. An

Elizabethan collar was placed around each cat's neck and the cat returned to its cage. The

placement of the catheter introducers was completed in less than thirty minutes and took

place 24 hours before the beginning of the experiment.

On the day of the experiment (Day 0), the cat was returned to the laboratory. A

physical exam was performed and the Elizabethan collar and bandages removed. A 4F

Swan-Ganz thermodilution catheter (Arrow International Inc.) was advanced through the jugular vein catheter introducer and positioned so that its distal tip was approximately 1

cm distal to the pulmonary valve. A fluid-filled catheter (PE 160; Becton, Dickinson,

and Company, Franklin Lakes, New Jersey, USA) was advanced through the right carotid

artery introducer and positioned with the distal tip in the ascending aorta. Both catheters

were connected to a computer-based data acquisition system (PO-NE-MAH Data

Acquisition Computer; Gould Instruments Systems Inc, Valley View, Ohio, USA),

permitting continuous analog display and digital recording of data. A lead II

electrocardiogram and the pulmonary artery temperature were continuously recorded for

each cat.

A handheld volumeter (Medishield; Fraser Harlake, Orchard Park, New York,

USA) was attached to a tight-fitting facemask placed over the cat's muzzle to measure

and record respiratory rate and tidal volume in conscious cats. Following induction with

test article, this instrument was attached to the end of an endotracheal tube for continued

respiratory monitoring.

20 Heparinized anaerobic blood samples were collected from the right carotid artery catheter and analyzed for arterial blood pH, blood gas (Pa02, PaC02), and bicarbonate

(cHC03) values (Model ABL 725; Radiometer America, Inc., Westlake, Ohio, USA).

Heart rate (beats/minute) and rhythm, aortic systolic, diastolic, and mean blood pressures

(mmHg), pulmonary artery (mmHg) and right atrial mean blood pressure (mmHg), cardiac output (mL/kg/minute; thermodilution), respiratory rate (breaths/minute), tidal volume (mL), minute volume (mL), body temperature (C), arterial pH, blood gases

(Pa02, PaC02; mmHg), and bicarbonate were recorded before and after intravenous

5 Alfaxan® administration. Systemic vascular resistance (SVR; dynes/second/cm- ) and rate pressure product (RPP; beat/mmHg) were calculated.

The anesthetic parameters of quality of induction, maintenance, and recovery from anesthesia and the response to noxious stimulation during anesthesia were categorized. Two different noxious stimuli were used to evaluate analgesia: a 10 cm

Rochester-Pean forcep was applied to the second digit of the left rear leg for 10 seconds

(or until the first signs of limb withdrawal) and to the base of the tail for 60 seconds (or until the first signs of tail withdrawal or head lift). The cat's response to the toe and tail pinches were categorically rated as: no response = O; minimal movement= 1; limb withdrawal= 2; limb withdrawal and lifting of the head= 3. Induction to anesthesia, maintenance, and recovery from anesthesia were categorically rated as described in Table

2.1.

If a cat's arterial P02 fell below 60 mmHg, positive pressure ventilation was initiated with 100% oxygen at 4-6 breaths per minute and an inspiratory pressure of 20 cmH20 until spontaneous breathing efforts produced a Pa02 greater than 80 mmHg.

21 Data from cats receiving 100% oxygen were excluded from statistical analysis during the duration of ventilation. The duration of apnea was calculated as the difference in minutes and seconds between the time at which the cat ceased to breathe and the time of the first spontaneous inspiratory effort. The duration of apnea was calculated as the difference in minutes and seconds between the last inspiratory effort and the onset of positive pressure ventilation in cats that received positive pressure ventilation. The times (minutes) to first head lift, swallowing, and sternal recumbency following Alfaxan® CD-RTU administration were recorded.

Experimental Plan-Placebo and two (5, 15 mg/kg) IV doses of Alfaxan® CD­

RTU (10 mg/mL) were administered in sequential order on experimental day 0, and 50 mg/kg of Alfaxan® CD-RTU was administered 24 hours later on experimental day 1 . A

1 hour washout period separated the placebo and 5 mg/kg doses, and a 3 hour washout period separated the 5 mg/kg and 15 mg/kg doses. The doses and washout intervals were chosen based upon the selection of 5 mg/kg IV Alfaxan® CD-RTU as the labeled dose in the cat and a drug half-life of approximately 45 minutes.[27] The 50 mg/kg IV dose was equivalent to 10 times the anticipated labeled dose. Data were collected approximately one hour (-60) and 5 (-5) minutes before (baseline) Alfaxan® CD-RTU administration and at 1, 3, 5, 10, 15, and 30 minutes after dosing and subsequently at 10 minute intervals until the cat regained sternal recumbency. Arterial pH and blood gas data were recorded at -5 and 1, 5, 15, and 30 minutes after dosing and subsequently at 10 minute intervals until the cat regained sternal recumbency.

Numerical variables were analyzed separately using analysis of variance for repeated measures, with dose as the independent variable. If differences were found, post

22 hoc analysis (Dunett's, Tukey's) was conducted to identify differences compared to baseline values and among dose groups. Categorical data were analyzed using non­ parametric procedures. A p<0.05 was considered significant.

23 2.2

RESULTS

Hematology (CBC) and serum biochemistry values were within normal values for all cats. There were no differences in any cardiovascular, respiratory, pH, or blood gas

(P02, PC02) baseline (-60, -5 minute) value (Tables 2.1-2.2). The administration of

Alfaxan® CD-RTU produced dose-dependent changes in cardiovascular, respiratory, pH, and blood gas (P02, PC02) values (Tables 2.1-2.2). Induction, maintenance, and recovery from anesthesia were uneventful. The duration of anesthesia increased with increasing doses of Alfaxan® CD-RTU.

Hemodynamic parameters-- Heart rate, arterial blood pressure, and cardiac output decreased after the administration of Alfaxan® CD-RTU. These effects were dose­ dependent and most pronounced in cats administered 50 mg/kg IV (Table 1). Heart rhythm remained sinus in origin and no cardiac arrhythmias attributable to the administration ofIV Alfaxan® CD-RTU were observed. Mean systolic arterial blood pressure was greater than 80 mmHg at all times in cats administered 5 mg/kg IV

Alfaxan® CD-RTU. Mean pulmonary artery pressure did not change following the administration of 5 and 15 mg/kg IV Alfaxan® CD-RTU (Table 2.2). In contrast, there was a marked decrease in systolic arterial blood pressure and a decrease in pulmonary arterial pressure in cats administered 50 mg/kg IV Alfaxan® CD-RTU. Systolic arterial

24 blood pressure did not return to a mean value greater than 80 mmHg for approximately

15-30 minutes (Table 2.2). Mean right atrial pressure did not change following the administration of any dose ofIV Alfaxan® CD-RTU. Systemic vascular resistance and pressure rate product decreased following the administration of the 15 and 50 mg/kg doses (Table 2.2). Systemic vascular resistance values returned to baseline values between 30 and 100 minutes. The RPP was decreased until recovery from anesthesia was complete (Table 2.2).

Respiratory, pH, and blood gas parameters and temperature-- Baseline (-60, -5 minute) values for respiratory rate, tidal volume, minute volume and arterial pH, P02,

PC02, bicarbonate and core body temperature were within normal limits for cats (Table

2.3). Respiratory rate and minute volume decreased after the administration ofIV

Alfaxan® CD-RTU (Table 2.3). Tidal volume increased compared to baseline values following the administration of 5 mg/kg, increased minimally after the administration of

15 mg/kg, and decreased for 15 minutes after the administration of 50 mg/kg IV

Alfaxan® CD-RTU (Table 2.3).

