WO2003008637A2

WO2003008637A2 - Use of genotyping in the individualization of therapy

Info

Publication number: WO2003008637A2
Application number: PCT/CA2002/001103
Authority: WO
Inventors: Brian Leyland-Jones
Original assignee: Xanthus Life Sciences, Inc.
Priority date: 2001-07-17
Filing date: 2002-07-17
Publication date: 2003-01-30
Also published as: WO2003008637A3

Abstract

The invention relates to the individualization of therapy. More specifically, the present invention relates to the use of genotyping for the individualization of therapy and/or individualization of drug dosing. More specifically, the present invention relates to the use of genotyping in the individualization of therapy with a therapeutic agent or a class of therapeutic agents.

Description

USE OF GENOTYPING IN THE INDIVIDUALIZATION OF THERAPY

BACKGROUND OF THE INVENTION

(a) Field of the Invention The invention relates to the individualization of therapy. More specifically, the present invention relates to the use of genotyping in the individualization of therapy and/or individualization of . drug dosing. More specifically, the present invention relates to the use of genotyping in the individualization of therapy with a therapeutic agent or a class of therapeutic agents.

(b) Description of the Prior Art

For the majority of drugs (or xenobiotics) administered to humans, their fate is to be metabolized in . the liver, into a form less toxic and lipophilic with their subsequent excretion in the urine. Their metabolism involves two systems that act consecutively: the cytochrome P450 system, which includes at least 20 enzymes catalyzing oxidation reactions and localized in the microsomal fraction, and the conjugation system which involves at least 5 enzymes. An enzyme of one system can act on several drugs and drug metabolites. Likewise, one drug or drug metabolite can be acted on by several enzymes. The rate of metabolism of a drug differs between individuals and between ethnic groups, owing to the existence of enzymatic polymorphism within each system. Furthermore, the rate of metabolism may be influenced by the concurrent metabolism of other drugs and substrates. As a result, a particular drug will be metabolized on an individual basis according to a variety of factors, including but not limited to individual genetic polymorphisms and metabolic capacity.

A metabolic phenotype is a functional profile of an individual's metabolic capacity. Metabolic phenotypes have been generally characterized for a plurality of enzymes, as poor metabolizers (PM) , . extensive metabolizers (EM) , and ultra-extensive metabolizers (UEM) .

To date, the ability to characterize individual phenotypes for the purpose of assessing drug treatment compatibility on an individual basis has been limited by the complexities of multiple metabolic pathways, and the lack of efficient and effective procedures for making these determinations . Currently, the determination of an individual's phenotype for a given metabolic enzyme can be performed either via direct metabolic phenotyping or indirect extrapolation of an individual's genotype to a given phenotype.

Direct phenotyping involves the use of a probe substrate known to be metabolized by a given enzyme. The rate of metabolism of the probe substrate is measured and this rate of metabolism is used to determine a metabolic phenotype. Although labor intensive and costly procedures for direct phenotyping have been known for many years these procedures are not readily adaptable for a clinical environment, nor are they practical for measuring multiple phenotypic determinants. For example, enzymatic phenotypes may be determined by measurements of the molar (or chiral) ratio of metabolites of a drug or a probe substrate in a urine sample from an individual by high-pressure liquid. chromatography (HPLC) , capillary electrophoresis (CE) or stereo-selective capillary gas chromatography. These determination methods are time-consuming, onerous, and employ systems and equipment that are not readily available in a clinical laboratory. Methodologies for the rapid determination of multiple determinants of a metabolic phenotypic are not available, and as a result, valuable information concerning an individual's phenotype is not considered on a routine basis in a clinical environment .

Indirect phenotyping is performed by analyzing the genetic sequence of a gene coding for a specific enzyme often by a polymerase chain reaction assay (PCR) or a PCR with a restriction fragment length polymorphism assay (PCR-RFLP) . The gene is examined for the presence of genetic mutations that can be linked to increased or decreased enzyme levels or activity, which in turn result in a specific phenotype, i.e. a poor metabolizer vs. an extensive metabolizer. The genotype is a theoretical measurement of what an individual ' s phenotype should be . Indirect phenotyping may be limited by several factors that can result in an alteration in the theoretical phenotype, such as enzyme inhibition or induction. Furthermore, the process of performing a complete genotyping can be quite complex. The mutation sequence must first be identified before they can be examined in a genotyping assay. Subsequent to identification, the mutation must be linked to a definitive effect on phenotype. For some enzymes, there appear to be very few mutations and those found have been well characterized, while for other enzymes multiple mutations are present with new mutations being found regularly (e.g. CYP2D6 has over 53 mutations and 48 allelic variants) . Therefore, while genotyping for CYP2C19 might be performed with relatively few measurements, a complete and accurate genotyping of CYP2D6 may be complex ^■ and require multiple measurements.

As indirect phenotyping suffers from complexity and the direct phenotyping techniques are time consuming and not readily accessible in clinical settings, physicians routinely prescribe treatment regimes without knowledge of an individual's metabolic capability (phenotype) or genotype for drug-specific metabolism. Accordingly, a trial and error treatment regime is initiated, often at the expense of severe side effects and loss of valuable treatment time. Furthermore, promising drug treatments often fail to meet regulatory approval due to isolated occurrences of toxicity in individuals with impaired metabolic capacity within a treatment group.

In order to gain approval from a governing regulatory body (e.g. FDA) a drug must be proven to be safe and effective. This currently involves the testing of the drug in normal healthy volunteers and in individuals with the disease the drug is designed to treat. Huge numbers of individuals are involved and these trials can take upwards of 7 years to complete.

The reason for the large number of individuals is to obtain statistical significance to prove the safety and efficiency of the drug.

New drug entities go through rigorous clinical trials prior to their approval for use in humans. These clinical trials ^■ are extremely lengthy and costly. During the course of clinical- testing, many promising new drug candidates are abandoned due to unacceptable toxicity profiles. In some cases the unacceptable toxicity occurs only in a minority of the general individual population. Often the occurrence of isolated toxicity is the result of a specific metabolic phenotype. Unfortunately, the ability to select defined individual populations for clinical trials has not been available on a routine basis, and this has resulted in the early termination of trials on otherwise promising new drug candidates .

As a result of the highly variable inter- individual rates of metabolism for some drugs, in which the variability is related to side-effects and/or efficacy, the inclusion of individuals with a phenotype or genotype making them prone to decreased drug response or to increased adverse effects can result in an overall decrease in the drug efficiency ratings or safety profiles of the trials. If this decrease in response rate or decreased safety profile is significant the drug may not be able to gain regulatory approval .

The ability to quickly and accurately screen individuals for their metabolic capacity prior to admission in a clinical trial could reduce the number of individuals required for participation and potentially allow the approval of promising drug treatments for a selective segment of the population, that otherwise would lack satisfactory response rates or safety profiles. Furthermore, the ability to perform metabolic screening in a clinical environment would provide physicians with a means for individualizing treatment regimes whereby an individual ' s genotypic and/or phenotypic metabolic profile could be used to determine a compatible drug treatment regime, and a corresponding individualized dose of that drug specific to that individual .

The ability to rapidly and accurately identify metabolic profiles on an individual basis would provide valuable individual-specific information that could be readily applied in the individualization of therapy. The ability to determine metabolic profiles on an individual basis may further support the selective use of otherwise unacceptable drug treatments having failed regulatory approval due to isolated toxicity as a result of metabolic inefficiencies.

SUMMARY OF THE INVENTION

One aim of the present invention is. 'to provide a method for selecting an individual treatment regime.

Accordingly, another aim of the present invention is to provide a method for the individualization of therapy. Yet another aim of the present invention is to provide a method for the individualization of therapy using genotyping.

Still another aim of the present invention is to provide a means for individualizing the dose of a desired treatment regime corresponding with an individual's metabolic profile.

The present invention is generally directed to the individualization of therapy. According to one embodiment, the present invention employs genotyping to identify individuals having risk factors for a given therapy. Preferably, the present invention employs genotyping to identify an individual's genotypic metabolic profile for use in determining an individual dosage regime for a given therapy or treatment. In this manner, an individual's genotype may be quantified with respect to one or more metabolic factors for the individualization of therapy. Alternatively, phenotyping may employed, together with genotyping or alone for the purpose of individualization of therapy according to the present invention.

According to one aspect of the present invention, metabolic determinants are employed in the individualization of therapy. These metabolic determinants may be genotypic and/or phenotypic determinants. According to an embodiment of the present invention, a metabolic profile based on at least one metabolic determinant may be provided for use in the individualization of therapy and/or drug dosing. The present invention may be employed in connection with the individualization of a variety of therapies and/or treatments characterized by a metabolic factor of interest, such as enzymatic and/or metabolic pathway activity.

In accordance with one aspect of the present invention there is provided a method of individualizing drug treatment for an individual, wherein an individualized dosage of a drug selected from a drug or class of drugs known for treating a condition is determined for said individual, said method comprising: determining a metabolic profile of said individual corresponding to at least one metabolic factor known to influence the metabolism of said class of drugs; and calculating said individualized dosage of said drug according to metabolic determinants specific for said at least one metabolic factor; wherein said metabolic determinants are correlated to a rate of drug metabolism specific of said individual and said individualized dosage is calculated therefrom.

In accordance with another aspect of the present invention there is provided a use of genotyping for the individualization of therapy and/or treatment, wherein an individual is genotyped for a specific metabolic factor and a corresponding genotypic determinant is characterized. In accordance with yet another aspect of the present invention there is provided a genomic assay for use in the individualization of therapy and/or treatment, said assay comprising: a means for identifying a genetic . marker corresponding to an individual's capacity for the metabolism of a given drug or class of drugs; a means for quantifying said genetic marker to provide an indicator of metabolic capacity specific for said drug or class of drugs; and a means for correlating said indicator with a therapeutically-effective dosage of said drug or class of drugs for said individual .

In accordance with another aspect of the present invention there is provided a method of using a genomic assay specific to a plurality of genotypic determinants for the individualization of therapy and/or treatment with a drug or class of drugs, said method comprising: a) genotyping a biological sample obtained from an individual to identify said plurality of genotypic determinants corresponding to metabolic factors of interest; b) calculating a rate of drug metabolism according to said plurality of genotypic determinants; and c) determining an individual dosage of said drug or class of drugs corresponding to said rate of drug metabolism; wherein said rate of drug metabolism is indicative of the rate of metabolism of said drug or class of drugs in said individual.

In accordance with still another of the present invention there is provided a method of selectively treating an individual with a drug or class of drugs; said method comprising: genotyping an individual to identify at least one allelic polymorphism known to influence the metabolism of said drug or class of drugs; phenotyping said individual to confirm their phenotypic capacity to metabolize said at drug of class of drugs; calculating a therapeutically-effective amount of said^' drug or class of drugs specific for said individual based on said genotyping and phenotyping; and selectively treating said individual with the same.

For the purpose of the present invention the following terms are defined below. The term "metabolic determinant" is intended to mean a qualitative or quantitative indicator of an metabolic-specific capacity of an individual.

The term "individualization" as it appears herein with respect to therapy is intended to mean a therapy having specificity to at least an individual's phenotype as calculated according to a predetermined formula on an individual basis .

The term "biological sample" is intended to mean a sample obtained from a biological entity and includes, but is not to be limited to, any one of the following: tissue, cerebrospinal fluid, plasma, serum, saliva, blood, nasal mucosa, urine, synovial fluid, microcapillary microdialysis and breath.

The term "therapeutic agent" is intended to mean an agent (s) and/or medicine (s) used to treat the symptoms of a disease, physical or mental condition, injury or infection.

The term "treatment" is intended to mean any administration of a pharmaceutical compound to an individual to treat, cure, alleviate, improve, diminish or inhibit a disease, physical or mental condition, injury or infection in the individual.

The term "individual treated" is intended to mean any individual being subjected to the administration of i) a pharmaceutical compound, for treating, curing, alleviating, improving, diminishing or inhibiting a disease, physical or mental condition, injury or infection, or ii) a probe substrate for determining multi-determinant metabolic phenotype.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates metabolites of the CYP2D6 enzymatic pathway according to another embodiment of the present invention; Fig. 2 illustrates metabolites of the CYP3A4 enzymatic pathway according to an embodiment of the present invention;

Fig. 3 illustrates metabolites of the NAT1 enzymatic pathway according to an embodiment of the present invention;

Fig. 4 illustrates metabolites of the CYP1A2 enzymatic pathway according to another embodiment of the present invention;

Fig. 5 illustrates metabolites of the CYP2A6 enzymatic pathway according to another embodiment of the present invention;

Fig. 6 illustrates metabolites of the CYP2C19 enzymatic pathway according to another embodiment of the present invention; Fig. 7 illustrates metabolites of the CYP2C9 enzymatic pathway according to another embodiment of the present invention; Fig. 8 illustrates metabolites of the CYP2E1 enzymatic pathway according to another embodiment of the present invention;

Fig. 9 illustrates metabolites of the NAT2 enzymatic pathway according to another embodiment of the present invention;

Fig. 10 illustrates the synthetic routes for the production of AAMU and IX derivatives used in accordance with one embodiment of the present invention;

Figs. 11 to 14 show other AAMU and IX derivatives which can be used for raising antibodies in accordance with another embodiment of the present invention; Fig. 15 illustrates the absorbance competitive antigen ELISA curves of AAMU-Ab and lX-Ab in accordance with one embodiment of the present invention;

Fig. 16 is a histogram of molar ratio of AAMU/IX; Fig. 17 illustrates an ELISA array in accordance with an embodiment of the present invention;

Fig. 18 illustrates an ELISA array in accordance with an embodiment of the present invention;

Fig. 19 illustrates an ELISA detection system in accordance with another embodiment of the present invention;

Fig. 20 illustrates a rapid immunoassay system in accordance with another embodiment of the present invention; and Fig. 21 illustrates individualized dosing schemes for direct vs . indirect phenotyping in accordance with yet another embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the individualization of therapy and/or treatment. In particular, the present invention relates to the individualization of therapy and/or treatment with a given drug or drugs . Based on a genotypic and/or phenotypic characterization of an individual's capacity to metabolize a given drug or drugs, the present invention also provides a system and method for determining an individualized dosage of that drug(s) based thereon.

