MQP-BIO-DSA-0262
Validation of Cytochrome P450 2C8 Inhibition Assay
A Major Qualifying Project Report
Submitted to the Faculty of the
W ORCESTER POLYTECHNIC INSTITUTE
In partial fulfillment of the requirements for the
Degree of Bachelor of Science
in
Biology and Biotechnology
By
______
Matthew Schulze
April 24, 2008
APPROVED:
______Adrian Sheldon, Ph.D. David Adams, Ph.D. Associate Director (Invitro ADMET) Biology and Biotechnology Charles River Laboratories Preclinical Services W PI Project Advisor Major Advisor ABSTRACT
One of the most important pharmaceutical properties of a drug candidate to be determined during any pre-clinical discovery is the effect of the specific test compound on the activity of Cytochrome P450, a key enzyme involved in drug metabolism. The purpose of this project was to validate a Cytochrome P450 2C8 (CYP2C8) enzyme inhibition assay in development at Charles River Labs (W orcester). The assay quantitates the in vitro inhibition of
CYP2C8 by a test compound as measured by the amount of specific metabolite generated by
CYP2C8-specific drug substrates Amodiaquine (AMOD) and Paclitaxel (PACL). The data presented here validate the assay.
TABLE OF CONTENTS
Abstract...... - 2 -
Table of Figures ...... - 4 -
Acknowledgements...... - 6 -
Background...... - 7 -
Drug Metabolism & Biotransformation ...... - 7 -
Cytochrome P450...... - 11 -
History...... - 12 -
Biochemistry...... - 14 -
Cytochrome P450 2C8 ...... - 19 -
Structure of CYP2C8...... - 20 -
Metabolism of CYP2C8 ...... - 21 -
Project Purpose ...... - 24 -
Methods...... - 25 -
Enzyme Titration...... - 26 -
Substrate Titration...... - 27 -
CYP2C8 Inhibition...... - 27 -
Results...... - 28 -
Discussion...... - 33 -
Bibliography...... - 35 -
TABLE OF FIGURES
Figure 1: Definitions of Specificity. (Monosson, 2008)...... - 1 -
Figure 2: Visual Description of Oxidation and Reduction (Phase I) reactions. ("Mnemonic: Oxidation and Reduction.“, 13 Feb 2008) ...... - 1 -
Figure 3: Model of the conserved tertiary structure of CYP monooxygenase. The heme is orange, substrate recognition site is red, and the heme coordinating I and L helices are green. (Urlacher & Eiben, 2006)...... - 1 -
Figure 4: —Carbon monoxide-difference spectra for NADH-reduced (- - -) and Na2S2O4-reduced (–) rat liver microsomes.“ (Sato & Omura, 1978) ...... - 1 -
Figure 5: Drug discovery and development process. (—KinomeScan“, 13 Feb 2008)...... - 1 -
Figure 7: epoxidation of styrene to styrene oxide. (Urlacher & Eiben, 2006)...... - 1 -
Figure 8: Hydroxylation of fatty acids to hydroxyl-fatty acids. (Urlacher & Eiben, 2006) ...... - 1 -
Figure 6: Hydroxylation of an aromatic compound naphthalene to 2-naphol/1-naphol. (Urlacher & Eiben, 2006)...... - 1 -
Figure 9: The fold and active site for CYP. Black displays the substrate recognition sites (SRS). (Denisov et al., 2005)...... - 1 -
Figure 10: Predicted secondary structure of Cytochrome P450 2C8 Dimer Enzyme. Light blue=turns; Pink=coils; Green=helices; Purple=strands; Magenta(center)=two palmitic acid molecules; Magenta(top and bottom)= hemes (Produced in MBT Protein W orkshop, Jan 2008)..- 1 -
Figure 12: Metabolism of PACK to 6M-hydroxypaclitaxel. (Separations of Paclitaxel and Its Metabolite 6a-Hydroxypaclitaxel.)...... - 1 -
Figure 11: Metabolism of AMOD to DEAQ. (Li et al., 2002) ...... - 1 -
Figure 13: IC50 plots of amodiaquine inhibition in CYP2C8 by montelukast (•), candesartan cilexetil (¢), zafirlukast (Å), quercetin (Æ), and gemfibrozil (Ç). (W alsky et al., 2005) ...... - 1 -
Figure 15: IS Peak Area vs. Index of DEAQ using DESA-d3 as an IS...... - 1 -
Figure 14: IS Peak Area vs. Index of DESA-d3 using RSRP as an IS...... - 1 -
Figure 16: Graphs of Average Area Ratio (Analyte Peak Area / IS Peak Area) vs. Enzyme Concentration for T=30 on the left and T=0 on the right...... - 1 -
Figure 17: Graph of Average Area Ratio (Analyte Peak / IS Peak) vs. substrate concentration (µM)...... - 1 -
Figure 18: Graph of Average Area Ratio (Analyte Peak / IS Peak) vs. substrate concentration (µM)...... - 1 -
Figure 20: IC50 of QRCT is 4.2 µM...... - 1 -
Figure 19: IC50 of MONT is 0.3 µM...... - 1 -
ACKNOW LEDGEM ENTS
First I would like to thank Adrian Sheldon for letting me use the Invitro ADMET lab at
Charles River Laboratories. I would also like to thank Sarah Mitchell, Patty W alton,
Susan Dearborn, and Ali Maarouf for showing me procedures that I was initially unfamiliar with, and getting me accustomed to the lab, for helping with my report writing, and most importantly for their guidance and direction. Thank you also to the Charles River Laboratories Preclinical
Facilities in Shrewsbury and W orcester for providing the necessary methods, materials and laboratory space respectfully. Finally, I would like to thank Professor David Adams for his assistance in setting this project up, his help writing the report, and his continued support.