The administration of Alfaxan® CD-RTU produced dose-dependent decreases in arterial P02 that persisted for 15-30 minutes in cats administered 5 mg/kg and were most pronounced, lasting from 30-60 minutes, in cats administered 15 and 50 mg/kg IV

Alfaxan® CD-RTU, respectively. The duration of apnea was dose-related and was most prolonged in cats administered 15 (2 of 8) and 50 mg/kg. One of the 8 cats receiving 5 mg/kg IV Alfaxan® CD-RTU demonstrated a period of apnea lasting approximately 3 minutes. Cats with an arterial P02 less than 60 mmHg were administered 100% oxygen and controlled ventilation until the arterial P02 increased to greater than 80 mmHg. Five

25 of 8 cats administered 5 mg/kg IV Alfaxan® CD-RTU, 7 of 8 cats administered 15 mg/kg oflV Alfaxan® CD-RTU, and 7of7 cats administered 50 mg/kg IV Alfaxan® CD-RTU required supplemental oxygen administration.

The arterial PC02 increased after the administration of 50 mg/kg IV Alfaxan®

CD-RTU, although the increase was not considered to be clinically significant. Arterial pH and bicarbonate were decreased at the 50 mg/kg dose, but returned to baseline values by 90-100 minutes (Table 2.3). Core body temperature remained within normal values

for cats administered 5 and 15 mg/kg of Alfaxan® CD-RTU, but decreased for the

duration of anesthesia in cats administered 50 mg/kg Alfaxan® CD-RTU. Core body temperature remained within normal values for anesthetized cats and returned to baseline values during recovery from anesthesia.

Induction, Maintenance and Recovery Criteria; Response to Noxious Stimuli-­

The quality of induction, maintenance, and recovery from anesthesia was considered to

be good to excellent following the administration of IV Alfaxan® CD-RTU, as evidenced

by a smooth transition to lateral recumbency and complete muscle relaxation with

minimal or no response to noxious stimuli (Table 2.4). Induction to anesthesia was

characterized as quiet, uneventful, and relaxed. All three doses of IV Alfaxan® CD-RTU

were administered over one minute, making the time to lateral recumbency inversely

proportional to the dose of Alfaxan® CD-RTU administered. The average times to lateral

recumbency ranged from approximately 15-30 seconds. Cats administered the 5 mg/kg

dose took longer to intubate than those administered larger doses (Table 2.4). Larger

doses of Alfaxan® CD-RTU produced longer average durations of anesthesia and

unresponsiveness to a noxious stimulus (Table 2.4). The qualities of induction and

26 maintenance of anesthesia were excellent across all doses. Recovery scores for the 5 and

15 mg/kg doses of Alfaxan® CD-RTU were excellent and not different from each other

(Table 2.3). Five of the 7 cats administered 50 mg/kg ofIV Alfaxan® CD-RTU did not recover from anesthesia after 5 hours and were euthanized. No cats demonstrated cardiac arrhythmias or signs of histamine release.

27 2.3

TABLES

Table 2.1: Description of anesthetic induction, recovery, and maintenance scores

Parameter Score Description Quality of No outward signs of excitement, rapidly assumes lateral recumbmcy, good musrular relaxation, Induction easily intubated within 60 seconds of finished dosing

2 Mild signs of excitement, some struggling, may or may not be intubated within 60 seconds of finished dosing

3 Hyperkinesis, obvious signs of excitement, vocalizaion, defecation, or urination, cannot be intubated at all

Quality of No tongue flicking or head shaking, maintains lateral recumbency and immobilization, minimal Maintenance muscle tremors or twitching, no response to noise

2 Occasional tongue flicking and head shaking, frequent movement, short duration oflateral recumbency and numerous attempts to rise after assuming lateral recumbency, some muscle tremors and twitching

3 Constant tongue flicking and head shaking, does not become laterally recumbent or assumes lateral recumbency briefly, muscle rigidity accompanied by twitching, vocalization, and defecation, responds to noise

Quality of Assumes sternal recumbency with little or no struggling and may attempt to stand and walk with Recovery 1ittle or no difficulty

2 Some struggling, requires assistance to sternal recumbency or standing, responsive to external stimuli, becomes quiet in sternal recumbency

3 Prolonged struggling, unable to assume sternal recumbency or diffirulty in maintaining sternal or standing position, becomes hyperkindic when assisted, prolonged paldling and swimming motion

28 Table 2.2: Hemodynamic average and standard deviation values for cats Variable Time (minutes) Dose (mg/kg) -60 -5 1 5 10 15 30 60 90 120 150 HR 0 203 ± 32 214 ± 2S 5 201±14 205 ± 20 199±241• IS6 ± 33 IS7 ± 30 IS7 ± 39 17S ± 15 177 ± 25 15 213 ± 27 203 ±IS IS3 ± 1s1 173 ± 27 171±22 177 ± 19 177 ± 19'" 192 ± 37• 1S7 ± 20 1S4 50 215 ± 31 193 ± 35 165 ± 24• 166 ± 43 157 ± 34 143 ± 35 141±24'" 159 ± 19'" 161±19 167 ± 23 165 ± 39 SAP(mmHg) 0 174 ± 45 167 ± 26 5 153 ± 29 161±37 129 ± 39• 130 ± 60 130 ± 36. 131 ±45. 142 ± 71 153 ± Sl 15 170 ± 32 171±16 89 ±27 117 ±46'" !03 ± 23'" 117±17'" 127 ± 33 145 ± 23t 13S ± 33 201 50 165 ± 24 151 ±47 69 ± 27• 51±24'" 64 ± 21·• 71±15"'" 99 ± s 131±17t 135 ± 34 144 ± 23 13S ± 37 DAP(mmHg) 0 124 ± 27 124 ± 24 5 116 ± 19 124 ± 31 94 ± 36. 90 ± 33 SS± 32• S3 ± 20· S2 ± 17 Sl ± 23 15 130 ± 32 129 ± 15 60 ± 30 S3 ± 44• 66 ± 16'" so± 15'" S5 ± 22'" 9S ± zot S5 ± 12 91 50 107 ± 9 97 ± 2S 41±16. 30 ± 24'" 33 ± 21 ·• 37 ± 20·• 55 ± 13'" SI± 21t S2 ±2S S4 ± 21t 77 ± 32 MAP(mmHg) 0 146 ± 29 144 ± 17 N 5 132 ± 17 139 ± 2S IOS ± 37• lOS ± 42 !06 ± 34• 102 ± 26· 106 ± 3S I IO± 45 \0 15 14S ± 27 149 ± 6 71±27 97 ± 45• so± 14'" 96 ± 12'" 103 ± 25 111±1st IOS ± lS 134 50 133 ± 12 122 ± 33 53 ± 19• 40 ± 22• 42 ± 19•• 47 ± ls·• 72 ± 101 102 ± 19t !05 ± 29 109 ±1st 102 ± 31 PAP(mmHg) 0 16 ± 5 14 ± 6 5 15 ± 6 15 ±6 15 ± 6 15 ± 7 15 ± 6 12 ±3 15 ±4 15 ±I 15 lS ± 6 17 ±3 15 ±3 16 ± 7 14 ±3 15 ± 3 14 ± 6 15 ±4 12 ± 3 IS 50 16 ± 5 15 ± s 15 ± 7 12 ±6t II± 5 II ±6 13 ±4 16 ± 5 16 ±4 17 ±4 15 ±4 CO (mL/kg/min) 0 52S ± 66 556 ± 131 5 529 ± 160 53S ± 170 470 ± 15S 463 ± 114!• 441±105• 394 ± 57" 394 ± 2S 397 ± 33 15 503 ± 117 53S ± 149 397 ± 113 369 ±I IS1 431±135'" 432 ± 126'" 39S ± 113 417 ± 55 403 ± 75 435 50 45S ±IOI 427 ± 147 35S ± 121 274 ± 67. 251±49•• 2S5 ± 57•• 402 ± 52 431±77 414 ± 66 37S ± 47 3SO ± 72 5 SVR ( dynes•sec•cm- ) 0 493S ± 1346 4999 ± 2016 5 4S25 ± IS30 4627 ± 1046 4S06 ± 2302 4719 ± 2420· 4535 ± 2os1• 471S ± 2040• 4560 ± 1622 4250 ± 1925 15 5456 ± 17S2 5371 ± 212S 3595 ± 2672 504S ± 3322 3776 ± 2156 4320 ± 1S49'" 5001 ± 244ot 5173±1706 450S ± 949 5154 50 5313±1543 5262 ± 2229 3132±2472 2511±1502' 2992 ± 2072• 3122 ± 2472•• 3109 ± 602 4117 ± 793 4347 ± 709 5026 ± 77S 4737 ± 1763 RPP (bpm• mmHg) 0 29770 ±7409 30604± 3659 5 26543 ±3766 2SSl5± 7730 21252± S067' 20342 ± I 0446 19S9S ±7014• 19713 ± S310· 19004 ±704S I 9S7S±I0573 15 3093S ±40S4 30195± 3116 13161± 5926 17170 ± S904'" 1407S± 3952'" 16980± 2942 .. I S36S± 5633 .. 2276S ±7047t• 201S6±4731 24656 50 2S41l±4325 23724 ±S007 S76S± 3236. 6424 ± 3636'" 6382 ± 267s·• 6895± 3691 ... 1041S ± 3159'" 16464 ± 45511• 17252±6261 IS576± 5329 17367± Sl29