For example, a majority of antihistamine agents are metabolized by either the CYP3A4 enzyme, (e.g. astemizole, ebastine, epinastine, loratadine, and terfenadine) or the CYP2D6 enzyme (e.g. chlorpheniramine, mequitazine, promethazine, cinnarizine, and flunarizine) . According to one aspect of the present invention, a method for quickly and accurately determining a metabolic determinant (s) for at least one enzyme and/or metabolic pathway known to factor into the metabolism of a given drug(s), that can be used to characterize an individual's metabolic profile specific for the metabolism of that drug is provided. In doing so, a characterization of an individual's ability to metabolize a given treatment agent or drug can be made and a corresponding drug dosage specific to the metabolic capacity of that individual can be determined. However, the present invention is not limited to any one treatment or therapy or class of drugs, but may be employed in accordance with any treatment, therapy or class of drugs that may be characterized in accordance with a metabolic factor, such as enzymatic and/or metabolic pathway activity. Table 1 exemplifies, without limitation, a plurality of drugs and/or classes of drugs and metabolic factors associated therewith that may be employed in connection with the individualization of therapy of the present invention.

Table 1

DRUG OR CLASS OF DRUGS METABOLIC FACTOR

Amonafϊde NAT2; CYP1A2

Irnrnunosuppressants CYP3A4

Antidepressants CYP2D6; CYP3A4

Antipsyc otics CYP2D6

Anxiolitics CYP3A4

Antiarryhthmics CYP2D6; CYP3A4

Hyperlipidemia CYP3A4

Erectile Dys uction Agents CYP3A4

GERD Agents CYP3A4

Antineoplastic Agents CYP3A4

Analgesics CYP3A4; CYP2D6

Antibiotics CYP3A4

Alzheimer's Disease Agents CYP3A4

Antihistamines CYP3A4; CYP2D6

Anesthetics CYP2E1 Further, the present invention provides a method for determining multiple metabolic determinants that can be used to characterize a metabolic profile of an individual that^" will exemplify that individual's ability to metabolize a given drug or group of drugs. Although most drugs are metabolized by a primary enzymatic pathway, such as CYP3A4 or CYP2D6 metabolize many antihistamine agents (drugs) , for example, it is often the case that a given drug may be metabolized by multiple enzymes and/or metabolic pathways. As a result, it may be preferred to characterize an individual's metabolic profile for a plurality of metabolic enzymes prior to selecting a corresponding drug treatment regime . Knowledge of an individual's metabolic profile, as determined by genotyping, may be applied clinically in determining a specific drug dosage based on the individual's capacity to metabolize the drug. Alternatively, a metabolic profile as determined by phenotyping, or a combination of phenotyping and genotyping may be employed in accordance with the present invention for purpose of individualization of therapy or treatment. Other factors representing an individual's capacity to metabolize a .drug may also find application in the present 'invention, together with a metabolic profile for providing the individualization of therapy.

According to one aspect of the present invention, an individual's metabolic profile is characterized on the basis of metabolic factors specific to a given therapy or treatment by genotyping. For example, genotyping may be employed to identify an allelic variation in an individual corresponding to a metabolic factor of inter.est, such as enzymatic activity specific to a candidate drug treatment or therapy. Metabolic determinants based on the identified allelic variations are subsequently characterized and used to quantify an individual dosage regime for that individual. In this regard, genotyping may be employed alone or in combination with phenotyping for the purpose of (1) individualizing a dosage regime for a given treatment or therapy; and (2) identifying a metabolic profile representative of a high risk individual with respect to a given treatment or therapy. A system of the present invention is exemplified in accordance with a protocol for determining phenotypic determinants for NAT2. This protocol is adapted to provide a system for determining phenotypic determinants for a specific enzyme or metabolic pathway, such as for example CYP3A4 or CYP2D6, identified as a metabolic factor for a specific drug or drug class of interest, in accordance with the present invention. The determination of at least one metabolic determinant for a specific enzyme or metabolic pathway may be performed as a single determination or in combination with methods for determining a metabolic profile including other metabolic factors of interest. A metabolic profile, according to an embodiment of the present invention may be specific to at least one of the following enzymes, without limitation: NAT1 , NAT2 ,

CYP1A2, CYP2A6, CYP2D6, CYP3A4 , CYP2E1, CYP2C9 and CYP2C19, the metabolites of which are illustrated in Figs. 1-9 and/or metabolic pathways related thereto. These enzymes are involved in the metabolism of a large number of drugs, and as a result have important implications in the outcome of many individual drug treatment regimes, as well as clinical trial studies. These enzymes and their corresponding metabolic determinants as described herein are provided as a representative example of determinants for the purposes of exemplifying the present invention. However, the present invention is not limited thereto. The present invention further provides a corresponding protocol for providing genotypic determinants for drug-specific metabolic factors, including without limitation: NAT1, NAT2, CYP1A2, CYP2A6, CYP2D6, CYP3A4 , CYP2E1, CYP2C9 and CYP2C19, and/or metabolic pathways related thereto.

One aspect of the present invention provides the characterization of a plurality of metabolic determinants specific to the metabolism of a plurality of drugs and/or classes of drugs for use in the individualization of treatment therewith. The present invention provides a method for individualizing a dosage regime that corresponds to an individual's metabolic capacity for a selected drug. As mentioned above, metabolic determinants may be characterized by genotyping, phenotyping or a combination thereof. In accordance with the present invention, a metabolic determinant may be employed in identifying an individual's metabolic capacity for a given drug, or drug class. Both CYP3A4 and CYP2D6 play a major role in the metabolism of many classes of drugs and hence, CYP3A4 and CYP2D6 are often considered when identifying an individual's capacity for the metabolism of many drugs. As such, metabolic determinants employed by the present invention for the individualization of therapy often encompass CYP3A4 and/or CYP2D6-specific metabolic factors, for example. Other enzymes and/or metabolic pathways may also be involved in the metabolism of a given class of drugs, such as CYP1A2 and CYP2C9, for example. As such, the present invention is not intended to be limited to any one metabolic factor but provides a means for determining metabolic determinants of any enzyme and/or metabolic pathway known to influence the metabolism of a given drug or class of drugs.

The present invention may further include the use of genotyping to identify individuals having a particular allelic variation known to influence the ability to effectively metabolize a given drug or class of drugs. For example, such an allelic variation may be associated with extremely high risks of toxicity from a given drug treatment regime . According to one embodiment of the present invention, those individuals without the "high risk" genotype may be subsequently phenotyped and an individualized dose determined according a at least one phenotypic determinant while the high risk individuals can be readily identified and removed from consideration for the given drug treatment regime. By employing genotyping in combination with phenotyping to screen individual's for treatment with a given treatment regime, those individuals found to be carrier of a high risk genotype can be eliminated as candidates for such treatment without the necessity of phenotyping .

Alternatively, genotyping may be employed alone, in accordance with an embodiment of the present invention, to characterize a metabolic determinant and identify an individualized dosage for a given drug treatment regime .

The clinical use of a genotypic or phenotypic screening method of the present invention provides the ability to individualize treatments according to metabolic profiles. In particular, dose specific determinations corresponding to a calculated rate of metabolism is possible on an individual basis. The integration of genotyping and/or phenotyping tests into the drug development process may also provide for a decreased number of individuals participating in a drug treatment testing trial, as individual screening can be conducted prior to the trial to select those individuals displaying the capability to metabolize the drug of interest safely and effectively. In particular, those individuals identified as being metabolically incompatible with the drug treatment trial can be screened out before undergoing treatment with the drug. This aspect of the present invention provides a means to selectively treat only those individuals identified as having an ability to safely metabolize the drug. In addition, the decrease in individual number will result in decreased costs and allow the drug to reach the market faster. Likewise, the present invention provides a method for identifying those individuals who would be at risk with a particular drug treatment regime.

Pre-trial or pre-treatment screening according to the present invention would include the phenotyping and/or genotyping of all individuals. The metabolic profiles for these individuals could then be used to identify those at high risk for serious adverse events (SAE's) and ensure that they were not included in. the trial or did not receive the given drug treatment regime. The individuals found not to be at risk with respect to ^■ the given drug treatment regime would then be treated with individualized drug doses corresponding to at least one metabolic determinant associated with a metabolic factor for the given drug treatment. The individualized dose would ensure that the each individual received a safe efficacious treatment, corresponding to their ability to safely metabolize the drug. Similarly, according to the present invention, individualized treatment has application in the clinical environment where drug treatment dosages will be customized according to an individual's metabolic profile or calculated rate of metabolism.

Every individual is genetically predisposed for metabolic function and accordingly, everyone does not metabolize a given drug to^' the same extent and efficiency. Furthermore, certain enzymes may be inhibited by the metabolism of other agents administered on a coterminous basis, thus effecting an individual's metabolic capacity at any given time. Despite the factors effecting an individual's metabolic capacity with respect to a given enzyme, the phenotypic result will be a decreased clearance or efficiency in the metabolism of certain agents (drugs) , and hence the possibility of adverse side effects. In turn, this result can have serious, if not life threatening effects for an individual being treated with an agent for which they are not capable of effective metabolism. For example, certain individuals having a genetic variation resulting in the absence or reduction of CYP2D6 will display an inability to effectively metabolize many drugs such as codeine, tramadol . Clearly, individual variations in CYP2D6 metabolic activity at any given time can have significant impacts on the rates of clearance of CYP2D6-specific drugs and hence the efficacy thereof. For these reasons, the utility of a reliable screening test specific to a metabolic factor such as an enzyme (s) and/or metabolic pathway (s) involved in the metabolism of a given drug or class of drugs, such as CYP2D6 and/or other enzymes, is evident . According to the present invention, metabolic determinants for one or more of the following enzymes may be characterized to provide a metabolic profile on an individual basis:

CYP2D6

CYP2D6 constitutes 1-3% of the total CYP 450 enzymes in the human liver. CYP2D6 has been postulated as participating in approximately 20% of drug metabolism. POLYMORPHISM

CYP2D6 was the first P450 enzyme to demonstrate polymorphic expression in humans. Three metabolic phenotypes can be distinguished: poor, PM, extensive (EM) and ultraextensive (UEM) phenotypes. The CYP2D6 gene is extensively polymorphic, for example, a 1997 study documented 48 , mutations and 53 alleles of the CYP2D6 gene in a screen of 672 unrelated individuals. Examples of alleles with normal (extensive, wild-type function) CYP2D6*1, CYP2D6*2A, CYP2D6*2B; alleles resulting in an absence of function CYP2D6*3, CYP2D6*4A, CYP2D6*4B, CYP2D6*5, CYP2D6*6A, CYP2D6*6B, CYP2D6*7, CYP2D6*8, CYP2D6*11 & CYP2D6*12; and alleles resulting in a reduced function, CYP2D6*9, CYP2D6*10A, CYP2D6*10B. The ultra-extensive phenotype appears to arise from the presence of multiple copies of the CYP2D6 gene (for example, one individual was identified with 13 copies of the gene) .

CYP2D6 metabolizes a large variety of drugs and dietary constituents including, but not limited to the following:

Antihistamines : Chlorpheniramine, mequitazine, promethazine, cinnarizine, flunarizine, loratadine, and terfenadine . Analgesics: codeine, tramadol, ethylmorphine , oxycodone, dihyrocodeine, norcodeine, acetaminophen, phenacetin, methadone .

Psychotropic drugs : amiflamine, amitryptyline, clomipramine, clozapine, desipramine, haloperidol, imipramine, maprotiline, methoxyphenamine , minaprine, norti-iptyline, paroxetine, perphenazine, remoxipride, thioridazine, tomoxetine, trifluperidol, zuclopenthixol , risperidone, fluoxetine.

Cardiovascular agents : aprindine, buf ralol , debrisoquine, encainide, flecainide, guanoxan, indoramin, metoprolol, mexiletin, n-propylamaline, propafenone, propranolol, sparteine, timolol, verapamil .

Miscellaneous agents: chlorpropamide, dextromethorphan, methamphetamine, perhexilene, phenformin.

In addition, CYP2D6 is involved in the metabolism of many carcinogens, however as yet is not reported as the major metabolizer for any. In one study it has been shown that individuals who are fast CYP2D6 metabolizers and slow N-acetylators are at a greater risk for hepatocellular cancer (OR=2.6 ; 95% CI=1.6-4).

INDUCTION AND INHIBITION

CYP2D6 is inhibited in vi tro by quinidine and by viral protease inhibitors as well as by appetite suppressant drugs such as D- and L-fenfluramine.