BACKGROUND
Drug Metabolism & Biotransformation A drug is defined as a substance taken for either medicinal or recreational use. After digestion, the substance circulates, enters the circulatory system, and begins to undergo metabolism or biotransformation. This process can be best described as —breaking down“ the drug whatever it may be, but it always involves the chemical modification or degradation of the drug. These changes can occur in any tissue, but most of them occur in hepatocytes. The liver produces numerous enzymes that alter drugs and toxins in order to clear them from the circulatory systems and excrete them in the bile. These enzymatic systems use a variety of reactions to metabolize drugs, such as oxidation, reduction, and hydrolysis. As these changes occur new metabolites are produced that may or may not remain active in vivo. However, the majority of these metabolites will enter the kidney where they can be processed and expelled in the urine, solid waste, or perspiration (—Liver“, 2005).
Biotransformation is vital to the survival of an organism because it allows for necessary nutrients to be absorbed as they are transformed, metabolize drugs into metabolites that may be the therapeutic, and of course are a mechanism of defense against xenobiotics, chemicals in the body that are not normally present. These processes aid in the elimination of hydrophobic substances by modifying them from one chemical to another by means of different chemical reactions. Since most toxicants are hydrophobic, many of the enzyme systems in the liver can transform these toxicants so that they pass without harming cells. W hile the process is not perfect, biotransformation produces metabolites that are less toxic. However, one example of
how a metabolite can harm the human body is where vinyl chloride is altered to vinyl chloride epoxide, which then binds to DNA and RNA and can potentially cause cancer (Monosson, pp.
28, 2008).
Chemical reactions are constantly taking place in vivo, and are a normal aspect of the human body processes, however many of them are catalyzed by enzymes and enzyme systems which accelerate the reactions. Nearly all the reactions that occur in the body are supported by enzymes, and many health problems occur due to genetic mutations causing a change in enzymes or drug interactions that inhibit enzymes from metabolizing certain substrates. Many biotransforming enzymes are composed of chains of amino acids linked together and are of high
Figure 1: Definitions of Specificity (M onosson, 2008). molecular weight such as
53,988 daltons that Cytochrome
P450 2C8 (CYP2C8) weighs in
at (Schoch et al., 2003). The
specificity of enzymes falls into
three categories; absolute specific, group specificity, and linkage specificity. The most relevant application to CYP 2C8 is group specificity since the enzyme is a nonspecific monooxygenase (—EC 1.14.14.1“, 2007).
W ith hundreds of thousands of reactions occurring in vivo, enzymes involved in biotransformation are defined and categorized not only by their reaction type, but also by the sequence in which they interact with a xenobiotic. The classification can be divided into two groups of major transformations involved with xenobiotics, Phase I and Phase II. Phase I reactions are commonly described as reactions in which modification involves altering a
chemical by adding a functional structure. After a Phase I reaction, the intermediate many times
—contains a reactive chemical group“ such as hydroxyl, amino, and carboxyl (Monosson, 2008).
These often cannot sufficiently clear the body, and as a result Phase II reactions bring about further biotransformation. Phase II reactions tend to need those functional groups present to act upon a substance usually requiring conjunction with another substance. After conjugation, the products tend to be larger and polar allowing them to dissolve more readily in water. The
Figure 2: Visual Description of Oxidation and Reduction (Phase I) Reactions ("M nemonic: Oxidation and Reduction.“, 13 Feb 2008). process of eliminating xenobiotics proceeds through Phase I type reactions if they do not have a functional group that allows them to be conjugated immediately. Phase I reactions tend to be the least complex of the two phases, and encompass a number of reactions such as hydrolysis, cyclization, reduction and oxidation, all of which expose or add a small polar group to the toxicant. Hydrolysis is the process where the addition of H2O separates the xenobiotic into two smaller pieces. On one piece a hydroxyl group is gained (-OH) and on the other a hydrogen (-H).
Reduction is the process where the substrate acquires electrons. The advantage of reduction is that it can occur in oxygen (O2) depleted environments; however many times reduction concludes with the creation of a xenobiotic instead of degrading the toxicant and eliminating it
from the body. Oxidation works in the opposite fashion of reduction where the substrate loses electrons. An enzyme can catalyze this reaction by adding O2 or if O2 is not present, dehydrogenation can occur where hydrogen is removed from the substrate. Another example of oxidation is electron transfer which is just a transfer of an electron from the substrate
(Monosson, 2008). Of the Phase I metabolic pathways, Cytochrome P450 (CYP) systems, are the most important and are incredibly diverse. CYP is a protein containing heme which is located in the phospholipid bilayer of the smooth endoplasmic reticulum of many cell types; however they are most abundant in the liver due to the liver‘s role in removing toxins from the body and more than 90% of the Phase I metabolism is carried out by CYP‘s (Lewis et al., 1998). The efficiency and diversity of these enzyme systems makes them responsible for the oxidation of many different chemicals and xenobiotics (Monosson, 2008).