1 Significant difference from minute I during a dose I Significant difference between 5 mg/kg and 15 mg/kg doses for a reading • Significant difference between 5 mg/kg and 50 mg/kg doses for a reading '"Significant difference between 15 mg/kg and 50 mg/kg doses for a reading Table 2.3: Respiratory and blood gas average and standard deviation values for cats Variable Time (minutes) Dose (mg/kg) -60 -5 1 5 10 15 30 60 90 120 150

RR(bpm) 0 39 ± 13 52 ± 36 5 38 ± 16 55 ± 28 12 ±9 11 ± 8° 9 ± 6° 11±7 18 ± 5 24 ±0 15 37 ± 18 41±19 6±5 8 ± 6"' 12 ± 9 12 ± 5"' 17 ± 12 17 ± 61 18 ± 6 36 50 41±7 51±24 0±0 2 ± 6""' 2 ± 6° 2 ± 6"' 5±7 13 ± 111 10 ± 5 13 ± 7 17 ± 9 Tidal Volume (mL) 0 39 ± 20 25 ± 12 5 32 ±II 30 ± 15 60 ± 52 81±73 72 ±41 75 ±47 44 ±23 29 ± 16 15 31±7 28 ± 16 27 ± 18 43 ±21 33 ± 18 34 ± 14"' 47 ±26 44 ± 17 35 ± 24 60 50 31±14 22 ± 8 16±4 16 ±4 18 ± 8 19 ±9 24 ±II 44 ±33 41±17 42 ± 17 38 ± 17 Minute Volume(mL) 1376 ± 1052 0 1518 ± 872 1574 ± 1052 494 ± 380 792 ± 799 5 1278 ± 640 1140±760 213 ±282 303 ± 170 15 1104 ± 564 1212 ± 888 0±0 57 ± 151 606 ± 428 1098 ± 1184 878 ± 702 696 ± 373 50 1285 ± 727 422 ± 433 357 ± 115"' 918 ± 1263 735 ± 406"' 580 ± 444 2160 80 ± 212 91±242"' 169 ± 224 361±198"'1 376 ± 208 484 ± 2541 526 ± 220 pH w 0 7.375 ± 0.046 NA NA 0 5 7.387 ± 0016 NA 7.335 ± 0.033 7.310 ± 0.090 NA 7 .335 ± 0.087 7 .309 ± 0.093 7.372 ± 0.048 15 7.362 ± 0.046 NA 7.334 ± 0.041 7.301 ± 0.080 NA 7 .279 ± 0.074 7.328 ± 0.056 7.395 ± 0.047 7.368 ± 0.040 7.423 50 7.384 ± 0.036 NA 7.296 ± 0.066 7.261±0.035 NA 7.164 ±0.096 7.170 ± 0.129 7 .270 ± 0.068 7.293 ± 0.067 7.343 ± 0.052 7.383 ± 0.047

pC0 2 (mmHg) 0 28.9 ± 5.2 NA NA 5 25.9 ± 1.8 NA 30.3 ± 4.8 34.1 ±JO.I NA 30.0 ± 10.9 29.7 ± 6.9 31.9 ± 1.6 15 26.2 ± 3.2 NA 29.3±3.1 33.6 ± 9.6 NA 33.2 ± 8.6 27.9 ± 6.9 22.8 ± 5.21"' 26.9 ± 6.9 26.0 50 26.7±4.1 NA 33.7 ± 6.7 37.8 ± 6.7 NA 47.7±11.4 49.9 ± 19.7 37.1 ± 8.7"' 34.8 ± 8.1 32.5 ± 8.0 26.7 ± 6.6 0 p0 2 (mmHg) 0 98.6 ± 16.2 NA NA 5 101.4 ± 6.4 NA 59.2 ± 12.2 64.8 ± 10.4 NA 81.1 ± 23.6 125.0 81.5 ± 2.0 15 94.6 ± 10.4 NA 58.8 ± 25.1 61.5±29.0 NA 73.5 ± 22.7 77.5 ± 12.9 89.8 ± 17.6 84.0 ± 20.4 62.2 50 99.6 ± 16.0 NA 35.5 ± 17.6 64.3 NA 80.9 89.9 ± 25.2 90.0 ± 20.8 90.0 ± 14.3 89.0 ± 16.4 93.8 ± 12.0 cHC0 3 (P, st)c 0 18.6 ± 1.7 NA NA 5 17.9 ± 1.2 NA 17.1±2.2 17.0 ± 1.3 NA 16.7 ± 2.1 16.0 ± 2.1 19.6 ± 1.6 15 17.1±2.1 NA 16.7 ± 1.7 16.6 ± 2.2 NA 15.8±2.4 15.9 ± 1.9 16.3 ± 1.6 17.0±1.4 19.2 50 18.1±1.5 NA 16.5 ± 1.9 16.7 ± 1.2 NA 15.3±1.7 15.5 ± 1.8 16.7 ± 1.8 17.0 ± 2.0 18.0 ± 1.6 17.7 ± 1.7 Temperature (0 C) 0 37.9 ± 0.9 38.2 ± 0.8 5 38.2 ± 0.6 38.3 ± 0.6 37.8 ± 0.7 37.8 ± 0.6 37.7 ± 0.6 37.6 ± 0.7 37.6±0.1 37.6 ± 0.5 15 38.1 ± 0.4 38.2 ± 0.4 38.0 ± 0.3 37.8 ± 0.4 37.7 ± 0.4 37.5 ± 0.4 37.4 ± 0.41 37.3 ± 0.51 37.6 ± 0.3 37.4 50 38.4 ± 0.6 38.4 ± 0.4 38.0 ± 0.4 38.0 ± 0.5 37.9 ± 0.5 37.8 ± 0.6 37.3 ± 0.7' 36.8 ± 0.81 36.6 ± 1.1 36.5 ± 1.2 36.6 ± 1.4 f Significant difference from minute I during a dose •Significant difference between 5 mg/kg and 50 mg/kg doses for a reading "'Signficant difference between 15 mg/kg and 50 mg/kg doses for a reading °Calculations based on cats breathing room air Table 2.4: Summary of time-related and categorical, descriptive variables for Alfaxan­ CD R TU® anesthesia in cats Dose 5 mg/kg 15 mg/kg 50 mg/kg Parameter Average± Standard Deviation Time until Lateral• 33 ± 24 20±4 17 ± 3 Time until Intubationb 27 ± 24 19± 7 13 ±30 Duration of Non-responsiveness' 17.76±9.45 54.68 ± 23.89 103.92 ± 56.21 Duration of Anesthesiad 26 ± 10.7 82.92 ± 23.92 125.55 ± 66.29 Duration of Initial Apnea• 0.35 ± 1.00 1.05 ± 2.01 28.27 ± 16.24 Duration of Initial Oxygenation• 9.37 ± 3.99 9.41±5.36 29.64 ± 14.14 Quality of Inductionr 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 Quality of Anesthesiar 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 Quality of Recoveryr 1.0 ± 0.0 I.I± 0.4 1.5 ± 0.7 Overall Anesthesia Score 3.0± 0.0 3.1±0.35 3.5 ± 0.71

• Seconds from start of injection b Seconds from end ofinjection 'Minutes from induction d Minutes from intubation to extubation •Minutes r Scored from 1-3 (description in Table 1)

31 CHAPTER3

CARDIORESPIRATORY AND ANESTHETIC EFFECTS OF ALFAXALONE IN~­ CYCLODEXTRIN IN THE CANINE

3.1

MATERIALS AND METHODS

Eight adult purpose-bred mixed breed dogs (4 male; 4 female) were used for this study.