INTER ETHNIC DIFFERENCES

The activity of CYP2D6 varies broadly in a given population. Poor (PM) , extensive (EM) and ultraextensive (UEM) phenotypes of CYP2D6 have been distinguished. The CYP2D6 gene is inherited as an autosomal recessive trait and separates 90 and 10% of the white European and North American population into extensive (EM) and poor (PM) metabolizer phenotypes respectively. In another study the percentage of PM in different ethnic populations was observed, and white North Americans and Europeans have 5-10% PM's, American blacks, 1.8%, Native Thais, 1.2%, Chinese 1%, Native Malay population, 2.1%, while the PM phenotype appears to be completely absent in the Japanese population.

It is reasonable that, in drug metabolism studies, each ethnic group can be .studied separately for evidence of polymorphism and its antimode should not be extrapolated from one ethnic population to another.

DEXTROMETHORPHAN/ANTIDEPRESSANTS An example of the need for the individualization of therapy and/or drug dosing is the case of dextromethorphan . According this example, CYP2D6 is identified as a metabolic factor known to influence the metabolism of dextromethorphan. Dextromethorphan is a nonopioid antitussive with psychotropic effects.

However, dextromethorphan doses range from 0 to 6 mg/kg based on individual subject tolerance. Dextromethorphan is activated via the CYP2D6 metabolizing system.

^■ Dextromethorphan produced qualitatively and quantitatively different objective and subjective effects in poor vs. extensive metabolizers (mean performance +/-SE, 95+/-0.5% for EMs vs. 86+/-6% for PMs; p<0.05) .

Another important drug that exemplifies the need for the individualization of therapy and/or drug dosing where CYP2D6 is a suitable metabolic factor is in the case of tricyclic antidepressants . Both the PM and UEM phenotypes of CYP2D6 are at risk of adverse reactions. PM individuals given standard doses of these drugs will develop toxic plasma concentrations, potentially leading to unpleasant side effects including dry mouth, hypotension, sedation, tremor, or in some cases life- threatening cardiotoxicity. Conversely, administration of these drugs to UEM individuals may result in therapeutic failure because plasma concentrations of active drugs at standard doses are far too low. For, these reasons, the utility of a reliable methodology for the individualization of therapy and/or drug dosing is evident.

PHENOTYPIC DETERMINANTS OF CYP2D6

Different probe substrates can be used to determine the CYP2D6 phenotype (dextromethorphan, debrisoquine, bufuralol, antipyrine, theophylline and hexobarbital) . In accordance with the present invention, suitable probe substrates include without limitation, dextromethorphan, debrisoquine, bufuralol.

Of these dextromethorphan is the preferred probe. The structure of dextromethorphan and its demethylated metabolite dextrorphan are illustrated in Fig. 1.

In accordance with the present invention, the molar ratio of dextromethorphan and its metabolite is used to determine the CYP2D6 phenotype of the individual as follows: dextromethorphan dextrorphan

An antimode of 0.30 is used to differentiate between extensive and poor metabolizers whereby an antimode of less than 0.30 indicates an extensive metabolizer and greater than 0.30 indicates a poor metabolizer.

GENOTYPIC DETERMINANTS OF CYP2D6 (GENOTYPING)

As mentioned previously the CYP2D6 gene is extensively polymorphic with one study identifying 48 mutations and 53 alleles. An example of a procedure for genotyping CYP2D6 involves the amplification of the entire CYP2D6 coding region (5.1kb product) by XL-PCR using specific primers. This product is then used for a series of polymerase chain reaction - restriction fragment length polymorphism reactions designed to detect nucleotide point mutations, deletions and insertions compared with the functional CYP2D6*1 allele (Garcia-Barcelό et a.1 . (2000) Clinical Chemistry 46 (1) :18-23) . For example, to detect the C188T transition mutation the following primers can be used to first amplify the CYP2D6 gene and then the specific region of the mutation:

Full CYP2D6 gene

5 ' -CCAGAAGGCTTTGCAGGCTTCA-3 ' SEQ ID NO . 1 5" -ACTGAGCCCTGGGAGGTAGGTA-3 ' SEQ ID .NO. 2 C188T Mutation

5'-CCATTTGGTAGTGAGGCAGGTAT-3' SEQ ID NO . 3 5'-CACCATCCATGTTTGCTTCTGGT-3' SEQ ID NO . 4

The presence of the C188T mutation is then detected by digestion with the Hphl restriction enzyme.

In general, the most frequent mutations are examined and these correspond to the most frequent alleles and genotypes. Those individuals with at least one allele encoding a functional enzyme are identified as extensive metabolizers, while individuals lacking two or more functional CYP2D6 alleles are identified as poor metabolizers.

CYP3A4

The CYP3A family constitutes approximately 25% of the total GYP 450 enzymes in the human liver.

POLYMORPHISM

A large degree of inter-individual- variability in the expression of the CYP3A4 isoenzymes has been shown in the human liver (>20 fold) . However, the activity of CYP3A4 metabolism is distributed unimodally and as a result, there is currently no categorical classifications for distinct subsets of this population. Further, there is currently no evidence of a common allelic variant in the coding region of the gene. Recently, a rare allelic variant was identified in exon 7 (CYP3A4*2) . Limited data suggested that this mutation may result in altered substrate dependant kinetics compared with the wt CYP3A gene. It has been considered that the large inter-individual variability in the activity of CYP3A may reflect differences in transcriptional regulation. Another allelic variant in the 5 '-flanking region of CYP3A has been identified

(CYP3A4*1B) that involves an A—»G transition at position

-290 from the transcriptional initiation site. It has been speculated that this nucleotide substitution may be associated with a reduced level of CYP3A activity.

Ongoing studies are investigating the existence of a common allelic variant linked to CYP3A4 activity.

CYP3A4 metabolizes several drugs and dietary constituents including:

Antihistamines : astemizole, ebastine, epinastine, loratadine, terfenadine, azelastine and rupatadine.

Analgesics : diclofenac, tazofelone, meloxicam, alfentanil, fentanyl and celecoxib. Hyperlipidemia agents: _. lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, rosuvastatin, benzafibrate, clofibrate, fenofibrate, ge fibrozil, and niacin.

Miscellaneous : benzydiamine, benzodiazepines, erythromycin, dextromethorphan dihydropyridines, cyclosporine, lidocaine, midazolam, nifedipine, and terfenadine .

In addition, CYP3A4 activates environmental pro- carcinogens especially N' -nitrosonornicotine (NNN) , 4-methylnitrosamino-l- (3- pyridyl- 1 -butanone) (NNK), 5 -Methyl chrysene , 4 , 4 ' -methylene-bis ( 2 -chloroaniline)

(tobacco smoke products) .

INDUCTION AND INHIBITION

CYP3A4 is induced by a number of drugs including dexamethasone , phenobarbital , primidone and the antibiotic rifampicin . Conversely, CYP3A4 is inhibited by erythromycin, grapefruit j uice , indinavir, ketoconazole , miconazole , quinine , and saquinavir .

INTER ETHNIC DIFFERENCES

Several studies have suggested that the activity of CYP3A4 varies between populations. Plasma levels of a CYP3A4 substrate drug after oral administration were reported to be twofold to threefold higher in Japanese, Mexican, Southeast Asian and Nigerian Populations compared with white persons residing in various countries. In addition, the CYP3A4*1B allelic has been reported to be more frequent in African-American populations as compared to European Americans or

Chinese populations (66.7% vs. 4.2% vs. 0%). The rare CYP3A4*2 allele was found in 2.7% of a white population and was absent in the black and Chinese subjects. It is reasonable that, in drug metabolism studies, each ethnic group can be studied separately for evidence of polymorphism and its antimode should not be extrapolated from one ethnic population to another.

Due to the variability in CYP3A4 activity within the population it would be advantageous to be provided with a system and method for quickly and easily determining an individual's CYP3A4 metabolic phenotype prior to administering a CYP3A4-dependant treatment thereto. In particular, such a system and method is believed to have' enormous benefit in the individualization of therapy.

CYCLOSPORINE

An example of the need for individualization of therapy and/or drug dosing is the case of cyclosporine in the treatment of organ transplant individuals. According to this example, CYP3A4 is characterized as a metabolic factor identified to influence the metabolism of cyclosporine. Cyclosporine is an immunosuppressant agent (drug) administered post transplant to protect the new organ from being rejected. Plasma levels of this drug are critical as high levels lead to renal toxicity but low levels can lead to organ rejection. Cyclosporine is metabolized via the CYP3A4 system. Several studies have indicated the importance of monitoring CYP3A4 activity in maintaining an effective and safe cyclosporine dose. For these reasons, the utility of a reliable methodology for the individualization of therapy and/or drug dosing is evident .

DIRECT PHENOTYPIC DETERMINANTS OF CYP3A4

Different probe substrates can be used to determine the CYP3A4 phenotype (dapsone, testosterone, nifedipine, midazolam, erythromycin, dextromethorphan) . In accordance with the present invention, suitable probe substrates include without limitation, midazolam, dextromethorphan, erythromycin, dapsone, testosterone, nifedipine .

Of these midazolam is the preferred probe. The structures of midazolam and its hydroxylated metabolite, 1 ' -hydroxymidazolam are illustrated in Fig. 2. In accordance with the present invention, the molar ratio of midazolam and its metabolite is used to determine the CYP3A4 phenotype of the individual as follows:

-hydroxymidazolam midazolam

An individual's ratio will be considered as indicative of CYP3A4 enzyme activity with a lower ratio indicating poorer metabolism and a higher ratio indicating more extensive metabolism. The activity of

^' CYP3A4 metabolism is distributed unimodally and hence no antimode is present. The levels of CYP3A4 activity as determined by direct phenotyping will be used.

GENOTYPIC DETERMINANTS OF CYP3A4 (GENOTYPING)

To date only two mutant alleles have been identified for the CYP3A4 gene (CYP3A4*1B & CYP3A4*2) . Studies have been unable to correlate these mutations with the large inter-individual variation in CYP3A4 activity. Despite confirmation in this regard to date, the use of genotyping is contemplated in accordance with the present invention. Ongoing studies continue to investigate this aspect of the present invention.

NAT1 The NAT1 enzyme catalyzes the N-acetylation of many compounds. It is expressed in the liver as well as in mononuclear leucocytes.

POLYMORPHISM The NAT1 gene was for a long time classified as monomorphic. However, it is now suggested that NAT1, like the other N-acetyltransferase gene (NAT2) , is polymorphic- Studies have demonstrated the presence of one wild type allele (NAT1*4) and six mutant alleles (NAT1*3, NAT1*5, NAT1*10, NAT1*11, NAT1*14 and NAT1*17) . NAT1 has two phenotypes of slow and rapid acetylators (e.g. NAT1*4 vs. NAT1*10 genotypes respectively) .

NAT1 metabolizes several drugs and dietary constituents including p-aminobenzoic acid, p-aminosalicylic acid, and dapsone.

In addition, NAT1 activates environmental pro- carcinogens especially diaminobenzidine, N-hydroxy-4- aminobiphenyl ; heterocyclic aromatic amines (MelQx and PhlP) . In one study it has been shown that individuals who have the NAT1*10 allele, and hence are rapid N-acetylators, are at a greater risk for colorectal cancer (0R=1,9; 95% CI=1.2-3.2), while in another study they have an increased risk for bladder cancer (metabolize benzidine) . INTER ETHNIC DIFFERENCES

The activity of NAT1 varies broadly in a given population. Slow, and rapid NAT1 phenotypes have been distinguished. The NAT1*10 genotype that is associated with rapid metabolic phenotype was monitored in three different ethnic populations, Indian, Malaysian and Chinese. The frequency of NAT1*10 allele was 17%, 39% and 30% respectively. While the NAT1*4 genotype associated with slow metabolizers had a frequency in the same populations of 50%, 30% and 35% respectively. Therefore, it is reasonable that, in drug metabolism studies, each ethnic group can be studied separately for evidence of polymorphism and its antimode should not be extrapolated from one ethnic population to another .

DAPSONE

A classical example of the need for the individualization of therapy and/or drug dosing is the case of dapsone. According to this example, NAT1 is characterized as a metabolic factor identified to influence the metabolism of dapsone. Dapsone is used in the treatment of malaria and is being investigated for the treatment of Pneumocystis carinii pneumonia in AIDS individual. Adverse effects include rash, anemia, methemoglobinemia, agranulocytosis, and hepatic dysfunction. Dapsone is cleared from the body via the

NAT1 metabolizing system. A study has shown a correlation between slow acetylation and increased adverse reactions to dapsone (46% vs. 17% for slow and fast acetylators respectively) . For these reasons, the utility of the present invention is evident .

PHENOTYPIC DETERMINANTS OF NAT1

Different probe substrates can be used to determine the NAT1 phenotype, such as (p-aminosalicylic acid (pASA) , p-aminobenzoic acid (pABA) ) . In accordance with the present invention suitable probe substrates include with out limitation p-aminosalicylic acid, p-amihobenzoic acid.