Cytochrome P450 The Cytochrome P450 (CYP) enzyme system is located mainly in hepatocytes and is one of the most important and diverse systems in prokaryotic and eukaryotic organisms. CYP is a heme containing protein that resides within the phospholipid bilayer of the endoplasmic reticulum throughout the body‘s cells, e.g. skin, adrenals, kidneys, heart, brain, etc. ("CYP 2C8
Figure 3: M odel of the conserved tertiary structure of CYP monooxygenase. The heme in the center of the diagram is orange, the substrate recognition site is red, and the heme coordinating I and L helices are green (Urlacher & Eiben, 2006).
Inhibitors - Pubdrug.", 13 Feb 2008). They are named from their spectrophotometric absorption peak at 450 nm when bound by carbon monoxide, and are thought to be as old as 3.5 billion years and exist in countless bacteria, fungi, plants, and animals. (Chang & Kam, 1999) CYP isozymes are part of a superfamily with greater than 4,000 members that are defined as hemoproteins and function as oxidase systems. W hile many are unique to certain substrates, some are observed to be more promiscuous and are responsible for the metabolism of exogenous substances. CYP families are named according to their amino acid sequence (CYP1, CYP2,
CYP3), their subfamily selected by their amino acid homology (CYP2A, CYP2B, CYP2C), and by individual enzyme (CYP2C8) ("CYP 2C8 Inhibitors - Pubdrug.", 13 Feb 2008).
Since the discovery of CYP in 1958, there has been a keen interest and constant focus on the research of this heme-protein. Understanding of this enzymatic system has evolved tremendously and help pharmacologist and biochemists elucidate its role and impact in oxidative transformations of a variety of xenobiotics.
History CYP was first observed in 1955 when G.R. W illiams detected a pigment in a carbon monoxide-binding spectrum in rat liver microsomes. This came about while he was researching the kinetics of microsome-bound cytochrome b5 and even though his observations were not
Figure 4: —Carbon monoxide-difference spectra for NADH-reduced (- - -) and
Na2S2O4-reduced (–) rat liver microsomes“ (Sato & Omura, 1978). published, the research was continued by M. Klingenburg. Klingenburg could not decipher what he saw because at that point there were no known hemoproteins that could account for the amount of protohemin. The basic properties of CYP were described by Omura and Sato in 1964
after they attempted to destroy the pigment with detergents, and instead observed absorbance at
420 nm. The name of this cytochrome was created due to the spectrum for NADH-reduced and
Na2S2O4-reduced rat liver microsomes and it is evident that the optical absorption is at 450nm
(Figure 4). The first evidence of CYP functioning as an oxygenase came with the development of the photochemical action spectrum technique where they showed CYP catalyze a C21- hydroxylation reaction. This process gave rise to vast amounts of work done by pharmacologists and endocrinologists who could use the carbon monoxide inhibition of given oxygenation reactions as a standard in their research. Lastly, discoveries were made by pharmacologists that showed specific drugs elevated drug-oxidizing activities in mammalian liver microsomes and began understanding CYP‘s role in the liver (Sato & Omura, 1978).
Due to the original discovery of CYP in liver microsomes, it was previously thought that it was only located in animal tissue. However, the contrary was found when the same pigment was discovered in mitochondria. Other tissues were examined, and yeast, nitrogen-fixing bacteria, and plants were found to have CYP in them as well. Frequently cytochrome was found in mircosomal fractions acquired from tissue homogenates which revealed that it functioned as a monooxygenase and —utilized either NADH of nicotinamide adenine dinucleotide phosphate
(NADPH) as a source of reducing equivalents for oxygenation reaction“ (Sato & Omura, 1978).
As the role of CYP in oxygenation reactions became clear, the mechanism of electron supply from NADPH needed to be understood. The first CYP-containing oxygenase system to be determined was the 11R-hydroxylase system of the adrenal cortex mitochondria. Since then there have been two distinct types of CYP-containing oxygenase systems, one containing a
NAD(P)H-linked flavoprotein (a dehydrogenase that contains a flavin and often a metal and
plays a major role in biological oxidations (—Flavoprotein, 13 Feb 2008)), an iron sulfur protein, and CYP; and one that is membrane-bound with NADPH-cytochrome c reductase and CYP
(Sato & Omura, 1978).
The extensive observation of CYP in different organisms and tissue, and its primary function in eliminating xenobiotics such as drugs, pesticides, carcinogens and other toxicants
Figure 5: Drug discovery and development process (—KinomeScan“, 13 Feb 2008). from the body, has made it one of the primary pharmaceutical properties to define within drug development. The FDA mandates the inclusion of in vitro CYP inhibition assays as part of a panel of assays (Figure-5) required for drug discovery and pre-clinical development.
Biochemistry Cytochrome P450 (CYP) isoenzyme is comprised of two protein chains, where each consists of its own heme group. CYP is responsible for the catalysis of xenobiotic compound metabolism, and this biotransformation of exogenous substrates alters them in such a way that they become hydrophilic or polar so that they can be cleared of the body through various means of excretion (Chang & Kam, 1999). These monooxygenases play a critical role in the metabolic
pathways and drug degradation. Many of these CYP‘s have the ability to introduce oxygen, some have limited substrate range where they can only synthesize steroids, and others that may be microsomal derived can react with many xenobiotics in their detoxification process (Urlacher &
Eiben, 2006).
The heme group reacts with oxygen after the electron transfer reactions from NADPH, and this reaction then integrates O2 onto the substrate. CYP typically catalyzes the reaction such that:
+ + NADPH + H + O2 + RH T NADP + H2O + R-OH
In the reaction, R corresponds to a substrate such a xenobiotic (Chang & Kam, 1999). The mechanism of CYP is described as a cascade of single steps that involve the protein redox couples and utilization of NADPH (Denisov et al., 2005). As the reactions are carried out with
O2 with an electron transfer system, different reactions are catalyzed such as aromatic hydroxylation (Figure 6), epoxidation of C=C double bonds (Figure 7), and the C-H activation of sp3 hybridized carbon atoms (Figure 8), among others (Urlacher & Eiben, 2006).