The dogs were between 10 and 30 kg body mass, eight months and ten years of age, not pregnant, and judged to be healthy based on unremarkable physical examination, electrocardiography, serum chemistry, and hematology analyses performed prior to experimentation. Serum chemistry analysis included assessment of total protein, albumin, sodium, potassium, chloride, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, creatinine kinase, creatinine, and blood urea nitrogen. Dogs were acclimated for 7 days prior to experimentation, during which time no medications or vaccinations were administered. The experimental protocol was approved by The Ohio

State University Institutional Laboratory Animal Care and Use Committee.

Experimental Preparation and Procedures:

The day before the administration of the test article (Day -1 ), all dogs were

anesthetized and surgically prepared for placement of right carotid artery and jugular vein

catheter introducers (Arrow International, Inc., Reading, PA, USA). Anesthesia was

induced with propofol (6 mg/kg IV once; PropoFlo™, Abbott Laboratories, North

32 Chicago, IL, USA) and maintained with isoflurane (2-3%; IsoFlo™, Abbott

Laboratories) in oxygen. The catheter introducers were flushed with heparinized saline, subcutaneously tunneled to exit the side of the neck, sutured in place with 1-0 silk, and bandaged. Local anesthetic (Ropivacaine HCl; Naropin™, AstraZeneca

Pharmaceuticals, Wilmington, DE, USA) was administered at the surgical site to reduce pam. An Elizabethan collar was placed prior to returning dogs to their cages.

On the day of the experiment (Day 0), dogs were returned to the laboratory and their bandages removed. A 7F Swan-Ganz thermodilution catheter (American Edwards

Laboratories, Irvine, CA, USA) was advanced into the right jugular vein through the catheter introducer and positioned such that its distal tip was 1-2 cm distal to the pulmonary valve. A fluid-filled catheter was advanced into the right carotid artery through the introducer and positioned so that its distal tip was in the ascending aorta.

Both catheters were electronically connected to the data acquisition system (PO-NE­

MAH Data Acquisition Computer; Gould Instruments Systems Inc, Valley View, OH,

USA) to allow continuous analog display and digital recording of data. Lead II electrocardiogram (PO-NE-MAH) and body temperature (Cardiac Output Computer,

American Edwards Laboratories) were continuously recorded for each dog. A hand-held volumeter (Medishield, Fraser Harlake, Orchard Park, NY, USA) was attached to a tight­ fitting facemask and placed over each dog's muzzle to measure respiratory rate and tidal volume. The same instrument was attached to the end of an endotracheal tube for continued measurement of respiratory rate and tidal volume following administration of the test anesthetic.

33 Heparinized anaerobic blood samples were collected from the right carotid artery catheter for arterial blood pH and blood gas analysis, including Pa02 and PaC02 (Model

ABL 500; Radiometer America, Inc., Westlake, OH, USA). Heart rate (beats/min) and rhythm, left ventricular end-diastolic pressure (L VEDP; mmHg), aortic systolic, diastolic, and mean blood pressures (mmHg), pulmonary artery and right atrial mean blood pressures (mmHg), cardiac output (mL/kg/min; thermodilution), respiratory rate

(breaths/min), tidal volume (mL), minute volume (mL/min), core temperature (°C), arterial pH, and blood gas (PaC02, Pa02; mmHg) analysis were recorded before and at predetermined times after test article administration. Systemic vascular resistance (SVR;

5 dynes/second/cm- ) and rate pressure product (RPP; beat/mmHg) were calculated prior to and following drug administration.

The times to first head lift, removal of the endotracheal tube, and return to sternal recumbency (minutes) were recorded. Two noxious stimuli were used to determine analgesia. First, a toe pinch using a 10 cm Rochester-Pean hemostat was applied to the second digit (middle phalanx) of the left rear leg for 30 seconds or until limb withdrawal.

In addition, two needle electrodes (Grass Medical Instruments, Quincy, MA, USA) were placed 1 cm apart in the buccal mucosa of the dog's mouth. The wire leads from the electrodes were attached to a stimulator preset to deliver a series of 10 ms, 5 Hz, 50 V electrical pulses for 30 seconds (Grass SD-9 Stimulator, Grass Medical Instruments).

Each dog's responses to toe pinch and buccal mucosal stimulation were categorically rated as: no response = O; minimal movement= 1; limb withdrawal = 2; limb withdrawal and lifting of the head = 3. The quality of anesthetic induction, maintenance, and

34 recovery were also determined categorically for each dog after anesthetic administration, as described in Table 2.1.

The duration of apnea was calculated as the difference in minutes and seconds between the onset of lateral recumbency and the first inspiratory effort. If positive­ pressure ventilation was used (initiated when Pa02 <60 mmHg), the duration of apnea was calculated as the difference in minutes and seconds between the onset of lateral recumbency and the onset of positive pressure ventilation.

Experimental Plan:

The study was conducted as a blinded four-way crossover randomized by dose.

Three intravenous doses (2, 6, and 20 mg/kg) of alfaxolone in cyclodextrin (Alfaxan®-CD

RTU, Jurox Pty. Ltd., Rutherford, NSW, Australia) or a control (0.9% NaCl; Baxter

Healthcare Corporation, Deerfield, IL, USA) were administered blinded and in random order. Each dose was separated by a three hour washout period. The doses and washout interval were chosen based on the selection of 2 mg/kg IV Alfaxan®-CD RTU as the labeled dose in the dog, with the 6 and 20 mg/kg doses equivalent to 3 and 10 times the anticipated dose, respectively. Each dose was infused over 60 seconds. The time of dosing was recorded as the time at which the administration of each dose was finished.

All data were collected one hour (-60 minutes) and 5 minutes (-5 minutes) before dosing and at 1, 5, 10, 15, and 30 minutes after dosing. Thereafter, data were collected at 10 minute intervals until the dog was responsive to noxious stimuli (score greater than 2).

Arterial pH and blood gas values were recorded at -5, 1, 5, 15, and 30 minutes after dosing and repeated at 10 minute intervals until the dog responded to a noxious stimulus.

After administration of Alfaxan®-CD RTU, all dogs were intubated and allowed to

35 breathe 100% oxygen. Positive pressure ventilation was instituted with 100% oxygen at

6 breaths/min if Pa02 fell below 60 mmHg and maintained until spontaneous breathing supported a Pa02 >80 mmHg.

Dogs were removed from the study if concomitant therapies were administered, or if an adverse drug experience occurred that required interruption of treatment, or if death occurred.

Statistical Analysis:

Numerical variables were analyzed separately using analysis of variance for repeated measures, with dose as the independent variable. If differences were found, post hoc analysis (Dunett's, Tukey's) was performed to identify differences compared to baseline values and among dose groups. Categorical data were analyzed using non­ parametric procedures. A p<0.05 was considered significant.

36 3.2

RESULTS

All dogs finished all phases of the study and were judged to be in excellent health based upon the results of a physical examination, ECG, and CBC the day after study.