Of these pASA is the preferred probe. The structure of pASA and its acetylated metabolite p-acetylaminosalicylic acid are illustrated in Fig. 3. In accordance with the present invention, the molar ratio of pASA and its acetylated metabolite is used to determine the NAT1 phenotype of the individual as follows : pASA pAcetyl-ASA

GENOTYPIC DETERMINANTS OF NAT1 (GENOTYPING)

The NAT1 alleles NAT1*4 (wt) and the mutant NAT1*14 can be determined either by PCR-RFLP and allele specific PCR (Hickman, D. et al . (1998); Gut 42:402- 409) . The PCR-RFLP methodology requires the amplification of the fragment of gene containing the A560G mutation. This is performed with the following primers : 5'-TCCTAGAAGACAGCAACGACC-3' SEQ ID NO. 5

5'-GTGAAGCCCACCAAACAG-3 ' SEQ ID NO . 6

This PCR amplification produces a 175 bp fragment that is incubated with the Bsal restriction enzyme. The Natl*4 allele is cleaved and produces a 155 bp fragment, the mutant NAT1*14 is uncleaved.

The NAT1*14 allele is confirmed using an allele specific PCR, with the following primers:

5'-TCCTAGAAGACAGCAACGACC-3' SEQ ID NO. 5 5 ' -GGCCATCTTTAAAATACATTTT-3 ' SEQ ID NO . 7

CYP1A2

CYP1A2 constitutes 15% of the total CYP 450 enzymes in the human liver.

POLYMORPHISM

CYP1A2 may be polymorphic although it remains to be established firmly. To date no mutant alleles have been identified, however, research continues in accordance with this aspect of the present invention. Three metabolic phenotypes can be distinguished: rapid, intermediate and slow metabolizers. CYP1A2 metabolizes several drugs and dietary constituents including acetaminophen, phenazone, naproxen, anti pyrine, 17 β-estradiol, caffeine, cloipramine, clozapine, flutamide (antiandrogenic) , imipramine, paracetamol, phenacetin, tacrine and theophylline .

In addition, CYP1A2 activates environmental pro- carcinogens especially heterocyclic amines and aromatic amines. In one study it has been shown that individuals who are fast N-acetylators and have high CYP1A2 activity are at a greater risk for colorectal cancer (35% of cases vs. 16% of controls, OR=2.79 (P=0.00-2).

INDUCTION AND INHIBITION

CYP1A2 is induced by a number of drugs and environmental factors such as omeprazole, lansoprasole, polyaromatic hydrocarbons and cigarette smoke. CYP1A2 is inhibited by oral contraceptives, ketoconazole, α-napthoflavone, fluvoxamine (seronine uptake inhibitor), furafylline.

INTER ETHNIC DIFFERENCES The activity of CYP1A2 varies broadly (60 to 70 fold) in a given population. Slow, intermediate and rapid CYP1A2 phenotypes have been distinguished. The proportion of these three CYP1A2 phenotypes varied between ethnic groups and countries : % of intermediates: 50, 70, 60, >95, 60, 20 in U.S.A., African-American, China, Japan, Italy and Australia respectively. It is reasonable that, in drug metabolism studies, each ethnic group can be studied separately for evidence of polymorphism and its antimode should not be extrapolated from one ethnic population to another . THEOPHYLLINE -

A classical example of the need for the individualization of therapy and/or drug dosing is the case of theophylline. According to this example, CYP1A2 is characterized as a metabolic factor identified to influence the metabolism of theophylline. Theophylline is used in the treatment of asthma. However, theophylline toxicity continues to be a common clinical problem, and involves life-threatening cardiovascular and neurological toxicity. Theophylline is cleared from the body via the CYP1A2 metabolizing system. Inhibition of CYP1A2 by quinolone antibiotic agents or serotonine reuptake inhibitors may result in theophyline toxicity. For these reasons, the utility of a methodology for the individualization of therapy and/or drug dosing is evident .

DIRECT PHENOTYPIC DETERMINANTS OF CYP1A2

Different probe substrates can be used to determine the CYP1A2 phenotype (caffeine, theophylline) . In accordance with the present invention suitable probe substrates include without limitation, caffeine, theophylline or acetaminophen.

Of these caffeine is the preferred probe. Caffeine is widely consumed and relatively safe. The structure of caffeine and its metabolites

1,7-dimethylxanthine (1,7 DMX) and 1 , 7-dimethyluric acid (1,7 DMU) are illustrated in Fig. 4.

In accordance with the present invention, the molar ratio of caffeine metabolites is used to determine the CYP1A2 phenotype of the individual as follows :

1,7-dimethylxanthine (1,7 DMX) + 1, 7-dimethyluric acid (1,7 DMU) /caffeine

Molar ratios of 4 and 12 separate slow, intermediate and fast CYP1A2 metabolizers (Butler et al . (1992) Pharmacogenetics 2:116-117).

GENOTYPIC DETERMINANTS OF CYP1A2 (GENOTYPING)

Despite the lack of mutant alleles identified for the CYP1A2 gene to date, the use of genotyping to identify allelic variants with respect to CYP1A2 is contemplated in accordance with the present invention.

CYP2A6

CYP 2A6 constitutes 4% of the total CYP 450 enzymes in the human liver. CYP2A6 is estimated as participating in 2.5% of drug metabolism.

POLYMORPHISM

CYP 2A6 is functionally polymorphic with two mutant alleles, CYP2A6*2 & CYP2A6*3, resulting in an inactive enzyme or the absence of the enzyme respectively. Two metabolic phenotypes can be distinguished: poor and extensive metabolizers. CYP2A6 metabolizes several drugs including neuroleptic drugs and volatile anaesthetics as well as the natural compounds, coumarin, nicotine and aflatoxin Bl .

In addition, CYP2A6 activates several components of tobacco smoke (e.g. NNK) , as well as 6-aminochrysene. The role of- activation of tobacco smoke and the metabolism of nicotine have suggested a role for CYP2A6 in the development of smoking related cancers .

INDUCTION AND INHIBITION

CYP2A6 is induced by barbiturates, antiepileptic drugs and corticosteroids .

INTER ETHNIC DIFFERENCES CYP2A6 demonstrates marked inter-individual variability and has demonstrated ethnic related differences. The proportion of the two phenotypes varied between ethnic groups and countries : % of wt genotype (extensive metabolizers) : 85, 76, 52, 83, 97.5 in Finnish, English, Japanese, Taiwanese and African- American populations respectively. It is reasonable that, in drug metabolism studies, each ethnic group can be studied separately for evidence of polymorphism and its antimode should not be extrapolated from one ethnic population to another.

NICOTINE

An example of the need for the individualization of therapy and/or drug dosing is in the delivery of nicotine, for a smoking cessation program. According to this example, CYP2A6 may be characterized as a metabolic factor of interest . CYP2A6 is the primary means of nicotine metabolism. Extensive CYP2A6 metabolizers will eliminate nicotine at a much higher rate. Identification of individuals with an increased CYP2A6 activity and hence increased nicotine metabolism may identify those individuals that will require higher doses of nicotine at the onset of their attempt to quit smoking with the assistance of a nicotine delivery system. Alternatively, these individuals may benefit from non-nicotine delivery systems for assisting in quitting smoking.

DIRECT PHENOTYPIC DETERMINANTS OF CYP2A6

A probe substrate can be used to determine the CYP2A6 phenotype, such as coumarin, for example. In accordance with the present invention suitable probe substrates include without limitation, coumarin. The structure of coumarin and its metabolite 7-hydroxycoumarin are illustrated in Fig. 5.

In accordance with the present invention, the molar ratio of coumarin and its metabolite, 7-hydroxycoumarin is used to determine the CYP2A6 phenotype of the individual as follows:

7-hydroxycoumarin coumarin GENOTYPIC DETERMINANTS OF CYP2A6 (GENOTYPING)

Currently three alleles have been identified for the CYP2A6 gene, the wild type allele (CYP2A6*1) and two mutant alleles (CYP2A6*2, CYP2A6*3). The wt allele codes for a fully functional enzyme. The CYP2A6*2 mutant al ele codes for an inactive enzyme and the CYP2A6*3 allele does not produce any enzyme.

Determination of an individual genotype can be performed by a combined LA-PCR & PCR-RFLP procedure . In this procedure, specific oliogonucleotide primers were used to amplify the CYP2A6/7 gene. The amplified CYP2A6/7 gene is then used as the PCR template to amplify exons 3 and^' 4 using specific oligonucleotide primers to amplify a 544 bp fragment. This fragment is then digested with the Fspl restriction enzyme and a 489 bp fragment re-isolated. This 489 bp fragment is then incubated with both Ddel and Xcml . The digestion patterns were determined by electrophoresis . The wildtype allele produces 330, 87 and 72 bp fragments, the CYP2A6*2 allele yields 189, 141, 87 and 72 ' bp fragments, and the CYP2A6*3 allele yields 270, 87, 72, 60 bp fragments (Nakaj ima et al . (2000) Clin Pharmacol & Ther. 67(1) :57-69) .

PRIMERS

CYP2A6/7 LA-PCR

5 ' -CCTCCCTTGCTGGCTGTGTCCCAAGCTAGGC-3 ' SEQ ID NO . 8

5 ' -CGCCCCTTCCTTTCCGCCATCCTGCCCCCAG-3 ' SEQ ID NO . 9 Exon 3 /4 PCR

5'-GCGTGGTATTCAGCAACGGG-3 ' SEQ ID NO . 10

5' -TGCCCCGTGGAGGTTGACG-3 ' SEQ ID NO. 11

CYP2C19

CYP2C19 accounts for about 2% of oxidative drug metabolism. CYP2C19 has been postulated as participating in approximately 8% of drug metabolism.

POLYMORPHISM

Individuals are genetically polymorphic with respect to CYP2C19 metabolism. Two metabolic phenotypes can be distinguished: extensive and poor metabolizers. Two genetic polymorphism have been identified (CYP2C19*2 and CYP2C19*3) that together explain all of the oriental poor metabolizers and about 83% of Caucasian poor metabolizers. Both of these mutations introduce stop codons resulting in a truncated and nonfunctional enzyme. CYP2C19 metabolizes a variety of compounds including the tricyclic antidepressants amitriptyline, imipramine and clomipramine, the sedatives diazepam and hexobarbital, the gastric proton pump inhibitors, omeprazole, pantoprazole, and lansoprazole, as well as the antimalarial drug proguanil and the β-blocker propanolol . INDUCTION AND INHIBITION

CYP2C19 is inhibited. by fluconazole, fluvoxamine, fluoxetine, sertraline, ritonavir and induced by rifampin.

INTER ETHNIC DIFFERENCES

The occurrence of the poor metabolizer phenotype for CYP2C19 shows a large inter ethnic variability. Poor metabolizers make up less than 4% of the European and white American populations. While the Korean population has a poor metabolizer frequency of 12.6%, the Chinese 17.4% and the Japanese 22.5%. In addition, the CYP2C19 mutant- alleles demonstrate interethnic variability with CYP2C19*2 frequency ranging from 28.9% in the Chinese population to only 13% in European- American population. The CYP2C19*3 allele is absent from the European-American or African-American populations, while occurring at a frequency of 11.7% in both the Korean and Japanese populations. It is reasonable that, in drug metabolism studies, each ethnic group can be studied separately for evidence of polymorphism and its antimode should not be extrapolated from one ethnic population to another .

OMEPRAZOLE

As an example, the benefit of individualization of therapy and/or drug dosing is evident in the case of omeprazole. According to this example, CYP2C19 is characterized as a metabolic factor identified to influence the metabolism of omeprazole. Omeprazole is a drug used in the treatment of H. pylori infections in conjunction with amoxicillin, and is cleared from the body via a CYP2C19 metabolic pathway. Studies have observed higher eradication rates of H pylori in CYP2C19 poor metabolizers. Therefore, extensive metabolizers may require higher doses of omeprazole to achieve the same level of H. pylori eradication observed in poor metabolizers. For these reasons, the utility of a reliable methodology for the individualization of therapy is evident. In particular, an accurate and convenient clinical assay would allow physicians to quickly identify safe and effective treatment regimes for individuals on an individual basis.

DIRECT PHENOTYPIC DETERMINANTS OF CYP2C19

In accordance with an embodiment of the present invention, the ratio of S-mephenytoin and R-mephenytoin in an urine sample may be used to provide a determination of an individual's CYP2C19 phenotype. These metabolites are used as quantitative markers in the determination of a CYP2C19 phenotype on the basis of the use of the preferred probe substrate mephenytoin. However, it is fully contemplated that the present invention is not limited in any respect thereto .

The structure of R-(-) and S- (+) mephenytoin and 4-hydroxymephenytoin are illustrated in Fig. 6. The chiral ratio of S-mephenytoin and R-mephenytoin metabolites, used to determine the CYP2C19 phenotype of the individual, is as follows:

S-Mephenytoin R-Mephenytoin

Chiral ratios of close to unity (>0.8) are indicative of fast CYP2C19 metabolizers.