Figure 8: Hydroxylation of an aromatic compound naphthalene to 2-naphol/1-naphol. (Urlacher & Eiben, 2006)
Figure 6: epoxidation of styrene to styrene oxide. (Urlacher & Eiben, 2006)
Figure 7: Hydroxylation of fatty acids to hydroxyl-fatty acids. (Urlacher & Eiben, 2006) These wide varieties of oxidation reactions can result in the activation or inactivation of the compound, and just like other enzymes can reach saturation. CYP uses co-factors in their reactions and can be induced or inhibited as well (Chang & Kam, 1999). Even though the applications are varied, CYP has a disadvantage due to its dependence on equimolar amount of
NADPH, but can be overcome by using whole cell systems (Urlacher & Eiben, 2006).
All CYP‘s are similar in their active site where the substrate binds to the enzyme. The core of CYP is made up of three parallel helices: D, L, and I, while helix E is anti-parallel
(Figure 9). CYP folds tend to be highly conserved, but also have the flexibility for binding of substrates of differing sizes. Although they have overlapping tendencies and diversity, the specificity varies such that stereospecificity can affect oxidation, while others such as CYP3A4 can metabolize more than 50% of pharmaceuticals on the market. There are six substrate recognition sites (SRS) regarded as the proteins most flexible regions. The sites are as follows:
Figure 9: The fold and active site for CYP. Black displays the substrate recognition sites (SRS) (Denisov et al., 2005). the B‘ helix, SRS 1; helices F and G, SRS 2 and 3; a region of helix I, SRS 4; the R2 connecting region of the K helix, SRS 6; and the R4 hairpin, SRS 5 (Denisov et al., 2005).
As the substrate binds to CYP, the low-spin ferric enzyme agitates water in coordination with the sixth legend of the Fe heme and the spin state changes to high-spin. The high-spin Fe3+ reduction potential is now more positive and is now easier to reduce to a ferrous state. Then O2 binding leads to a CYP-O2 complex. This intermediate which is relatively stable is reduced to a peroxide-ferric intermediate where it then is protonated to form hydroperoxo-ferric intermediary.
A second protonation occurs at the distal O2 and heterolysis of the O-O bind forms Compound I, the result is water and oxygenation of the substrate. During the enzymatic reaction cycle there are three possible abortive reactions (Denisov et al., 2005).
W hile the majority of biotransformation regarding CYP takes place in the hepatocytes
(70%), CYP isoenzymes are abundant in other regions of the body as well. There currently are
influxes of differing CYP‘s as the amount varies when observing the mucosa of the gut, and
CYP has been identified in the brain where high concentrations are thought to be relevant in regulating progesterone and corticosterone concentrations. It is important to remember that all somatic cells carry the genetic information to transcribe and translate DNA into functioning enzymes. Genetic polymorphism often times results in organisms that are poor or slow metabolizers, however the frequency of this recessive trait is less than 1%. Some examples of
CYP‘s that are usually affected by polymorphism are CYP2D6, CYP2C19, and CYP2E1 (Chang
& Kam, 1999).
CYP has always been an enzyme system centered on Phase I biotransformation as it is the main defense against toxicants within the body. In recent years however there has been a rise in observation of CYP in other physiological roles. Not only is it evident that CYP is responsible for the biotransformation of xenobiotics but also the biosynthesis or degradation of endogenous compounds like steroids, cholesterol, and fatty acids. Other areas where there is evidence of CYP is in the brain where it is thought that it may have physiological role in signal transduction and that CYP2D6 may regulate the metabolism of neurotransmitters. Throughout the rest of the human body CYP systems play an important role in adrenal and gonadal steroidogenesis, as well being responsible for reproductive function and sex differentiation (Change & Kam, 1999).
In the pharmaceutical industry, the CYP inhibition assay has become a standard for scientists in evaluating the metabolism of new drug candidates which may be able to treat diseases or other maladies. Being able to measure activity in vitro can be very important in developing drugs. The data that is generated from in vitro drug interaction studies is invaluable to designing clinical drug interaction studies. Drug-drug interactions are very central to drug
research, regulatory guidelines, and patient safety. One of the most powerful tools that a scientist can use is information about interactions that result in the inhibition of CYP. The development of these assays will help avoid producing new chemicals that may have a high potential for drug-drug interactions. W hile the data collected does not have to be done by Good
Laboratory Practice (GLP), organizations may be subject to audit from the Food and Drug
Administration (FDA). W ith that in mind, it is important for the analytical methods and assays to be validated (W alsky & Obach, 2004).
Cytochrome P450 2C8
D C I
Figure 10: Predicted secondary structure of Cytochrome P450 2C8 Dimer Enzyme. Light blue=turns; Pink=coils; Green=helices; Purple=strands; M agenta(center)=two palmitic acid molecules; M agenta(top and bottom)= hemes (Produced in M BT Protein W orkshop, Jan 2008).
The CYP2 family is quite large and includes CYP‘s 2A, 2B, 2C, 2D, and 2E sub families.