There were no differences in any cardiovascular, respiratory, pH, or blood gas (Pa02,

PaC02) value at baseline-60 and baseline-5 minutes (Tables 3.1-3.2). The administration of Alfaxan®-CD RTU produced dose-dependent changes in cardiovascular, respiratory, pH, and blood gas (P02, PC02) values (Tables 3.1-3.2). The duration of anesthesia was increased with increasing doses of Alfaxan®-CD RTU (Table

3.3). Induction, maintenance, and recovery from anesthesia were judged to be good to excellent (Table 3.3).

The administration of Alfaxan®-CD RTU increased heart rate and produced dose­ dependent decreases in systemic (systolic, diastolic, mean) and mean pulmonary arterial pressures. Cardiac output and mean right atrial pressure did not change. The 2 mg/kg IV dose caused minimal decrease and the 6 and 20 mg/kg doses caused more apparent decreases in systemic vascular resistance and the rate-pressure product. These changes were most apparent 10 minutes after drug administration. Most hemodynamic variables returned to within baseline values by 15 minutes (2 mg/kg IV) and 30 minutes (6, 20 mg/kg IV) after drug administration. The mean pulmonary artery pressure remained

37 decreased for over 60 minutes following the 20 mg/kg dose. This dose produced consistent decreases in all hemodynamic variables except heart rate. Several cardiovascular variables increased above baseline values during recovery from anesthesia. Changes in hemodynamic variables are summarized in Table 3.1.

The administration of Alfaxan®-CD RTU produced dose-dependent decreases in respiratory rate, minute volume, and Pa02. This effect was most pronounced after the administration of the 20 mg/kg IV dose and persisted for approximately 15 minutes.

Tidal volume remained relatively unchanged from baseline-5-minute values. Some dogs demonstrated variable, dose-dependent periods of apnea lasting for 1 to 3 minutes after administration of the 6 and 20 mg/kg doses (Table 3.3). Apnea was more frequent in dogs administered higher doses (Table 3.3). The PaC02 did not change after the administration of the 2 mg/kg dose, but increased after the administration of higher doses.

Dose-dependent decreases in pHa were most pronounced between 5 and 15 minutes after the administration of the 20 mg/kg IV dose, and returned to within baseline values by 50 minutes. The base excess did not change following any dose of Alfaxan®-CD RTU.

Respiratory and blood gas values are summarized in Table 3.2.

The qualities of induction, maintenance, and recovery from anesthesia were judged to be good to excellent following the administration of Alfaxan®-CD RTU.

Transition to lateral recumbency and recovery from anesthesia were smooth, excitement­ free and uneventful. All doses were administered over one minute, with time to lateral recumbency inversely proportional to the dose administered. Induction to anesthesia was characterized by quiet, uneventful relaxation to sternal, then lateral recumbency in all but one dog. The average time to lateral recumbency for the 2, 6, and 20 mg/kg doses was

38 0.9 ± 0.3, 0.5 ± 0.1, and 0.5 ± 0.4 minutes from the beginning of drug injection, respectively (Table 3.3). One dog experienced a longer time to lateral recumbency (1.6 minutes) after being administered the 20 mg/kg dose. The range of induction times in the remaining dogs was 0.3-0.5 minutes. There were no significant differences among the three drug doses for time to intubation. One dog demonstrated a longer time to intubation after administration of the 2 and 20 mg/kg doses of Alfaxan®-CD RTU. This dog also displayed a brief period (> 10 seconds) of head shaking immediately after the 2 mg/kg dose. The dog relaxed and within 10-15 seconds was easily intubated. Quality of induction scores are summarized in Table 3.3.

The maintenance phase of anesthesia was characterized by good muscle relaxation and little or no response to noxious stimuli. The anesthetic maintenance score improved with increases in drug dose. The duration of anesthesia was 9.3 ± 2.9, 32.0 ±

7 .1, and 69. 7 ± 23 .5 minutes after the administration of 2, 6, and 20 mg/kg doses of

Alfaxan®-CD RTU, respectively. Recovery scores for the high and low doses were not different from each other, 1.4 ± 0.5 and 1.3 ± 0.5, respectively, but were different from 6 mg/kg dose, 1.6 ± 0.5. Quality of maintenance and recovery scores are summarized in

Table 3.3.

39 3.3

TABLES

Table 3.1: Hemodynamic average and standard deviation values for dogs Time (minutes) Variable -S l 10 15 __Q_o~- (mg/kg) -60 s 30 60 90 -----~-- HR(bpm) 0 146 ± 25 124±21t 2 126± 13 120±21 155 ± 18 143±17 148±7 6 124± 26 112±28 158 ± 33t 150 ±20t 144±18t 137 ± 22t 128 ± 3gt 20 127 ±28 128 ± 33 162 ± 16t 131±9t 139 ± 19t 134 ± 12t 128 ± 15t 12s ± 2st 170± 12 SAP (mmHg) 0 152± 14 145± 19 2 140± 11t 143 ± 14t 139±24t 133 ± 13t 122 ±25 1 6 152± 11t 130 ± 14t 145 ± 26tt 122 ± 23tl 125 ± 22tt 125 ± 16t· 123 ± 15t· 20 145 ± 11t 147± 1st 118±34tl 79 ± 12tt 93 ± 27tl 116 ± 19t· 121±19t· 131±22t 173 ±46 DAP(mmHg) 0 109±21 102± 15 2 99± 12 100±13 99± 15t 98± gt 84 ± 1s1 6 112± 14 100± 12 108 ± 3ot 87± 1st 93± 161 94± 12· 92±6" 20 101±14 103 ± 15 77 ± 2gtt 56 ± 14tt 67 ± 25tl 83 ± 12t· 87 ± 14t· 91±1lt 132±23 MAP(mmHg) ~ 0 126± 15 120 ± 12 0 2 117±9 120± 12 11s ±1st 114 ± 91 100 ± 2ot 6 128± 10 113 ± 12 122 ± 2gtl 101±1gtt 107 ± 17tt 107 ± 12t· 105 ±gt· 20 121±11 123 ± 13 94 ± 2911 65 ± 13tt 77 ± 25tl 97 ± 13t· 102 ± 151" 112 ± 13t 146±31 PAP (mmHg) 0 20± 5 19±7 2 18 ± 3 17±5 16± 6 15±4 15 ± 7 6 16±5 21 ±4 15 ± 4t 14±5 1 13 ± 3t 13±4t 18 ± 3t 20 15 ±4 18±4 19 ± 61 12 ± 5t 13 ±5 1 13±6 1 13 ± 5t 12±4t 20±2 CO (mUkg/min) 0 240± 53 246±48 2 247±46 229± 60 245 ± 53 231±77 250 ± 30 6 240±44 218 ± 62 23s ± so1 222 ± 71t 222 ± 66t 247 ± 511 215 ± 781 20 267 ± 28 246± 71 221±6ot 190 ± 641 222 ± 56t 236 ± 411 228 ± 45t 196 ± 46 357 ± 66 ·Significant difference between 6 mg/kg and 20 mg/kg dose groups I Significant difference between all dose groups t Significant difference between times during a dose Table 3.2: Respiratory and blood gas average and standard deviation values for dogs Time (minutes) Variable Dose (mg/kg) -60 -5 I 5 IO 15 30 60 90 RR(bpm) 0 35 ±35 22± 8 2 33 ± 36 32 ±36 25± 14 20± 10 27±2 6 17 ± 7 17 ± 6 9±9 1" 13 ±gt· 16± 9t· 20 ± 2t· 30 ± 26t· 20 18±9 31 ±37 6±4 1" 9 ± 5t· 11±9t· 12 ±gt· 15 ± 11t· 19± IO 32±6 Tidal Vol. (mL) 0 204± 72 217 ± 55 2 228 ± 102 212±46 186 ± 117 210± 119 190 ± 125 6 223 ± 55 234 ± 78 198± 92 216 ± 139 254± 161 238 ± 101 208 ± 93 20 274 ± 69 230 ± 68 179±71 1 179±99 1 254 ±21st 296 ±205t 311 ± 17gt 246 ± 128 325 ± 25 Minute Vol. (mL) 8355 ± 11353 4740 ±2191 0 8083 ± 11382 5890 ± 5106 3739 ± 1778 3280 ± 732 4880 ± 2763 2 3918 ± 2063 3789 ± 1668 1526 ± 9861" 2318 ± 1049t· 3095 ± 1268t· 3790 ± 1049t· 4586 ± 2086t· 6 5665±3155 6008 ± 6186 1136 ± 759t· 1330 ± 594t· 1953 ± 145lt· 2520 ± 746t· 3363 ± 1517t· 3820 ± 886 10300 ± 707 20 ~ pH ...... 0 7.38 ± 0.02 ND ND 2 7.39 ± o.02t ND 7.36 ± o.o4t 7.38 ± 0.03t ND 6 7.39 ± o.02t ND 7.35 ± 0.01 t 7.33 ± o.03t ND 7.36 ± o.02t· 7.38 ± o.05t· 20 7.39 ± o.02t ND 7.35 ± o.02t 7.29 ± o.o5t ND 7.29 ± o.04t· 7.34 ± o.03t· 7.39 ± 0.01 7.41±0.01 pC0 2(mmHg) 0 32±2 ND ND 2 32± 2t ND 34±3 1 33 ± 31 ND 6 32 ± 31 ND 35 ± 71 39±4 1 ND 36±2t 33 ± 31 20 31±3 1 ND 39± 21 43 ± 51 ND 43 ± 71 36±4 1 32±2 28±3 p02(mmHg) 0 100±4 ND ND 2 109 ± 1811 ND 79 ±JOI! 87 ± 15 ND 6 97 ± 9t! ND 58 ± 131! 73 ± 10 ND 89 ± 9t· 94 ± 9t· 20 99±4 11 ND 43 ± 171! 352± 151' ND 87 ± 27t· 101±20t· 107±21 95 ± 5 Significant difference between times during a dose 1 Significant difference between all dose groups · Significant difference between 6 mg/kg and 20 mg/kg dose groups