GENOTYPIC DETERMINANTS OF CYP2C19 (GENOTYPING)

As mentioned previously the CYP2C19 has two predominant variant alleles, which account for all Japanese poor metabolizers and 83% of Caucasian poor metabolizers. Studies have demonstrated an excellent correlation between a homozygous presence of mutant alleles and poor metabolizer status. An example of a procedure for genotyping CYP2D6 involves the a series of polymerase chain reaction - restriction fragment length polymorphism reactions designed to detect nucleotide point mutations, deletions and insertions compared with the functional CYP2C19*1 allele (Furuta et al . (1999) Clin Pharmacol Thera 65 (5) :552-561; Tanigawara et al . (1999) Clin Pharmacol Thera 66(5) : 528-5534) . PCR amplification of exon 5 or exon 4 for CYP2C19*2 and CYP2C19*3 respectively are performed using the following primers : CYP2C19*2 Exon 5 Primers

5 ' -AATTACAACCAGAGCTTGGC-3 ' . SEQ ID NO. 12

5 ^■ -TATCACTTTCCATAAAAGCAAG-3 ' SEQ ID NO. 13

CYP2C19*3 Exon 4 Primers 5 ' -AACATCAGGATTGTAAGCAC-3 ' SEQ ID NO. 14 5 ' -TCAGGGCTTGGTCAATATAG-3 ' SEQ ID NO. 15

The presence of the G681A mutation in CYP2C19*2 is then detected by digestion with the Smal restriction enzyme. The wild type allele will produce a 120 and a 49 bp fragment, while the CYP2C19*2 allele will remain uncleaved. The CYP2C19*3 allele is detected by incubating the exon 4 PCR product with BamHl . The wild type allele will produce a 233 bp and a 96 bp fragment while the CYP2C19*3 allele will remain uncleaved.

Those individuals with at least one allele encoding a functional enzyme are identified as extensive metabolizers, while individuals lacking two or more functional CYP2C19 alleles are considered poor metabolizers .

CYP2C9

The CYP2C9 family of metabolic enzymes accounts for approximately 8% of the metabolic enzymes in the liver. CYP 2C9 has been postulated as participating in approximately 15% of drug metabolism. POLYMORPHISM

Individuals are genetically polymorphic with respect to CYP 2C9 metabolism. Two metabolic phenotypes can be distinguished: extensive^' and poor metabolizers. Three genetic polymorphism have been, definitively identified, one wild type (CYP2C9*1) and two mutant

(CYP2C9*2 and CYP2C9*3) . The CYP2C9*2 allele was found to result in 5-10 fold increase in expression of mRNA and have 3 -fold higher enzyme activity for metabolism of phenytoin and tolbutamide. Conversely, this genotype appears to have a lower level of activity for the metabolism of S-warfarin. The CYP2C9*3 allele appears to demonstrate decreased metabolic activity against all three of these substrates . CYP2C9 metabolizes a variety of compounds including S-warfarin, phenytoin, tolbutamide, tienilic acid, and a number of nonsteroidal anti-inflammatory drugs such as diclofenac, piroxicam, tenoxicam, ibuprofen, and acetylsalicylic acid.

INDUCTION AND INHIBITION

CYP2C9 is inhibited by fluconazole, metronidazole, miconazole, ketoconazole, itaconazole, ritonavir, clopidrogel, amiodarone, fluvoxamine, sulfamthoxoazole, fluvastatin and fluoxetine. It is induced by rifampin and rifabutin.

INTER ETHNIC DIFFERENCES

The CYP2C9 genotypes demonstrate marked inter ethnic variability. The CYP2C9*2 is absent from Chinese, Taiwanese and present in only 1% of African American populations, but accounts for 19.2% of the British population and 8% of Caucasians. CYP2C9*3 is rarer and is present in 6% of Caucasian, 2% of Chinese, 2.6% of Taiwanese and 0.5% of African-American populations .

It is reasonable that, in drug metabolism studies, each ethnic group can be studied separately for evidence of polymorphism and its antimode should not be extrapolated from one ethnic population to another .

S-WARFARIN

As an example, the benefit of the individualization of therapy and/or drug dosing is evident in the case of S-warfarin. According to this example, CYP2C9 is characterized as a metabolic factor identified to influence the metabolism of S-warfarin. S-warfarin is an anticoagulant drug. Studies have demonstrated that the presence of either CYP2C*2 or

CYP2C9*3 haplotypes results in a decrease in the dose necessary to acquire target anticoagulation intensity. In addition, these individuals also suffered from an increased incidence of bleeding complications. Therefore, the CYP2C9 gene variants modulate the anticoagulant effect of the dose of warfarin prescribed. For these reasons, the utility of the present invention is evident. In particular, an accurate and convenient clinical assay would allow physicians to quickly identify safe and effective treatment regimes for individuals on an individual basis .

DIRECT PHENOTYPIC DETERMINANTS OF CYP2C9 In accordance with an embodiment of the present invention, the ratio of diclofenac and its hydroxylated metabolite, 4 ' -hydroxydiclofenac in an urine sample may be used to provide a determination of an individual ' s CYP2C9 phenotype. These metabolites are used as quantitative markers in the determination of a CYP2C9 phenotype on the basis of the use of the preferred probe substrate diclofenac. The structures of diclofenac and its metabolite 4 -hydroxydiclofenac are illustrated in Fig. 7. However, it is fully contemplated that the present invention is not limited in any respect thereto. In fact, due to the nature of the substrate specific alterations caused by the individual CYP2C9 mutations, multiple probe substrates may be necessary for a completely informative phenotypic determination of CYP2C9.

The molar ratio of diclofenac and its 4 ' -hydroxydiclofenac metabolite, used to determine the CYP2C9 phenotype of the individual, is as follows:

diclofenac

4 ' -hydroxydiclofenac

GENOTYPIC DETERMINANTS OF CYP2C9 (GENOTYPING)

As mentioned previously the CYP2C9 has two predominant variant alleles, CYP2C9*2 and CYP2C9*3. An example of a procedure for genotyping CYP2C9 involves a series of polymerase chain reaction - restriction fragment length polymorphism reactions designed to detect nucleotide point mutations, deletions and insertions compared with the functional CYP2C9*1 allele (Taube et al.^' (2000) Blood 96 (5) : 1816-1819) . PCR amplification of exon 3 for CYP2C9*2 is performed using the following primers:

CYP2C19*2 Exon 3 Primers

5'-CAATGGAAAGAAATGGAAGGAGGT-3' SEQ ID NO. 16

5 ' -AGAAAGTAATACTCAGACCAATCG-3 ' SEQ ID NO . 17

A forced mismatch was included in the penultimate base of the forward primer to create a control site for the Avail digestion. The PCR product from this amplification is 251 bp in length. After Avail digestion the CYP2C9*1 (wt) allele produces 170- and 60 bp fragments. The CYP2C*2 allele produces a 229 bp fragment.

The CYP2C9*3 allele does not naturally destroy or produce a restriction site. Therefore, a restriction site was forced into the forward primer such that A1061 in combination with the mismatch creates a restriction site for Nsil restriction enzyme. The CYP2C9*3 A1061C mutation removes this restriction site. This primer also includes a natural Avail restriction sequence. This reverse primer also has a forced mismatch at 1186 to provide a control for the Nsil restriction enzyme. The PCR product for this set of primers prior to restriction enzyme digest is 160 bp in length. Following restriction digest with Nsil and Avail, the CYP2C9*1 allele produces a 130 bp fragment, the CYP2C9*3 allele have 140 bp fragments.

CYP2C19*3 Primers

5'-TGCACGAGGTCCAGAGATGC-3 ' SEQ ID NO . 18

5'-AGCTTCAGGGTTTACGTATCATAGTAA-3 ' SEQ ID NO. 19

Due to the substrate specific alterations in enzyme activity resulting from the two allelic variants, the metabolic determination will be correlated on an individual substrate as is.

CYP2E1

CYP2E1 constitutes approximately 5% of the total CYP 450 enzymes in the human liver.

POLYMORPHISM The CYP2E1 gene has been demonstrated to be polymorphic in the human population. Studies have demonstrated the presence of 10 CYP2E1 (one wt CYP2E1*1, and 9 mutant, CYP2E1*2, CYP2E1*3, CYP2E1*4, CYP2E1*5A, CYP2E1*5B, CYP2E1*6, CYP2E1*7A, CYP2E1*7B, and CYP2E1*7C. The exact relationship of these polymorphisms to CYP2E1 enzyme activity has not been clarified, however some studies suggest that the mutant allele CYP2E1*5A & CYP2E1*5B, results in increased transcription and increased enzyme activity. CYP2E1 metabolizes several drugs and dietary constituents including ethanol, acetone, acetaminophen, nitrosamines, nitrosodimethylamine, p-nitrophenol .

In addition, CYP2E1 activates environmental pro- carcinogens especially nitrosodimethylamine, nitrosopyrrolidone, benzene, carbon tetrachloride,

3-hydroxypyridine (tobacco smoke product) . In one study it has been shown that individuals who have high CYP2E1

(CYP2E1*5A or CYP2E1*5B) activity are at a greater risk for gastric cancer (0R=23.6-25.7) .

INDUCTION AND INHIBITION

CYP2E1 is induced by a number of drugs and environmental factors such as cigarette smoke as well as by starvation, chronic alcohol consumption and in uncontrolled diabetes. CYP2E1 is inhibited by chlormethiazole, trans-1 , 2-dichloroethylene, disulferan

(cimetidine) and by the isoflavonoids genistein and equol . This level of induction or inhibition by environmental factors can severely alter an individual's capacity to metabolize certain drugs.

INTER ETHNIC DIFFERENCES

The proportion of CYP2E1 phenotypes varied between ethnic groups and countries: The frequency of the rare c2 (CYP2E1*5A or CYP2E1*5B) allele is about 4% in Caucasians and 20% in the Japanese and a study of a separate polymorphism described a rare C allele (CYP2E1*5A or CYP2E1*6) that has a frequency of about 10% in Caucasian and 25% in Japanese population. In one study it was shown that Japanese males had much lower levels of CYP2E1 activity as compared to Caucasian males. Therefore, it is reasonable that, in drug metabolism studies, each ethnic group can be studied separately for evidence of polymorphism and its antimode should not be extrapolated from one ethnic population to another.

ACETAMINOPHEN An example of the need for the individualization of therapy and/or drug dosing is the case of acetaminophen. According to this example, CYP2E1 is characterized as a metabolic factor of interest. Acetaminophen is a widely used painkiller. However, acetaminophen causes hepatotoxicity at low frequency. The hepatotoxicity is due to its transformation via CYP2E1, to a reactive metabolite (N-acetyl-p- benzoquinoneimine) , which is capable of binding to nucleophiles . For these reasons, the utility of a reliable methodology for the individualization of therapy and/or drug dosing is evident .

DIRECT PHENOTYPIC DETERMINANTS OF CYP2E1

In accordance with the present invention a suitable probe substrate is, without limitation, chlorzoxazone .

In accordance with the present invention, the molar ratio of chlorzoxazone and its metabolite is used to determine the CYP2E1 phenotype of the individual as follows: 6-hydroxychlorzoxazone chlorzoxazone

The structures of chlorzoxazone and its metabolite 6-hydroxychlorzoxazone are illustrated in Fig. 8.

GENOTYPIC DETERMINANTS OF CYP2E1 (GENOTYPING) As mentioned previously the CYP2E1 gene has multiple polymorphisms. An example of a procedure for genotyping CYP2E1 for the most common mutations those termed the Pst/Rsal and Draϊ mutations (allows genotyping of CYP2E1*5A, CYP2E1*5B & CYP2E1*6) involves the amplification of a fragment containing either the Pstl and i?sal sites or the Dral site using specific primers (Nedelcheva et al . (1996) Methods in Enzymology 272:218-225) . The amplified product is then incubated with the appropriate restriction enzyme (Pstl or Rsal/Dral ) and separated on electrophoretically. In an allele with wt sequence at the Pstl or Rsal site, the 510 bp fragment produced by PCR is cleaved to a 360 bp and a 150 bp fragment. In the mutant allele the 510 bp fragment remains uncleaved. In the Dral mutation, the 370 bp PCR amplified fragment is cleaved to a 240 bp and 130 bp pair of fragments in the wt allele, and is uncleaved in the mutant allele. Pstl/Rsal Primers

5'-CCCGTGAGCCAGTCGAGT-3' SEQ ID NO. 20

5'-ATACAGACCCTCTTCCAC-3' SEQ ID NO . 21 Dral Primers 5 ' -AGTCGACATGTGATGGATCCA-3 ' SEQ ID NO. 22

5'-GACAGGGTTTCA-TCATGTTGG-3' SEQ ID NO. 23

The CYP2E1*5A mutant allele contains both the Rsal and the Dral mutations, the CYP2E1*5B contains the .Rsal mutation alone. The .Rsal mutation has been associated with an increased expression and increased enzyme activity. Therefore, an individual with two copies of either CYP2E1*5 allele could be considered an extensive metabolizer. Conversely, the CYP2E1*2 mutation has been associated with decreased protein expression and decreased enzyme activity. Therefore, a person homozygous for the CYP2E1*2 allele could be assigned a poor metabolizer.

NAT2

POLYMORPHISM

Individuals are genetically polymorphic in their rate of N-acetylation of drugs via the N-acetyltransferase (NAT2) pathway (Meyer, U.A. (1994) Proc. Natl. Acad. Sci. USA, 91:1983-1984) . Two major metabolic phenotypes can be distinguished: fast and slow N-acetylators. Drugs that are subject to N-acetylation polymorphism include sulfonamides (sulfamethazine) , antidepressants (phenelzine) , antiarrhymics (procainamide) , and antihypertensives

(hydrazine) . Some adverse therapeutic consequences of the acetylator phenotype are peripheral neuropathy and hepatitis. In an opposite manner, the N-acetylation of procainamide produces a therapeutically active metabolite with reduced toxicity. N-acetylation polymorphism has also been linked to detoxification pathway of some environmental carcinogenic arylamines and there is a higher frequency of bladder cancers among chemical dye workers who are slow N-acetylators.