More specifically the CYP2C enzyme subfamily demethylates diazepam, tricyclic antidepressants and oxidizes omeprazole (Chang & Kam, 1999). CYP2C8 is one of the most
principal hepatic-drug metabolizing enzymes that oxidize cervastatin, amodiaquine (AMOD), and paclitaxel (PACL), as well as endobiotics like retinoic acid and arachidonic acid. Some key characteristics of CYP2C8 is an extended active site, it is crystallized as an asymmetric dimer which are bound by two palmitic acid molecules (Schoch et al., 2004).
CYP2C8 has a pivotal part in metabolism of many drugs and is expressed significantly within the liver where much of drug clearance occurs. CYP2C8 actively clears taxols such as
PACL, the antimalarial drug AMOD, as well as others such as troglitazone, rosiglatazone, amiodarone, and verapamil. The research and development of the CYP2C8 assays are important for discovery of different drug-drug interactions like that of cerivastatin and gemfibrozil, where
CYP2C8 is the primary enzyme that metabolizes cerivastatin and gemfibrozil is an inhibitor and when mixed causes a toxic interaction, rhabdomyolysis (the breakdown of muscle fibers resulting in the release of muscle fiber contents into the bloodstream) (Schoch et al., 2004).
Structure of CYP2C8 W hile CYP‘s exhibit selectivity in terms of region- and stereospecific reactions this tends to more evident on the diverse CYP2C subfamily. In order to observe the structure of CYP2C8 changes had to be made to the N-terminus where it was modified by replacing a hydrophobic domain with a short hydrophilic domain from residue 1 to 27. The protein was natural from the
28th residue till the C-terminus where a histidine tag was placed for purification purposes
(Schoch et al., 2004).
As the protein was purified it was analyzed through mass spectrometry and found that its molecular weight match its predicted weight, 53,979 daltons, at a weight of 53,988 daltons. The two molecules form an asymmetric dimer where the G and F helices interact between chains A
and B (Figure 10). The dimer is held together and stabilized by two palmitic acid molecules
(Figure 10). CYP2C8 also differs in its active site where it has been determined that it is about twice the size of other CYP‘s like CYP2C5 and further evidence is given by its ability to metabolize such a large substrate like taxol (Schoch et al., 2004).
M etabolism of CYP2C8 CYP2C8 is defined as an unspecific monooxygenase and its reaction with its substrates are similar to other CYP‘s but more specifically acts like this:
RH + reduced flavoprotein + O2 T ROH + oxidized flavoprotein + H2O
Basically CYP2C8 acts as a group of heme-thiolate proteins that act on a variety of substrates
Figure 12: M etabolism of AM OD to DEAQ. (Li et al., 2002) like steroids, fatty acids, vitamins prostaglandins and most importantly in the case of drug development, xenobiotics ("EC 1.14.14.1.", 29 Dec 2007). CYP2C8 as said before is specific to certain substrates. Two different substrates that are important to determining assays for CYP2C8
/òt/
Figure 11: M etabolism of PACK to 6J-hydroxypaclitaxel (Separations of Paclitaxel and Its M etabolite 6a-Hydroxypaclitaxel).
are amodiaquine (AMOD) and paclitaxel (PACL). As CYP2C8 metabolizes these they are oxidized and form N-desethylamodiaquine (DEAQ) (Figure 11) and 6M-hydroxypaclitaxel
(Figure 12). AMOD has been used widely as treatment of malaria due to an increased resistance and greater toxicity of a previous therapeutic, chloroquine. As it is taken orally it is metabolized quickly, so quickly that it was determined CYP2C8 was the main isoenzyme that cleared AMOD and catalyzed it into DEAQ (Li et al, 2002). PACL is known for its use as an anit-cancer drug treatment. W hen the drug is taken, it is hydroxylated into 6M-hydroxypaclitaxel and it has been observed that taxanes are chiefly metabolized within the liver. Research of taxanes uses in vitro
CYP2C8 assay to find inhibitors because of the rapid hepatic metabolism which shortens the turnover of the therapeutic PACL (—Method to Potentiate“…2008).
In the development of therapeutic drugs, it is important and necessary to have the ability to compare the test compounds at various concentrations. It has also become apparent that inhibition of CYP‘s are often times the most vital piece of knowledge in understanding drug- drug interactions. There are three kinds of reversible enzyme inhibitors: competitive, mixed, and non-competitive. Competitive inhibition occurs when the substrate and the inhibitor cannot bind to the enzyme at the same time, and basically the two compete for the active site of the enzyme.
Mixed inhibition happens when the inhibitor can bind while the substrate is bound but generally affects the enzyme-substrate complex allosterically. Non-competitive inhibition transpires as the inhibitor binds to the enzyme but the amount of substrate does not have an effect and the concentration of the inhibitor controls activity. In most cases, drug interaction occurs by alterations in the activity (mixed inhibition) of CYP‘s where one drug inhibits the activity and the other is affected by the change in metabolic clearance (W alsky et al. 2005).
Two of CYP2C8‘s most prominent inhibitors are montelukast (MONT) and quercetin
(QRCT). Many drugs have been tested as inhibitors for CYP2C8 and MONT, a therapeutic drug for asthma, inhibited the catabolism of AMOD and an inhibitor concentration resulting in 50% inhibition (IC50) value was found to be 19.6 nM. Out of the 209 drugs tested, MONT was identified as the most potent inhibitor. QRCT has also been identified as a moderate inhibitor and is often used as an inhibitor for other CYP‘s and its IC50 value was determined 3.94 µM (W alsky et al., 2005).
a h b Ç v w/Ç
Figure 13: IC50 plots of amodiaquine inhibition in CYP2C8 by montelukast (•), candesartan cilexetil (¢), zafirlukast (Å), quercetin (Æ), and gemfibrozil (Ç) (W alsky et al., 2005).