'All dogs were on I 00% 0 2 during this measurement ND= not done Table 3.3: Summary of time-related and categorical, descriptive variables for Alfaxan­ CD RTU® in dogs

2 mg/kg 6 mg/kg 20 mg/kg Parameter Average± Standard Deviation Time until Lateral" 0.9 ± 0.3 0.5 ± 0.1 0.5 ± 0.4 Time until Intubationb 0.7± 1.1 0.4 ± 0.4 0.6 ± 1.0 Duration of Apnea• 0.5 ±0 0.7 ± 0.3 1.0 ± 0.7 Number of dogs with Apnea 5 8

Duration of Anesthesia d 9.3 ± 2.9 32.0±7.1 69.7 ± 23.5 Time until Extubationb 9.8 ± 2.4 31.4 ± 6.9 75.1±18.9 Time until Sternalb 18.6 ± 10.4 39.5 ± 8.4 84.4± 17.8 Quality of Induction• 1.3 ± 0.5 1.0 ± 0 1.0 ± 0 Quality of Maintenance• 2.0 ± 0.5 1.4 ± 0.5 1.0 ± 0 Quality of Recovery• 1.4 ± 0.5 1.6 ± 0.7 1.3 ± 0.5

• Minutes from start of injection b Minutes from end of injection

42 CHAPTER4

DISCUSSION AND CONCLUSIONS

4.1

OVERVIEW

Alfaxan® CD-RTU produced anesthetic effects typical of hypnotic anesthetics, with a rapid loss of consciousness, loss of the swallowing reflex, and marked muscle relaxation. Anesthesia was of relatively brief duration at recommended doses for cats and dogs. The characteristics of anesthetic maintenance and recovery were good to excellent in nearly all cases, with the highest dose tested in the cat alone exhibiting unacceptably prolonged anesthesia. Moreover, Alfaxan® CD-RTU resulted in anesthesia with a relatively wide therapeutic index, particularly in dogs (up to 10 times the recommended dose).[3, 5, 6] Cardiovascular and respiratory depressant effects were comparatively mild, even at supraclinical doses. Significantly, no histaminoid reactions were observed following drug administration in either species, a notable improvement when Alfaxan® CD-RTU is compared to previous alfaxalone formulations.

In the following sections, the cardiovascular, respiratory, and anesthetic qualities of Alfaxan® CD-RTU will be compared to other commonly used injectable anesthetics, including propofol, thiopental, and ketamine in the dog and cat. The species-specific characteristics and therapeutic index of Alfaxan® CD-RTU will also be reviewed.

43 4.2

CARDIOVASCULAR EFFECTS OF ALF AXAN-CD RTU®

The effects of anesthetic drugs on cardiovascular function are frequently assessed by evaluation of heart rate and rhythm, arterial blood pressure, and cardiac output.

Cardiac output is dependent upon heart rate, venous return (preload), vascular impedance

(resistance+ reactance; afterload), and cardiac contractile force. Increases in heart rate, preload, and cardiac contractile force and decreases in afterload all result in increased cardiac output.

All currently available injectable anesthetic drugs except the dissociative anesthetics (ketamine, tiletamine) produce mild to marked cardiorespiratory depression at clinically relevant anesthetic doses.[6] Ketamine produces increases in heart rate, arterial blood pressure, and cardiac output when administered at low dosages (5 mg/kg).[38-40]

The mechanisms for cardiac stimulation following ketamine administration are uncertain, but likely relate to central nervous system and arterial baroreceptor effects.[41]

Peripherally, ketamine may inhibit the intraneuronal uptake of catecholamines, with increased norepinephrine levels detectable in blood after ketamine administration. [41]

However, ketamine dosages exceeding 10-15 mg/kg produce tachycardia and transient decreases in arterial blood pressure.[7] This hemodynamic depression has been linked to ketamine-induced inhibition of somatosympathetic reflexes in the medulla oblongata and spinal cord, whereas lower doses activate supra-midbrain regions.[42] Ketamine tends to

44 increase myocardial oxygen consumption, cardiac work, and pulmonary vascular resistance, making its use in critically ill patients controversial. [41]

The effects of thiopental on cardiovascular parameters are variable. Generally, thiopental results in a reduction in MAP and CO following anesthetic induction. Its effects on the myocardium are not well-defined, but it may affect myocardial contractility by reducing ventricular filling and depressing sympathetic outflow.[43] Canine studies do not support myocardial depression as an important factor in the circulatory depression associated with thiopental.[43] Thiopental administration can result in catastrophic circulatory depression in the face of relative or absolute hypovolemia, as blood volume is redistributed to the periphery through relaxation in the tone of venous capacitance vessels and impairment ofbarostatic reflexes.[43] Consistent with this, administration of thiopental in dogs was shown to increase heart rate and mean arterial pressures (transient) and to decrease stroke volume.[44] This effect was attributed to a decrease in vagal efferent tone and depressed baroreceptor reflex activity.[44] A study comparing the anesthetic activity of propofol and thiopental in dogs found significant increases in heart rate after thiopental administration compared to propofol and a tendency for heart rate to be increased compared to control values.[45] Mean arterial blood pressure was increased at 2 minutes following thiopental administration and decreased at 60 minutes compared to other time points.[45]

Propofol has been shown to decrease systolic, mean, and diastolic blood pressures by 25-40% following a 2.5 mg/kg dose in humans.[46] Similar to thiopental, hypotension is worsened in volume-depleted patients and is most likely related to vasodilation. Vasodilation after propofol administration is due to a decrease in

45 sympathetic activity, a direct effect on smooth muscle calcium flux, and potentially increased nitric oxide release from vascular endothelium. [46] Propofol, like thiopental, may also inhibit baroreceptor reflexes. [46] Propofol administration in dogs did not result in significant alteration in heart rate, cardiac output, stroke volume, arterial blood pressure, or left ventricular work.[45]

The hemodynamic effects of Alfaxan® CD-RTU in cats were dose-dependant and similar to those of other hypnotic anesthetics.[7, 9, 47] The targeted clinical dose of

Alfaxan® CD-RTU (5 mg/kg IV) produced clinically irrelevant decreases in hemodynamic values that coincided with the loss of consciousness, consistent with anxiolysis and sedation. At this dose, small but significant decreases in systemic vascular resistance without changes in cardiac output or rate pressure product were noted, suggesting minimal changes in cardiac contractility and myocardial 0 2 consumption.