The NAT2 gene is polymorphic, there have been 9 mutation detected and 14 mutant alleles. 6 mutant alleles are responsible for 99% of Caucasian slow acetylators (NAT2*5A, NAT2*5B, NAT2*5C, NAT2*6A, NAT2*7B, and NAT2*13) . The NAT2*4 allele is the wild- type allele.

INTER ETHNIC DIFFERENCES The frequency of PM (poor metabolizer) and EM

(extensive metabolizers) (autosomal recessive trait) show considerable inter ethnic differences for the N-acetylation polymorphism. In Caucasians, the frequencies are approximately 60% and 40%, while in Orientals, they are 20% and 80%, respectively (Meyer, U.A. (1994) Proc. Natl. Acad. Sci. USA, 91:1983-1984). It is reasonable that, in drug metabolism studies, each ethnic group is studied separately for evidence of polymorphism and its antimode should not be extrapolated from one ethnic population to another. DIRECT PHENOTYPING - PHENOTYPIC DETERMINANTS OF NAT2

Different probe substrates can be used to determine the NAT2 phenotype . In accordance with the present invention a suitable probe substrate is, without limitation caffeine. Caffeine is widely consumed and relatively safe. A phenotype may be generally determined from ratios of the caffeine metabolites 5-acetamino-6-amino-l-methyluracil (AAMU) or 5-acetamino-6-formylamino-l-methyluracil (AFMU) and 1-methylxanthine (IX) present in urine samples of an individual collected after drinking coffee. The structure of these metabolites are illustrated in Fig. 9. The ratio of these metabolites provides a determination of an- individual's N-acetylation (NAT2) phenotype .

In accordance with the present invention, the molar ratio of caffeine metabolites is used to determine the acetylation phenotype of the individual as follows. Individuals with a ratio less than 1.80 are slow acetylators.

GENOTYPIC DETERMINANTS OF NAT 2 (GENOTYPING)

An example of NAT2 genotyping involves the amplification of a 547 bp fragment which includes the 5 of the 6 mutant alleles which are responsible for 99% of Caucasian slow acetylators. Analysis of these 5 alleles and the wt^' allele can be performed by examining 4 mutations (Smith CAD et al . J Med Genet (1997) 34:758-760) . The PCR amplification is performed with the following primers:

5 ' -GCTGGGTCTGGAAGCTCCTC-3 ' SEQ ID NO. 24 5 ' -TTGGGTGATACATACACAAGGG-3 ' ^' SEQ ID NO.. 25

The analysis of this fragment with 4 restriction digestion enzymes allows the detection of 6 alleles (NAT2*4 (wt) and the mutants NAT2*5A, NAT2*5B, NAT2*5C, NAT2*6 and NAT2*7) . Each of the 6 alleles have distinct combinations of the mutations and as each mutation alters a specific restriction digestion enzyme site (Kpnl , Ddel , Taql or BamHl), the performance of 4 separate digestions of the 547 bp fragment will allow the identification of the different alleles.

CHARACTERIZATION OF MULTIPLE PHENOTYPIC DETERMINANTS

On the basis of the above enzyme-specific metabolic factors, several approaches to identifying metabolic determinants thereof have been developed in accordance with the present invention. The characterization of multiple phenotypes offers multiple applications. The determination of an individual's metabolic phenotype for a multitude of enzymes and/or metabolic pathways allows the use of this single profile for multiple applications. If a drug is metabolized by more than one enzyme, the phenotypic and/or genotypic status of each of the enzymes may be important for first, determining if the individual can safely ingest a given drug and second, determining the optimal dose for this individual if they are able to take the drug.

For example, in the case of amonafide, it is suggested that CYP1A2 may, in addition to NAT2 , play a minor but nonetheless significant role in the metabolism of this drug. Accordingly, it is contemplated that the ability to characterize multiple metabolic determinants may also play an important role in the individualization of therapy with amonafide on the basis of phenotyping and/or genotyping.

In addition, the knowledge of multiple metabolic determinants will facilitate the comparison of multiple drugs within the same class or genus, where different metabolic enzymes are involved in the metabolism of these drugs. For example, consider an individual requiring treatment with a certain class of drugs, of which there are three that are primarily prescribed. If one is metabolized by CYP1A2 , one by CYP2D6 and the remaining drug by CYP3A4, and all individuals that are poor metabolizers of these drugs are at risk for toxicity. Then the drug chosen for treating that individual may be determined on the basis of a metabolic profile of that individual, as determined by genotyping and/or phenotyping. If for example the individual is a poor metabolizer for CYP2D6 and CYP3A4 , then the first drug metabolized by CYP1A2 may be the first drug to consider for treating the individual .

Another advantage to the determination of an individual's metabolic profile for multiple phenotypic determinants is the effect of a drug on the metabolic status of enzymes not primarily involved in its metabolism. For example, a drug may be metabolized by CYP2C9 and inhibit the activity of CYP3A4. If an individual has very low levels of CYP3A4, or possesses an allelic variation specific for that enzyme then this inhibition may have little effect on that individual's capacity to metabolize a CYP3A4-specific drug. However, if the individual is an extensive CYP3A4 metabolizer this drug may profoundly alter the CYP3A4 metabolic status. This can cause enormous problems in the case of polypharmacy, where an individual may be taking multiple drugs, and the addition of one drug may affect the safety and efficacy of the pre-existing drug treatment (s) .

A metabolic profile can be determined by phenotyping (by measuring enzyme activity) or genotyping (by examining enzymes genetic sequence) . In general, for example, for phenotyping, a probe substrate or drugs, such as those exemplified in

Table 2 are administered to an individual to be phenotyped. A biological sample, such as a urine sample

■ is subsequently collected from the individual approximately 4 hours after administering the probe substrate (s) . The urine sample is analyzed according to an ELISA technology, as described hereinbelow, for metabolites corresponding to the probe substrate (s) and the molar ratios of the metabolites calculated to reveal the individual .phenotypes .

In general, for example, for genotyping, a blood sample of an individual is- obtained, and the genetic sequence of the enzyme (s) is examined for the presence or absence of specific mutations. A specific probe for a known allelic variation may be used to screen for a specific genotype known to effect an individual's specific enzymatic capacity. The combination of mutations on the two alleles is matched to known genotypes. Based on this information, a metabolic profile indicative of an individual's metabolic capacity for a specific metabolic factor is characterized and may be employed in the individualization of therapy as provided by the present invention.

Table 2 Examples ofEnzymes and Corresponding Probes Drugs

Enzyme Probe substrate

NAT1 p-aminosalicylic acid

NAT2 Caffeine

CYP1A2 Caffeine

CYP2A6 Coumarin

CYP2C9 s-ibuprofen

CYP2C19 Mephenytoin

CYP2D6 Dextromethorphan

CYP2E1 Chlorzoxazone

CYP3A4 Midazolam

In Example I, a detailed description of the synthesis of probe substrate and metabolite derivatives and the ELISA development for N-acetyltransferase (NAT2) are illustrated. The materials and methods, and the overall general process described for the development of the NAT2 ELISA method and kit for metabolic are adapted to the development of the metabolic phenotyping ELISA kits for other metabolic enzymes and/or metabolic pathways thereof, including, without limitation CYP3A4 , NAT1, CYP1A2, CYP2A6 , CYP2D6, CYP2E1, CYP2C9 and CYP2C19, as well as a multi-determinant metabolic phenotyping system and method. In particular, the 5 protocol as herein described for the development of an ELISA specific to NAT2 is adapted for the development of an ELISA corresponding to a drug-specific enzyme and/or metabolic pathway, in accordance with the present invention. According to an embodiment of the

10 present invention, an assay system is provided that is adapted for the characterization of metabolic determinants of at least one metabolic factor and can be used for individualizing treatment with a drug metabolically influenced by the metabolic factor.

15. Furthermore, the present invention may also be adapted to provide for the identification of other characteristics or determinants of drug clearance and drug toxicity known to vary on an individual basis.

20 EXAMPLE I

DETERMINATION OF PHENOTYPIC DETERMINANTS BY ELISA

NAT2

Different probe substrates can be used to 25 determine the NAT2 phenotype (Kilbane, A.J. et al . (1990) Clin . Pharmacol . Ther. , 47 :470-477 ; Tang, B-K. et al . (1991) Clin . Pharmacol . Ther . , 49:648-657). For the determination of the NAT 2 phenotype caffeine was the preferred probe because it is widely consumed and 30 relatively safe (Kalow, W. et al . (1993) Clin . Pharmacol . Ther. , 53:503-514). In studies involving this probe, the phenotype has been generally determined from ratios of the caffeine metabolites 5-acetamino-6- amino-1-methyluracil (AAMU) or 5-acetamino-6- formylamino-1-methyluracil (AFMU) and 1-methylxanthine

(IX) . In these studies, the subjects are given an oral dose of a caffeine-containing substance, and the urinary concentrations of the target metabolites determined by HPLC (Kilbane, A.J. et al . (1990) Clin . Pharmacol . Ther. , 47:470-477; Tang, B-K. et al . (1991)

Clin . Pharmacol . Ther. , 49:648-657) or CE (Lloyd, D. et al . (1992) J. Chrom . , 578:283-291).

The number of clinical protocols requiring the determination of NAT2 phenotypes is rapidly increasing and, an enzyme linked immunosorbent assay (ELISA) was developed for use in these studies (Wong, P., Leyland- Jones, B., and Wainer, I.W. (1995) J^". Pharm. Biomed. Anal . , 13:1079-1086). ELISAs have been successfully applied in the determination of low amounts of drugs and other antigenic components in plasma and urine samples, involve no extraction steps and are simple to carry out .

Antibodies were raised in animals against two caffeine metabolites [5-acetamino-6-amino-1-methyluracil (AAMU) or 5-acetamino-6-formylamino-1-methyluracil

(AFMU) and 1-methyl xanthine (IX)] present in urine samples of an individual collected after drinking coffee. Their ratio provides a determination of an individual's N-acetylation (NAT2) phenotype. Subsequently, there was developed a competitive antigen enzyme linked immunosorbent assay (ELISA) for measuring this ratio using these antibodies.

The antibodies of the present invention can be either polyclonal antibodies or monoclonal antibodies raised against two different metabolites of caffeine, which allow the measurement of the molar ratio of these metabolites .

The molar ratio of caffeine metabolites is used to determine the acetylation phenotype of the individual as follows. Individuals with a ratio less than 1.80 are slow acetylators.

MATERIALS AND METHODS

MATERIALS

Cyanomethylester, isobutyl chloroformate, dimethylsulfate, sodium methoxide, 95% pure, and tributylamine were purchased from Aldrich (Milwaukee, WI, USA); horse radish peroxidase was purchased from Boehringer Mannheim (Montreal, Que . , Canada); corning easy wash polystyrene microtiter plates were bought from Canlab (Montreal, Que., Canada); o-methylisourea hydrochloride was obtained from Lancaster Laboratories

(Windham, NH, USA) ; alkaline phosphatase conjugated to goat anti-rabbit IgGs was from Pierce ^' Chemical Co.

(Rockford, IL, USA) ; bovine serum albumin fraction V initial fractionation by cold alcohol precipitation

(BSA) , complete and incomplete Freund's adjuvants, diethanolamine, 1-methylxanthine, p-nitrophenol phosphate disodium salt, o-phenylenediamine hydrochloride; porcine skin gelatin, rabbit serum albumin (RSA) ,- Sephadex™ G25 fine, Tween™ 20 and ligands used for testing antibodies cross-reactivities were obtained from Sigma Chemical Co. (St. Louis, MO, USA) . Whatman™ DE52 diethylaminoethyl-cellulose was obtained from Chromatographic Specialties Inc. (Brockville, Ont . , Canada). Dioxane was obtained from A&C American Chemicals Ltd. (Montreal, Que., Canada) and was refluxed over calcium hydride for 4 hours and distilled before use. Other reagents used were of analytical grade.

SYNTHETIC PROCEDURES

The synthetic route for the production of AAMU-hemisuccinic acid (VIII) and 1-methylxanthine-8- propionic acid (IX) is presented in Fig. 10.

SYNTHESIS OF 2-METHOXY-4-IMINO-6-OXO-DIHYDROPYRIDINE (III) Compound III is synthesized according to the procedure of Pfeiderer (Pfeilderer, W. (1957) Chem.

Ber. , 90:2272-2276) as follows. To a 250 mL round bottom flask 12.2 g of o-methylisourea hydrochloride

(110.6 mmol), 11.81 mL methylcyanoacetate (134 mmol), 12.45 g of sodium methoxide (230.5 ,mmol) and 80 mL of methanol are added. The suspension is stirred and refluxed for 5 hours at 68-70°C. After cooling at room temperature, the suspension is filtered through a sintered glass funnel (Pyrex, 40-60 ASTM, 60 mL) , and the NaCl on the filter is washed with methanol. The filtrate is filtered by gravity through a Whatman™ no.