PROJECT PURPOSE
The aim of this project was to devise methods for performing an in vitro drug-based cytochrome P450 2C8 inhibition assay that could be used on test compounds. More specifically to discover an optimum concentration of cytochrome P450 2C8, an optimum concentration of amodiaquine and paclitaxel, and a constant range of concentrations of quercetin and montelukast in order to permit the measurement of inhibition by test compounds of cytochrome P450 2C8 mediating the conversion of specific drug substrates to corresponding specific metabolites. The validation of this inhibition assay would be done by using lab bench techniques, and then measuring specific drug substrates as they are converted by cytochrome P450 2C8 and are detected by standard LC/MS/MS methods. Cytochrome P450 2C8 inhibition by the test compounds would then be assessed by diminished production of the specific metabolites.
M ETHODS
W ith the Food and Drug Administration looking for more data in drug development and discovery, Charles River Laboratories looked to expand their repertoire of assays and develop a cytochrome P450 2C8 (CYP2C8) inhibition assay. The primary preparation methods were basic research in order to determine from literature what range of concentrations would be used to hone the assay constants.
The assay will test compounds for inhibition of cytochrome P450 2C8 (CYP2C8) involved in drug metabolism. Performing the in vitro process will measure the inhibition of test compounds of CYP2C8, and the alteration of the substrates amodiaquine (AMOD) and paclitaxel
(PACL) to their respective metabolites. It is important to point out that this differs from a fluorescence assay which relies on the products to fluoresce after the oxidation of artificial substrates. The drug-based CYP inhibition assays are more flexible in that they can use different types of enzymes like microsomes or supersomes. The assay will be held at a constant temperature and the CYP2C8 will be held at or near Km, 1.7 pmol/ml (W alsky et al., 2005). The reaction will be carried out in company of the CYP2C8 co-factor, R-nicotinamide adenine dinulceotide phosphate (NADPH) for a set amount of time that will be held constant throughout the entire validation. Stock solutions will be dissolved in dimethyl sulfoxide (DMSO) unless the stock cannot go into DMSO and then alternative solvents maybe used such as methanol (MeOH) or Acetonitrile (ACN).
To end the reaction, a quench solution will be used that consists of ACN, Formic acid and an internal standard (IS). After quenching the reactions, the plates that the assay is performed on
must be centrifuged at 3,000 rpm for 15 min at 2-8o C. This will precipitate in soluble material in order to stop interference with the LC/MS/MS analysis. The samples can then be stored in a
2-8o C environment before analysis.
The data gathered from the LC/MS/MS analysis will be used to calculate inhibition of the specific metabolite that is formed, either desethylyamodiaquine (DEAQ) or 6M-hyroxypaclitaxel, by known and inhibitors as percent control. Percent control is defined by comparing metabolite formation with and without fully inhibited CYP2C8. Percent control can then be used to calculate the IC50.
Enzyme Titration The first step in validating the CYP2C8 inhibition assay was determining the optimal enzyme concentration. This is also a pivotal step in determining the assay conditions, incubation, temperature and time. Since the CYP2C8 fluorescence assay is performed at 37o C, the starting conditions for this titration will be the same. The time range that will be employed will be between 30 minutes and 60 minutes. The literature states that the CYP2C8 inhibition assays were performed successfully at an enzyme concentration of 1.7 pmol/ml (W alsky et al.,
2005). The cytochrome stock that was used for the validation was Human rCYP 2C8 from BD
Gentest with a stock concentration of 1000 pmol/ml. Substrate concentrations had to be determined from the literature as well. AMOD was used at 2.5 µM as it was used from 0.24-
5.7µM in W alsky et al. and 2.4 µM in Donato et al. PACL was used at 10 µM from Donato et al. The other experimental conditions included 4 mM NADPH, 0.1% of DMSO for AMOD, 0.9
ACN for PACL, and several concentrations of Metabolic Stability Buffer (sodium phosphate)
due to the varying concentrations of CYP2C8. The assay was run in 96-well 2ml V-bottom plates with several concentrations of CYP2C8.
Initially the IS in the quench solution that was used was reserpine (RSRP) and a reserpine/desethylamodiaquine-d3 (DESA-d3) solution, and later only DESA-d3 was used for the AMOD metabolism.
Substrate Titration After determining the concentration of enzyme to be used, a constant concentration for the two substrates had to be established. The goal is to find a substrate concentration versus metabolite formed results in a graph that is linear. The results should show a plateau at higher concentrations of substrate. For this part of the validation, CYP2C8 was held at a constant concentration of 2 pmol/ml and sodium phosphate at 50 mM. AMOD and PACL were diluted from stock concentrations. The range was based on the literature in order to generate a curve where we can use the linear results to establish a concentration.
CYP2C8 Inhibition The concentrations of CYP2C8, AMOD, and PACL have been determined at this point.
The two major inhibitors of CYP2C8 are quercetin (QRCT) and montelukast (MONT). The concentrations chosen range around the IC50‘s. The assay should be able to generate an inhibition curve where percent inhibition versus inhibitor concentration can be seen. At this point we can see which inhibitor generates the better curve so once the validation is complete we can compare
CYP2C8 inhibition by QRCT or MONT to the desired test compound. The final concentrations at this point are 2 pmol/ml of CYP2C8, 4mM NADPH, 0.1% DMSO when using AMOD, 50 mM of sodium phosphate, and 1 µM of AMOD.