Larger doses (15 mg/kg and 50 mg/kg) produced decreases in heart rate, arterial blood pressure, cardiac output, and derived variables (systemic vascular resistance), suggestive of centrally-induced and direct cardiac depression and vasodilation.

Collectively, previous reports and our data suggest that the IV administration of a

5 mg/kg dose in cats causes mild vasodilatory effects resulting in minimal change in heart rate and cardiac output, while larger doses (> 15 mg/kg) cause vasodilation and negative inotrophic effects with subsequent decreases in arterial blood pressure and cardiac output.

More detailed analysis of load-dependent variables (preload, afterload) and evaluation of indices of cardiac contractility are necessary to determine the relative contributions of cardiac depression and vasodilation to these effects.

In dogs, all hemodynamic parameters remained within normal limits for dogs

46 administered 2 and 6 mg/kg doses of Alfaxan®-CD RTU. Cardiac output did not change or increased minimally after administration of2 and 6 mg/kg of Alfaxan®-CD RTU. Any effects were likely due to transient increases in heart rate and small decreases in peripheral vascular resistance (decreased afterload) secondary to peripheral vasodilation.

We did not assess cardiac contractility and are thus unable to determine its contribution to changes in cardiac output. We suspect that, if affected, contractility was minimally changed at the 2 and 6 mg/kg doses due to the relative stability of cardiac output.

Hemodynamic changes were more pronounced after the administration of 20 mg/kg, with the average systolic arterial blood pressure falling below 80 mmHg (79 ± 12) at the 5 minute recording, a value reported to be representative of hypotension in anesthetized dogs.[48] Heart rate was unchanged while cardiac output, rate pressure product, and systemic vascular resistance decreased to below baseline values during the same time period in our study, suggesting decreased cardiac contractile force and potentially decreased myocardial oxygen consumption. All hemodynamic changes returned to baseline values within 10 minutes after the administration of the 20 mg/kg IV dose of Alfaxan®-CD RTU, suggesting that peripheral vasodilation and decreased cardiac contractile force were causes for hypotension. As in the cat, additional evaluation of indices of cardiac contractility is necessary to determine its contribution.

Historically, investigators have reported mild to moderate (10-30%) decreases in arterial blood pressure in dogs and cats following IV doses (9-12 mg/kg) of an alphaxolone-containing drug combination (Saffan®) known to cause histamine release.[6-

9] The administration of the same product (Althesin®) to humans was associated with an initial decrease in systemic vascular resistance, followed by a delayed decrease in stroke

47 volume.[49] Unfortunately, the role of Cremophor® diluent-induced histamine release in the development of these effects cannot be determined from these studies.

48 4.3

RESPIRATORY EFFECTS OF ALFAXAN-CD RTU®

Respiratory depression and apnea are common following the administration of most intravenous anesthetics to cats and dogs.[50] This occurs as the slope of the minute volume-C02 curve is shifted down and to the right and inspiratory occlusion pressure, a measure of central nervous system respiratory drive, is decreased. [51] Respiratory depression has been linked to anesthetic depression of the cortex and brainstem, increases in the concentration of inhibitory GABA, and decreases in the amount of excitatory glutamate neurotransmitters in the central nervous system.[33]

Most anesthetic drugs that produce anxiolysis and anesthesia by interacting with

GABAA receptors are noted respiratory depressants.[34] Ketamine, an N-methyl-d­ aspartate (NMDA) receptor antagonist, does not produce significant respiratory depression unless a large dose is given rapidly, resulting in a transient decrease in respiratory rate.[41] After induction, minute ventilation is generally maintained.

Ketamine increases pulmonary compliance and decreases airway resistance due to increases in blood catecholamine levels and its direct smooth muscle relaxant effects. [41]

The effects of ketamine on pulmonary vascular resistance are variable, with some reports describing increased resistance and others suggesting a relaxant effect.[41] At a 5 mg/kg dose in cats, ketamine results in decreases in P02 and occasional increases in PC02.

These respiratory alterations are most likely due to the induction of an apneustic pattern 49 of breathing, leading to maldistribution of pulmonary blood flow and hypoventilation. [3 8-40] Ketamine dosages exceeding 10-15 mg/kg produce further decreases in P02 and increases in PC02. [7]

Thiopental and other barbiturate anesthetics are known to be potent central respiratory depressants. Thiopental administration to dogs at 19.4 mg/kg intravenously resulted in decreased Pa02 and pHa and increased PaC02 compared to baseline values at all measurement times in one study.[44] Similar respiratory depressant effects were also noted for thiamylal and methohexital, other short-acting barbiturate anesthetics.[44]

Thiopentone has been described as producing a biphasic effect on respiratory rate, with tachypnea noted during light anesthesia and progressive bradypnea with deepening anesthesia.[52]

Propofol is also known to reduce respiratory drive and diminish upper airway protective reflexes, limiting its safety with administration to non-intubated patients.[46]

Dogs receiving thiopental and propofol experienced apnea following induction, with dogs receiving propofol also demonstrating significant decreases in pH.[45] Studies investigating the respiratory effects of thiopental and propofol in cats suggest that apnea is a relatively common (60%) occurrence [9, 53, 54]. This effect has been attributed to the rate of drug administration, direct dose-dependent depression of CNS respiratory centers, and skeletal muscle relaxation.[47, 53, 54]

Respiratory depression occurred after all three doses of Alfaxan® CD-RTU in cats. We recorded moderate decreases in respiratory rate with increased or unchanged tidal volume following the administration of 5 and 15 mg/kg IV, respectively. P02 decreased rapidly in our cats, with minimal change in pH and PC02 from baseline values,

50 potentially due to altered distribution of pulmonary blood flow (ventilation-perfusion mismatch).

A dose-dependent increase in the frequency and duration of apnea was noted following the administration of Alfaxan® CD-RTU. One and two cats that were administered 5 and 15 mg/kg IV Alfaxan® CD-RTU, respectively, developed apnea, as did all 7 cats that were administered 50 mg/kg. We were unable to compare the frequency and duration of apnea we noted with studies comparing Saffan® with thiopental, propofol, or ketamine because the rates of administration in these reports could not be determined or were not reported.[7-9, 38, 40] Other authors have reported hyperventilation following Saffan® administration that could be attributed to hypotension and CNS stimulation or to CNS depression.[7] One study investigating the cardiorespiratory effects of anesthetic doses of several injectable anesthetics in cats suggested that Saffan® was less likely to produce respiratory depression than thiopental, methohexital, pentobarbital, and ketamine,[6] while another cautioned concerning the development of apnea.[8] Additional comparative trials will need to be conducted to determine whether Alfaxan® CD-RTU produces less respiratory depression and apnea than currently available injectable anesthetic regimens.

In the dog, 2 mg/kg IV Alfaxan®-CD R TU produced minimal changes in respiratory rate, variable effects upon tidal volume, and decreases in minute volume.

Minute volume remained within normal values for conscious dogs [51]. Apnea did not occur at this dose and PaC0 did not change, although Pa0 decreased minimally. The 2 2 administration of 6 and 20 mg/kg IV Alfaxan®-CD RTU did produce respiratory depression, with early decreases in respiratory rate and minute volume, increased

51 incidence of apnea, increased PaC0 , and decreased Pa0 . 2 2

Similar respiratory responses have been observed in normal dogs administered an anesthetic "induction dose" of 6 mg/kg IV thiopental or propofol. [5 5] We did not compare the anesthetic potency of Alfaxan®-CD RTU to other injectable anesthetics, but it is our opinion that it produced less respiratory depression than thiopental and propofol based upon the results of this and earlier studies. Clinical trials will need to be completed to further evaluate this issue.