1 paper in a 500 mL round bottom flask, and the solvent is evaporated under reduced pressure with a rotary evaporator at 50°C. The residue is solubilized with warm distilled water, and the product is precipitated by acidification to pH 3-4 with glacial acetic acid. After

2 hours (or overnight) at room temperature, the suspension is filtered under vacuum through a sintered glass funnel (Pyrex, 40-60 ASTM, 60 mL) . The product is washed with water, acetone, and dried. The product is recrystallized with water as the solvent and using charcoal for decolorizing (activated carbon, Norit^r A< 100 mesh, decolorizing) . The yield is 76 %.

SYNTHESIS OF l-METHYL-2 -METHOXY-4-IMINO-6-OXO- DYHYDROPYRIMIDINE (IV)

Compound IV is synthesized according to the procedure of Pfeiderer (Pfeilderer, W. (1957) Chem. Ber. , 90:2272-2276) as follows. To a 250 mL round bottom flask llg of compound III (77.0 mmol) and 117 mL of IN NaOH (freshly prepared) are added. The solution is stirred and cooled at 15°C, using a water bath and crushed ice. Then 11.7 mL dimethylsulfate (123.6 mmol) are added dropwise with a pasteur pipette over a period of 60 min. Precipitation eventually occurs upon the addition. The suspension is stirred at 15°C for 3 hours and is left at 4°C overnight . The product is recovered by filtration under vacuum through a sintered glass funnel (Pyrex, 40-60 ASTM, 60 mL) . The yield is 38 %. SYNTHESIS OF l-METHYL-4-IMINOURACIL (V)

Compound V is synthesized according to the procedure of Pfeiderer (Pfeilderer, W. (1957) Chem. Ber. , 90:2272-2276) as follows. To a 250 mL round bottom flask 11.26 g of compound IV (72.6 mmol) and 138 mL 12 N HCI are added, and the suspension is stirred at room temperature for 16-20 hours. The suspension is cooled on crushed ice, the product is recovered by filtration under vacuum through a sintered glass funnel (Pyrex, 40-60 ASTM, 60 mL) . The product is washed with water at 4°C, using a pasteur pipette, until the pH of filtrate is around 4 (about 150 mL) . The product is washed with acetone and dried. The yield is 73 %.

SYNTHESIS OF 1-METHYL-4-IMINO-5-NITROURACIL (VI)

Compound VI is synthesized according to the procedure of Lespagnol et al (Lespagnol, A. et al.(1970) Chim . Ther. , 5:321-326) as follows. To a 250 mL round bottom flask 6.5 g of compound V (46 mmol) and 70 mL of water are added. The suspension is stirred and refluxed at 100°C. A solution of 6.5 g sodium nitrite

(93.6 mmol) dissolved in 10 mL water is added gradually to the reaction mixture with a pasteur pipette. Then

48 mL of glacial acetic acid is added with a pasteur pipette. Upon addition, precipitation occurs and the suspension becomes purple. The suspension is stirred and heated for an additional 5 min. , and cooled at room temperature and then on crushed ice. The product is recovered by filtration under vacuum through a sintered glass funnel (Pyrex, 10-15 ASTM, 60 mL) . It is washed with water at 4 °C to remove acetic acid and then with acetone. Last traces of acetic acid and acetone are removed under a high vacuum. The yield is 59 %.

SYNTHESIS OF l-METHYL-4, 5-DIAMINOURACIL (VII)

Compound VII is synthesized by the procedure of Lespagnol et al . (Lespagnol, A. et al . (1970) Chim . Ther. , 5:321-326) as follows. To a 100 mL round bottom flask 2 g of compound VI (11.7 mmol) and 25 mL water are added. The suspension is stirred and heated in an oil bath at 60°C. Sodium hydrosulfite (88%) is gradually added (40.4 mmol), using a spatula, until the purple color disappears (approximately 5 g or 24.3 mmol) . The suspension is heated for an additional 15 min. The suspension is cooled on crushed ice and left at 4°C overnight. The product is recovered by filtration under vacuum through a sintered glass funnel

(Pyrex, 30-40 ASTM, 15 mL) . The product is washed with water and acetone, and dried. The last traces of acetone are removed under a high vacuum. The yield is 59%.

SYNTHESIS OF AAMU-HEMISUCCINIC ACID (VIII)

Compound VIII is synthesized as follows. To a 20 mL beaker 0.30 g of compound VII (1.92 mmol) and 5 mL water are added. The suspension is stirred and the pH is adjusted between 8 to 9 with a 3N NaOH solution. Then 0.33 g succinic anhydride (3.3 mmol) is added to the resulting solution, and the mixture is stirred until the succinic anhydride is dissolved. During this process, the pH of the solution is maintained between 8 and 9. The reaction is completed when all the succinic anhydride is dissolved and the pH remains above 8. The hemisuccinate is precipitated by acidification to pH 0.5 with 12N HCl. The product is recovered by filtration on a Whatman™ No. 1 paper, and washed with water to remove HCl. It is then washed with acetone and dried.

OTHER AAMU OR AFMU DERIVATIVES

The derivatives shown in Figs. 11 and 12 can also be used for raising antibodies against AAMU or AFMU that can be used for measuring the concentrations of these caffeine metabolites in urine samples.

SYNTHESIS OF 1-METHYLXANTHINE-8-PROPIONIC ACID (IX)

This product is synthesized according to a modified procedure of Lespagnol et al . (Lespagnol, A. et al.(1970) Chim. Ther. , 5:321-326) as follows. A 0.2 g sample of compound VIII (0.78 mmol) is dissolved in 2-3 mL of a 15% NaOH solution. The resulting solution is stirred at 100°C until all of the solvent is evaporated, and is then maintained at this temperature for an additional 5 min. The resulting solid is cooled at room temperature, and dissolved in 10 mL water. The product is precipitated by acidification to pH 2.8 with 12 N HCl. After cooling at 4°C for 2.5 hours, the product is recovered by filtration on a Whatman™ No. 1 paper, washed with water and acetone, and dried. It is recrystallized from water-methanol (20:80, v/v), using charcoal to decolorize the solution.

OTHER DERIVATIVES OF IX The other derivatives of IX, shown in Figs. 13 and 14, can also be used for raising antibodies against IX and thereby to allow the development of an ELISA for measuring IX concentration in urine samples.

SYNTHESIS OF AAMU

AAMU is synthesized from compound VII according to the procedure of Fink et al (Fink, K. et al . (1964)

J^". Biol . Chem . , 249:4250-4256) as follows. To a 100 mL round bottom flask 1.08 g of compound VII (6.9 mmol) and 20 mL acetic acid anhydride were added. The suspension is stirred and refluxed a 160-165 °C- for 6 min. After cooling at room temperature, the suspension is filtered under vacuum through a sintered glass funnel (Pyrex, 10-15 ASTM, 15 mL) . The product is washed with water and acetone, and dried. The product is recrystallized in water.

NMR SPECTROSCOPY

¹H and ¹³C NMR spectra of compounds VIII and IX are obtained using a 500 MHz spectrophotometer (Varian™

XL 500 MHz, Varian Analytical Instruments, San

Fernando, CA, USA) using deuterated dimethyl sulfoxide as solvent . CONJUGATION OF HAPTENS TO BOVINE SERUM ALBUMIN AND RABBIT SERUM ALBUMIN

The AAMU-hemisuccinic acid (VIII) and the 1- methylxanthine propionic acid (IX) are conjugated to BSA and RSA according to the following mixed anhydride method. To a 5 mL round bottom flask 31.7 mg of compound VIII (0.12 mmol) or 14.9 mg of compound IX (0.06 mmol) are added. Then 52.2 μL of tri-n- butylamine (0.24 mmol) and 900 μL of dioxane, dried over calcium hydride and freshly distilled, are added. The solution is cooled at 10°C in a water bath using crushed ice. Then 12.6 μL isobutyl chloroformate at 4°C (0.12 mmol, recently purchased or opened) are added and the solution is stirred for 30-40 min at 10-12°C. While the above solution is stirring, a second solution is prepared as follows. In a glass tube 70 mg BSA or RSA

(0.001 mmol) are dissolved in 1.83 mL water. Then 1.23 mL dioxane, freshly dried and distilled, is added and the BSA or RSA solution is cooled on ice. After 30- 40 min of the above stirring, 70 μL of 1 N NaOH solution cooled on ice is added to the BSA or RSA solution and the resulting solution is poured in one portion to the flask containing the first solution. The solution is stirred at 10-12°C for 3 hours and dialyzed against 1 liter of water for 2 days at room temperature, with water changed twice a day. The protein concentration of the conjugates and the amounts of moles of AAMU or IX incorporated per mole of BSA or RSA is determined by methods described below. The products are stored as 1 mL aliquots at -20°C 72

PROTEIN DETERMINATION BY THE METHOD OF LOWRY ET AL.

(Lowry, O.H. et al . (1951) J. - Biol . Chem. , 193:265-275)

A) SOLUTIONS Solution A: 2 g Na2Cθ3 is dissolved in 50 mL water,

10 mL of 10 % SDS and 10 mL 1 NaOH, water is added to 100 mL. Freshly prepared.

Solution B: 1 % NaK Tartrate Solution C: 1 % CuS0 .5H₂0

Solution D: 1 N phenol (freshly prepared) : 3 mL Folin & Ciocalteu's phenol reagent (2.0 N) and 3 mL water.

Solution F: 98- mL Solution A, 1 mL Solution B, 1 mL Solution C. Freshly prepared.

BSA: 1 mg/mL . 0.10 g bovine serum albumin (fraction V)/100 mL.

B) ASSAY

Standard curve Tubes # (13 x 100 mm)

Solution ϊ 2 3 4 5 6 7 BSAμL) 0 10 15 20 30 40 50

Water μL) 200 190 185 180 170 160 150

Solution F (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0

The solutions are vortexed and left 10 min at room temperature. Solution D μL) 200 200 200 200 200 200 200

The solutions are vortexed and left at room temperature for 1 hour.

The absorbance of each solution is read at 750 nm using water as the blank.

UNKNOWN .

Solution D.F.^a Tube # (13 x 100 mm)

1 2 3 Unknown (μL) x x x

Water (μL) y y y x + y = 200 μL

Solution F(mL) 2.0 2.0 2.0

The solutions are vortexed and left 10 min at room temperature.

Solution D(μL) 200 200 200 The solutions are vortexed and left 1 hour at room temperature .

The absorbance of each solution is read at 750 nm using water as the blank. The protein concentration is calculated using the standard curve and taking account of the dilution factor (D.F. ) . a. D.F. (dilution factor) . It has to be such so that the absorbance of the unknown at 750 nm is within the range of absorbance of the standards.

METHOD TO DETERMINE THE AMOUNTS OF MOLES OF AAMU OR IX INCORPORATED PER MOLE OF - BSA OR RSA

This method gives an approximate estimate. It is a useful one because it allows one to determine whether the coupling proceeded as expected.

A) SOLUTIONS

10% sodium dodecyl sulfate (SDS) - 1% SDS solution

0.5 or 1 mg/mL of AAMU-BSA (or AAMU-RSA) in a 1% SDS solution (1 mL) .

0.5 or 1 mg/mL of BSA or RSA in a 1% SDS solution (1 mL) . B) PROCEDURE

The absorbance of the AAMU conjugate solution is measured at 265 nm, with 1% SDS solution as the blank.

The absorbance of the BSA (or RSA) solution is measured at 265 nm, with 1% SDS solution as the blank.

The amount of moles of AAMU incorporated per mole of BSA (or RSA) is calculated with this formula:

A₂65 (AAMU-BSA)- A₂65^' (BSA) y =

8265 (AAMU) x [BSA]

Where: y is the amount of moles of AAMU/mole of BSA (or RSA) ;

8265 (AAMU) is the extinction coefficient of AAMU = 10⁴ M^-1cm^_1; and

[BSA] = BSA (mg/mL) /68, 000/mmole.

To calculate the amount of moles of IX incorporated per mole of BSA or RSA, the same procedure is used but with this formula:

^A252 (1X-BSA)- 52 (BSA) y = ε₂52 ^{(lχ) x} [^βSA] Where : y is the amount of moles of lX/mole of BSA (or RSA) ;

8252 (AAMU) is the extinction coeff icient of IX = 10⁴ M^{- 1}cm^{_ 1} ; and

[BSA] = BSA (mg/mL) /68, 000/mmole.

COUPLING OF HAPTENS TO HORSE RADISH PEROXIDASE

The AAMU derivative (VIII) and IX derivative (IX) are conjugated to horse radish peroxidase (HRP) by the following procedure. To a 5 mL round bottom flask 31.2 mg of compound VIII (or 28.3 mg of compound IX) are added. Then 500 μL of dioxane, freshly dried over calcium chloride, are added. The suspension is st^'irred and cooled at 10°C using a water bath and crushed ice. Then 114. μL tributylamine and 31 μL of isobutyl chloroformate (recently opened or purchased) are added. The suspension is stirred for 30 min at 10°C. While the suspension is stirring, a solution is prepared by dissolving 13 mg of horse radish peroxidase (HRP) in 2 mL of water. The solution is cooled at 4°C on crushed ice. After the 30 min stirring, 100 μL of a 1 N NaOH solution at 4°C is added to the HRP solution and the alkaline HRP solution is poured at once into the 5 mL flask. The suspension is stirred for 4 hours at 10-12° C. The free derivative is separated from the HRP conjugate by filtration through a Sephadex G-25™ column (1.6 x 30 cm) equilibrated and eluted with a 0.05 M sodium phosphate buffer, pH 7.5. The fractions of 1.0- 1.2 mL are collected with a fraction collector. During the elution two bands are observed: the HRP conjugate band and a light yellow band behind the HRP conjugate band. The HRP conjugate elutes between fractions 11- 16. The fractions containing the HRP conjugate are pooled in a 15 mL tissue culture tube with a screw cap. The HRP conjugate concentration is determined at 403 nm after diluting an aliquot (usually 50 μL+650 μL of buffer) .