RESULTS
The validation of the CYP2C8 inhibition assay is not yet completed. At the present time the inhibition assay (including the enzyme titration and substrate titration) for AMOD is nearly finished and will require additional assays with inhibitors, the validation using PACL will require additional method development due to unforeseen issues with the IS.
Figure 15: IS Peak Area vs. Index of DESA-d3 using RSRP as an IS
Figure 14: IS Peak Area vs. Index of DEAQ using DESA-d3 as an IS.
W hen the assay first began with the enzyme titrations, the hope was that many of the procedures and approaches would be similar to that of the other CYP assays. RSRP is a generic
IS which is used in the other CYP assays, however with the CYP2C8 assays RSRP did not prove to be a suitable IS. RSRP showed significant variability, including signal enhancement and suppression with metabolite concentration (Figure 14). The LC/MS/MS data of CYP2C8 metabolizing AMOD and using DESA-d3 as the IS gave much better results (Figure 15). In fact, the enzyme titration using the DESA-d3 as the IS generated and very nice curve with a linear regression up to 5 pmol/ml (Figure 16). After discussion regarding the substrate concentration to use for the assay, 2 pmol/ml was the final decision because it was on the linear portion of the substrate titration curve. The substrate concentration of 2 pmol/ml was also consistent with the
Figure 16: Graphs of Average Area Ratio (Analyte Peak Area / IS Peak Area) vs. Enzyme Concentration for T=30 (left) and T=0 (right.) literature values. The 60 min incubation does not significantly increase the turnover of substrate therefore the incubations of the assays will be kept at 30 min. Since RSRP did not prove to be a suitable IS for the assay a deuterated IS, such as 6M-hyrdoxypaclitaxel will be purchased and the assay will then be optimized using this IS.
The enzyme titration began with ranging the CYP2C8 concentration and looking for a linear region on the graph (Figure 17), while holding AMOD at 2 µM. The substrate titration began by holding the CYP2C8 concentration constant. The assay was performed at 37o C for 30 minutes and the range of AMOD that was analyzed was from 0.5 µM to 25.0 µM, while PACL will be analyzed from 0.5 µM to 50 µM. As stated in the methods, the goal of this titration is to find where the —substrate concentration versus the metabolite formed“ graph is linear and the graph should also display a plateau for higher concentrations of substrate.
Figure 17: Graph of Average Area Ratio (Analyte Peak / IS Peak) vs. substrate concentration (µM ).
The lower concentrations form a very linear region in the graph at about 1 µM of substrate and the formation of the metabolite begins to plateau as the substrate concentration reaches 6 µM (Figure 18). It is important to note that metabolism of AMOD was run twice, once with DESA-d3 as the IS and once with RSRP as the IS, and both yielded great results in metabolite formation but the background was still distorted by use of RSRP. The substrate titration of PACL did not generate a curve; XLfit fit a curve onto the points that is merely an average of the metabolite peak and the substantial drop in metabolite (Figure 18). W ith the
unknown rise and fall of PACL metabolites the next step was to try different IS‘s as well as to continue running CYP2C8 inhibition for PACL. The inhibition of AMOD metabolism yielded good results and it was determined to that the constants for CYP2C8 inhibition would be 2 pmol/ml of CYP2C8 and 1µM of AMOD.
Figure 18: Graph of Average Area Ratio (Analyte Peak / IS Peak) vs. substrate concentration (µM ).
The next was determining the range of the positive control inhibitors (IC50) of QRCT and
MONT. The final assay concentrations of the these two inhibitors ranged from 49 nM to 50 µM
(QRCT) and .03 nM to 30 µM. The CYP2C8 inhibition assay was run with AMOD as the substrate (1 µM) for 30 minutes at 37o C and was quenched with —Quench/DESA-d3“ solution.
The analysis took place on the LC/MS/MS (MS-27). The inhibition curves that were generated looked good as the IC50‘s were in line with the literature. The average IC50 of MONT was 0.3
µM and the average IC50 of QRCT was 4.2 µM (Figures 19 & 20).
Figure 20: IC of M ONT is 0.3 µM 50 Figure 19: IC50 of QRCT is 4.2 µM
DISCUSSION
Since 1955, CYP‘s have been studied extensively in their position in drug metabolism and drug- drug interaction and have come to be one of the most important enzymes in drug discovery and development. Inhibition of CYP‘s is largely responsible for drug-drug interactions and as the understanding of this process grows as fast as the pharmaceutical market, the need for quantitative
information to predict these mechanisms becomes imperative. The knowledge that is attained through a
validation such as this one can be used in the planning of clinical studies and is on the forefront of Charles
River Laboratories mission to —improve and expedite the research and discovery, development through
first-in-human evaluation and safe manufacture of new therapies for patients who need them.“
The validation currently is in the process of being finished for inhibition of CYP2C8 in its
metabolism of AMOD. After the initial change in IS from RSRP to DESA-d3, the results from the
enzyme and substrate titrations using AMOD encouraged us to continue onto the CYP2C8 inhibition
using QRCT and MONT. The initial results were promising after acquiring IC50 values of 0.3 µM
(QRCT) and 4.2 µM (MONT). The validation process for using PACL requires additional evaluation.