52 4.4

ANESTHETIC PROPERTIES OF ALFAXAN-CD RTU®

Ketamine is a racemic phencyclidine derivative that acts primarily as an NMDA receptor antagonist, though it interacts with more than one receptor type.[41] At the

NMDA receptor, it non-competitively inhibits binding of glutamate to the phencyclidine site. The NMDA receptor mediates neuronal signaling and neuronal gene expression and is important in pain processing, neuronal plasticity, and induction and maintenance of central sensitization after nociceptive stimuli.[41] Thus, NMDA receptor antagonists play a role in preventing the development of pain states. Additionally, it binds toµ and K opioid receptors in the brain, spinal cord, and at peripheral sites, providing another of its analgesic effect. [41] This binding pattern may also provide a mechanism for the dysphoria often noted with ketamine. Ketamine also affects non-NMDA glutamate, nicotinic and muscarinic cholinergic, monaminergic, Na+, and Ca++ channels.[41]

In addition to its relative lack of cardiorespiratory effects, these properties make ketamine a useful adjunct to analgesic and anesthetic protocols in many situations.

However, the anesthetic properties of ketamine when used alone are less desirable.

Administration of ketamine is commonly accompanied by excessive salivation and lacrimation. Muscle relaxation is poor. Animals are hyperresponsive and ataxic during recovery, potentially a manifestation of emergence deliriums, a phenomenon that is

53 common after ketamine administration in humans.[41] Tremors, tonic spasticity, and convulsions can occur at larger doses.[50]

Thiopental and other barbiturates, propofol, and neurosteroids potentiate GABA receptor activation by increasing the receptor affinity for GABA.[30] These drugs accomplish this by increasing the open time of the er channel associated with the GABA receptor. These effects are most pronounced at low concentrations of GABA, with peak er currents elicited by high GABA levels essentially unaffected.[30] Barbiturates do not have significant antinociceptive effects at subhypnotic dosages.[50] Despite its classification as an ultrashort-acting barbiturate, thiopental is not rapidly cleared from the body, but instead relies on redistribution to lean body tissues to limit their duration of action followed by redistribution to fat.[50] Metabolism of barbiturates occurs over hours. Repeated doses, obesity, and use in sighthounds are associated with prolonged recovery times. Liver disease, hypothermia, and depressed cardiovascular function may also delay recovery from barbiturate anesthesia.[50]

Though its major effect is in the potentiation of the GABAA receptor, propofol has a number of other mechanisms of action that are less fully understood.[46] Propofol also depresses excitatory neurotransmission by inhibiting the NMDA receptor, modulates ea++influx through slow calcium channels, and inhibits voltage-gated Na+ channels.[46]

Unlike thiopental, propofol is rapidly redistributed and eliminated through conjugation in the liver to glucuronide and sulfate, inactive metabolites that are then excreted in the urine.[46] Propofol does not have significant analgesic effects.

Propofol is characterized by rapid onset of unconsciousness and a smooth recovery. A comparison of propofol and thiopental in dogs demonstrated similar times to

54 extubation and head lifting, but found that dogs receiving propofol experienced a significantly shorter time to sitting stemally and walking unaided.[45] Dogs receiving thiopental experienced more struggling and vocalization during recovery than those receiving propofol.[45]

The qualities of anesthetic induction, maintenance, and recovery were judged to be good to excellent in all cats administered clinically relevant doses of Alfaxan ® CD­

RTU. The duration of anesthesia and unresponsiveness increased with dose. Only 1 of7 cats recovered from anesthesia after being administered 50 mg/kg IV Alfaxan® CD-RTU.

These cats were euthanized due to failure to recover within 5 hours, but, based on their cardiorespiratory status, were likely to ultimately recover. Even at this dose, Alfaxan-CD

R TU® did not produce the cardiac arrhythmias, cardiac arrest, or adverse neuromuscular responses (muscle rigidity, myoclonus, hyperreflexia) reported following the administration of clinically relevant doses of Saffan®. [3] Previous studies of Saffan ® administration reported a good quality of anesthesia, but noted sneezing, vomiting, and defecation prior to induction, involuntary movements during anesthesia, and hyperreflexia, excessive sneezing, nose rubbing, and paddling during recovery.[7] Other than occasional involuntary muscle movement, we did not observe any of the aforementioned side effects. We conjecture that most of the side effects previously reported are due to the rate of drug administration and to histamine release inherent to the earlier formulations, but more focused studies are required to confirm this speculation.

When challenged with a supramaximal noxious stimulus, all of the cats in the current study demonstrated a dose-dependent loss of responsiveness that lasted for approximately 60% of the duration of anesthesia. Studies investigating the

55 antinociceptive properties of neurosteroids have suggested that alphadolone' s analgesic effects are mediated through GABAA receptors at the level of the spinal cord. Alfaxalone is comparatively devoid of this activity, producing analgesia secondary to unconsciousness and the modulation of GABAA receptors in the brain.[32, 56] The additive or synergistic effects of analgesics such as opioids and alpha-2 agonists when administered in combination with Alfaxan® CD-RTU should be a focus of future investigations.

In dogs, Alfaxan®-CD RTU produced rapid and excitement-free induction to anesthesia, uneventful maintenance, good to excellent muscle relaxation and analgesia, and stress-free recovery from anesthesia. Unlike the cat, it was safe across a relatively wide dose range. Anesthetic induction was good to excellent in all dogs; only one dog demonstrated a temporary period of head shaking after administration of the 6 mg/kg dose. The maintenance of anesthesia was characterized by excellent muscle relaxation and good to excellent analgesia. The average time to sternal recumbency was relatively rapid, ranging from approximately 20-80 minutes following the 2 mg/kg and 20 mg/kg doses, respectively. These anesthetic effects were comparable to those reported for CT-

1341 in cats and in a safety trial investigating the effects of 9, 16 and 30 mg/kg IV alfaxalone in cyclodextrin in dogs.[57]

One study investigating the anesthetic, cardiovascular, respiratory, and adverse effects of CT 1341, thiopental, methohexital, pentobarbital, propanidid, and ketamine

suggested that CT 1341 produced better quality anesthesia with fewer adverse effects.[6]

We did not compare the effects of the IV doses of Alfaxan®-CD RTU to any of the

aforementioned drugs, but based upon our results and clinical experience, we were

56 impressed by the quality of anesthesia and relative absence of adverse effects recorded.

57 4.5

CONCLUSIONS

Our studies indicate that Alfaxan-CD RTU® produced good to excellent anesthesia in cats, characterized by rapid induction to anesthesia, excellent muscle relaxation, unresponsiveness to noxious stimuli, and smooth, uneventful recovery from anesthesia. Hypoventilation and apnea were uncommon at clinically relevant doses, but became the most important adverse effects when larger doses were administered rapidly

IV. Arterial blood pressure remained stable following the IV administration of clinically relevant doses (<5 mg/kg) of Alfaxan® CD-RTU, but caution is advised with larger doses or with administration to animals with cardiovascular compromise.

In dogs, Alfaxan®-CD RTU produced safe and effective anesthesia. Induction to anesthesia was rapid and uneventful. The maintenance and recovery periods were characterized by good to excellent muscle relaxation and analgesia. Cardiovascular status was well maintained when dosages of up to 6 mg/kg IV were administered.

Respiratory depression and apnea were the only notable disadvantages, and then only when larger dosages (>6 mg/kg IV) are administered. Alfaxan®-CD R TU should be an excellent alternative to currently available injectable anesthetics when administered for short surgical procedures or as induction to inhalant anesthesia.

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