Presently the enzyme titration for PACL heeded results that helped define the CYP2C8 concentration
when using PACL but because of the problems with finding a reliable IS inhibition study will not be able
to continue. Another issue with using the substrate PACL is the drop in metabolite expression in the
substrate titration (Figure 18). One evaluation of this event is possibly the prozone effect. W hile prozone
effect is largely present is anti-body experiments, it has been observed that at high concentrations there is
no aggulation. However, there isn‘t substantial evidence that this is the effect that we are seeing.
The following will be needed to finish the validation of CYP2C8 inhibition assay. Additional runs of CYP2C8 inhibition must be done with the substrate AMOD in order to hone the conditions and gather a consistent IC50 value. The inhibition of CYP2C8 using the substrate PACL needs additional
research and testing to determine a suitable IS that is inexpensive compared to 6M-hdroxypaclitaxel as
well as additional testing to determine why there is a drop in metabolite with increased concentrations of
PACL.
Due to the increasing mandates by the FDA on the process of drug discovery and development it
might prove beneficial for additional CYP inhibition assays to be validated outside of CYP1A2,
CYP3A4, CYP2C9, CYP2D6, and CYP2C19 and currently CYP2C8. W hile these more validations
would help with drug development, the possibilities of CYP is greater. The ability of these enzymes have
been rarely identified as possible use in biotechnology and may have the possibility with the help of
genetic engineering change their activity and use for uses in the life science industry and other
applications in the field of research and development.
BIBLIOGRAPHY
1. Chang, G W ., and P C. Kam. "The Physiological and Pharmacological Roles of Cytochrome P450 Isoenzymes." Anaesthesia (1999): 42-50.
2. "Charles River Labs." Charles River Jobs. 12 Apr. 2008
3. "CYP 2C8 Inhibitors - Pubdrug." Pubdrug. 13 Feb. 2008
4. Denisov, Ilia G., Thomas M. Makris, Stephen G. Sligar, and Ilme Schichting. "Structure and Chemistry of Cytochrome P 450." Chemical Reviews 105 (2005): 2253-2277.
5. Donato, M T., Nuria Jimenez, Jose V. Castell, and M J. Gomez-Lechon. "Fluorescence- Based Assays for Screening Nine Cytochrome P450 (P450) Activitiesin Intact Cells Expressing Individual Human P450 Enzymes." The American Society for Pharmacology and Experimental Therapeutics 32 (2004): 699-706.
6. "EC 1.14.14.1." IUBMB Enzyme Nomenclature. 29 Dec. 2007
7. "Flavoprotein." Merriam-W ebster. 13 Feb. 2008
8. "KinomeScan." Ambit Biosciences. 13 Feb. 2008
9. Lewis, David F., Costas Ioannides, and Dennis V. Parke. "Cytochromes P450 and Species Differences in Xenobiotic Metabolism and Activation of Carcinogen." Environmental Health Perspectives 106 (1998): 633-641.
10. Li, Xue-Quing, Anders Bjorkman, Tommy B. Andersson, Marianne Ridderstrom, and Collen M. Masimirembwa. "Amodiaquine Clearence and Its Metabolism to N- Desethylamodiaquine is Mediated by CYP2C8: a New High Affinty and Turnover Enzyme-Specific Probe Substrate." The Journal of Pharmacology and Experimental Therapeutics 300 (2002): 399-407.
11. "Liver." Microsoft Encarta Reference Library. 2005. CD-ROM.
12. "METHOD TO POTENTIATE THE THERAPEUTIC EFFICACY OF TAXANE AND DERIVATIVES THEREOF." W orld Intellectual Property Organization. 13 Feb. 2008
13. "Mnemonic: Oxidation and Reduction." Mnemonic. 13 Feb. 2008
14. "Monooxygenase." The Free Dictionary. 28 Dec. 2007
15. Monosson, Emily, ed. "Biotransformation." Encyclopedia of Earth. 28 Feb. 2008. National Library of Medicine. 29 Feb. 2008 http://www.eoearth.org/article/Biotransformation#.
16. Philips, Ian R., and Elizabeth A. Shephard, eds. Cytochrome P450 Protocols. Vol. 107. Totowa, New Jersey: Humana P, 1998. 95-126.
17. Sato, Ryo, and Tsuneo Omura, eds. Cytochrome P450. New York, N.Y.: Academic P, 1978. 1-233.
18. Schoch, Guillaume A., Jason K. Yano, Michael R. W ester, Keith J. Griffin, C D. Stout, and Eric F. Johnson. "Structure of Human Microsomal Cytochrome P450 2C8." The Journal of Biological Chemistry 279 (2003): 9497-9503.
19. Separations of Paclitaxel and Its Metabolite 6a-Hydroxypaclitaxel. Bellefonte, PA: Sigma-Aldrich Co.
20. Urlacher, Vlada B., and Sabine Eiben. "Cytochrome P450 Monooxygenases: Perspectives for Synthetic Application." TRENDS in Biotechnology 24 (2006): 325-329.
21. W alsky, Robert L., Emily A. Gannin, and R S. Obach. "Examination of 209 Drugs for Inhibition of Cytochrome P450 2C8." The Journal of Clinical Pharmacology (2005): 68- 78.
22. W alsky, Robert L., R S. Obach, Emily A. Gaman, Jean-Paul R. Gleeson, and W illiam R. Proctor. "Selective Inhibition of Human Cytochrome P4502C8 by Montelukast." The American Society for Pharmacology and Experimental Therapeutics 33 (2005): 413-418.
23. W alsky, Robert L., and R S. Obach. "Validated Assays for Human Cytochrome P450 Activities." The American Society for Pharmacology and Experimental Therapeutics 32 (2004): 647-660.