[HR -conjugate] (mg/mL) = A₄₀3 x 0.4 x D.F.

The ultraviolet (UV) absorption spectrum is recorded between 320 and 220 nm. The presence of peaks at 264 and 270 nm for AAMU-HRP and IX-HRP conjugates, respectively, are indicative that the couplings proceeded as expected.

After the above measurements, 5 μL of a 4 % thiomersal solution is added per mL of the AAMU-HRP or IX-HRP conjugate solution. The conjugates are stored at 4°C.

ANTIBODY PRODUCTION

Four mature females New Zealand white rabbits (Charles River Canada, St-Constant, Que., Canada) are. used for antibody production. The protocol employed in this study was approved by the McGill University Animal Care Committee in accordance with the guidelines from the Canadian Council on Animal Care. Antibodies of the present invention may be monoclonal or polyclonal antibodies .

An isotonic saline solution (0.6 mL) containing

^■ 240 mg of BSA conjugated antigen is emulsified with 0.6 mL of a complete Freund's adjuvant. A 0.5 mL aliquot of the emulsion (100 mg of antigen) is injected per rabbit intramuscularly or subcutaneously. Rabbits are subsequently boosted at intervals of three weeks with 50 mg of antigen emulsified in incomplete Freund's adjuvant. Blood is collected by venipuncture of the ear

10-14 days after boosting. Antisera are stored at 4°C in the presence of 0.01% sodium azide.

DOUBLE IMMUNODIFFUSION IN AGAR PLATE An 0.8% agar gel in PBS is prepared in a 60 x 15 mm petri dish. Rabbit serum albumin (100 μL of 1 mg mL^~ ¹) conjugated to AAMU (or IX) are added to the center well, and 100 μL of rabbit antiserum are added to the peripheral wells. The immunodiffusion is carried out in a humidified chamber at 37°C overnight and the gel is inspected visually.

ANTISERUM TITERS

The wells of a microtiter plate are coated with 10 μg mL^-1 of rabbit serum albumin-AAMU (or IX) conjugate in sodium carbonate buffer, pH 9.6) for 1 hour at 37°C (100 μL/per well) . The wells are then washed three times with 100 μL TPBS (phosphate buffer saline containing 0.05% Tween™ 20) and unoccupied sites are blocked by an incubation with 100 mL of TPBS containing 0.05% gelatin for 1 hour at 37°C. The wells are washed three times with 100 μL TPBS and 100 μL of antiserum diluted in TPBS are added. After 1 hour at 37°C, the wells are washed three times with TPBS, and

100 μL of goat anti-rabbit IgGs-alkaline phosphatase conjugate, diluted in PBS containing 1% BSA, are added. After 1 hour at 37°C, the wells are washed three times with TPBS and three times with water. To the wells are added 100 μL of a solution containing MgCl2 (0.5 mM) and p-nitrophenol phosphate (3.85 mM) in diethanolamine buffer (10 mM, pH 9.8). After 30 min. at room temperature, the absorbency is read at 405 nm with a micro- plate reader. The antibody titer is defined as the dilution required to change the^' absorbance by one unit (1 au) .

ISOLATION OF RABBIT IgGs

The DE52 -cellulose resin is washed three times with sodium phosphate buffer (500 mM, pH 7.50), the fines are removed and the resin is equilibrated with a sodium -phosphate buffer (10 mM, pH 7.50) . The resin is packed in a 50 x 1.6 cm column and eluted with 200-300 mL equilibrating buffer before use. To antiserum obtained from 50 mL of blood (30-32 mL) is added drop- wise 25-27 mL of a 100% saturated ammonium sulfate solution with a Pasteur pipette. The suspension is left at room temperature for 3 h and centrifuged for 30 min. at 2560 g at 20°C. The pellet is dissolved with 15 mL sodium phosphate buffer (10 mM, pH 7.50) and dialyzed at room temperature with the buffer changed twice per day. The dialyzed solution is centrifuged at 2560 g for 10 min. at 20°C to remove precipitate formed during dialysis. The supernatant is applied to the ion- exchange column. Fractions of 7 mL are collected. After application, the column is eluted with the equilibrating buffer until the absorbance at 280 nm becomes less than 0.05 au. The column is then eluted with the equilibrating buffer containing 50 mM NaCl. Fractions having absorbencies greater than 0.2 at 280 nm are saved and stored at 4°C. Protein concentrations of the fractions are determined as described above.

COMPETITIVE ANTIGEN ELISA Buffers and water without additives are filtered through millipore filters and kept for 1 week. BSA, antibodies, Tween™ 20 and horse radish peroxidase conjugates are added to these buffers and water just prior to use. Urine samples are usually collected 4 hours after drinking a cup of coffee (instant or brewed with approximately 100 mg of caffeine per cup) and stored at -80 °C. The urine samples are diluted 10 times with sodium phosphate buffer (620 mosm, pH 7.50) and are subsequently diluted with water to give concentrations of AAMU and IX no higher than 3 x 10^~6 M in the ELISA. All the pipettings are done with an eight-channel pipette, except those of the antibody and sample solutions. Starting with the last well, 100 μL of a carbonate buffer (100 mM, pH 9.6) containing 2.5 μ g mL-¹ antibodies are added to each well. After 90 min. at room temperature, the wells are washed three times with 100 L of TPB: isotonic sodium phosphate buffer (310 mos , pH 7.50) containing 0.05% Tween™ 20.

After the initial wash, unoccupied sites are blocked by incubation for 90 min. at room temperature with 100 μL TBP containing 3% BSA. The wells are washed four times with 100 μL TPB. The washing is followed by additions of 50 μL of 12 mg mL^-1 AAMU-HRP or IX-HRP conjugate in 2 x TPB containing 2% BSA, and 50 μL of either water, standard (13 standards; AAMU or IX, 2 x

10^-4 to 2 x 10^-8 M) or sample in duplicate. The micro- plate is gently shaken with an orbital shaker at room temperature for 3-4 hours. The wells are washed three times with 100 μL TPB containing 1% BSA and three times with water containing 0.05% Tween™ 20. To the washed plate is added 150 μL of a substrate buffer composed of citric acid (25 mM) and sodium phosphate dibasic buffer

(50 mM, pH 5.0) containing 0.06% hydrogen peroxide and

0.04% o-phenylenediamine hydrochloride. After 20 min. at room temperature with shaking, the reaction is stopped with 50 μL of 2.5 M HCl. After shaking the plate 3 min. , the absorbances are read with a microtiter plate reader at 490 nm.

RESULTS

Polyclonal antibodies against AAMU and IX could be successfully raised in rabbits after their conjugation to bovine serum albumin. Each rabbit produced antibody titers of 30,000-100,000 as determined by ELISA. This was also indicated by strong precipitin lines after double immunodiffusion in agar plates of antisera and derivatives conjugated to rabbit serum albumin. On this basis, a) IgGs antibodies were isolated on a DE-52 cellulose column and b) a competitive antigen ELISA for NAT2 phenotyping using caffeine as probe substrate was developed according to the methods described in the above section entitled Materials and Methods.

Contrary to current methods used for phenotyping, the assay involves no extraction, is sensitive and rapid, and can be readily carried out on a routine basis by a technician with a minimum of training in a clinical laboratory.

The present invention -will be more readily un- derstood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

A COMPETITIVE ANTIGEN ELISA FOR NAT2 PHENOTYPING USING CAFFEINE AS A PROBE SUBSTRATE

Buffers and water without additives were filtered through millipore filters and kept for 1 week. BSA, antibodies, Tween™ 20 and horse radish peroxidase conjugates were added to these buffers and water just prior to use. Urine samples were usually collected 4 hours after drinking a cup of coffee (instant or brewed with approximately 100 mg of caffeine per cup), and stored at -80 °C. They were diluted 10 times with sodium phosphate buffer (620 mosm, pH 7.50) and were subsequently diluted with water to give concentrations of AAMU and IX no higher than 3xlO^~6 M in the ELISA. All the pipettings were done with an eight-channel pipette, except those of the antibody and sample solutions. Starting with the last well, lOOμL of a carbonate buffer (100 mM, pH 9.6) containing 2.5μg mL-¹ antibodies was pipetted. After 90 min at room temperature, the wells were washed three times with 100 mL of TPB: isotonic sodium phosphate buffer (310 mosm, pH 7.50) containing 0.05% Tween™ 20. After the initial wash, unoccupied sites were blocked by incubation for 90 min at room temperature with 100 μL TBP containing 3% BSA. The wells were washed four times with 100 μL TPB. This was followed by additions of 50 mL of 12 mg mL^-1 AAMU-HRP or IX-HRP conjugate in 2xTPB containing 2% BSA, and 50 μL of either water, standard (13 standards; AAMU or IX,

2xl0-⁴ to 2xl0-⁸ M) or sample in duplicate. The microplate was gently shaken with an orbital shaker at room temperature for 3-4 hours. The wells were washed three times with 100 μL with TPB containing 1% BSA and three times with water containing 0.05% Tween™ 20. To the washed plate was added 150 μL of a substrate buffer composed of citric acid (25 mM) and sodium phosphate dibasic buffer - (50 mM, pH 5.0) containing 0.06% hydrogen peroxide and 0.04% o-phenylenediamine hydrochloride. After 20 min at room temperature with shaking, the reaction was stopped with 50 μL of 2.5 M HCl. After shaking the plate 3 min, the absorbances were read with a microtiter plate reader at 490 nm. The competitive antigen ELISA curves of AAMU-Ab and lX-Ab determinations obtained in duplicate are presented in Fig. 15. Each calibration curve represents the average of two calibration curves. The height of the bars measure the deviations of the absorbency values between the two calibration curves. Data points without bars indicate that deviations of the absorbency values are equal or less than the size of the symbols representing the data points. Under the experimental conditions of the ELISA: background was less than 0.10 au; the practical limits of detection of AAMU and IX were 2xl0-⁷ M and 2xl0-⁶ M, respectively, concentrations 500 and 50 times lower than those in urine samples from previous phenotyping studies (Kilbane, A.J. et al . (1990) Clin . Pharmacol . Ther. , 47:470-477); the intra-assay and interassay coefficients of variations of AAMU and IX were 15-20% over the concentration range of 0.01-0.05 mM.

A variety of conditions for the ELISA were tested and a number of noteworthy observations were made: gelatin, which was used in the competitive antigen ELISA determination of caffeine in plasma (Fickling, S.A. et al . (1990) J^". Immunol . Meth . , 129:159-164), could not be used in our ELISA owing to excessive background absorbency which varied between 0.5 and 1.0 au; in the absence of Tween™ 20, absorbency changes per 15 min decreased by a factor of at least 3, and calibration curves were generally erratic; absorbency coefficients of variation of samples increased by a factor of 3 to 4 when the conjugates and haptens were added to the wells as a mixture instead individually.

The cross reactivities of AAMU-Ab and IX-Ab were tested using a wide variety of caffeine metabolites and structural analogs (Table 3 below) . AAMU-Ab appeared highly specific for binding AAMU, while IX-Ab appeared relatively specific for binding IX. However, a 11% cross reactivity was observed with 1-methyluric acid (1U) , a major caffeine metabolite.

Table 3

Cross-reactivity of AAMU-Ab and IX-Ab Towards Different Caffeine Metabolites and Structural Analogs

% Cross-Reaction

Compound AAMU-Ab IX-Ab

Xanthine øa 0

Hypoxant ine 0 0

1 -Methyl Xanthine (IX) 0 100

3-Methyl Xanthine 0 0

7-Methyl Xanthine 0 0

8-Methyl Xanthine 0 0

1,3-Dimethyl Xanthine (Theophylline) 0 0.2

1,7-Dimethyl Xanthine (Paraxanthine) 0 0.5

3,7-Dimethyl Xanthine (Theobromine) 0 0

1,3,7-Trimethyl Xanthine (Caffeine) 0 0

1-Methyluric acid 0 11

1,7-Dimethyluric acid 0 0

Guanine 0 0

Uracil 0 0

5-Acetamino-6-amino-uracil 0.6 0 .

5-Acetamino-6-amino-l-methyluracil (AAMU) 100 0

5-Acetamino-6-amino-l,3-dimethyluracil 0 0 a. The number 0 indicates either an absence of inhibition or an inhibition no higher than 40% at the highest compound concentration tested in the ELISA

(5xl0-³ M) ; concentrations of 5-acetamino-6-amino-l- methyluracil (AAMU) and 1-Methyl Xanthine (IX) required for 50% inhibition in the competitive antigen ELISA were 1.5xl0-^s M and 10-⁵ M, respectively.

The relative high level of cross reactivity of 1U is, however, unlikely to interfere • significantly in the determination of IX and the assignment of NAT2 phenotypes, since the ratio of 1U:1X is no greater than 2.5:1 in 97% of the population (Tang, B-K. et al . (1991) Clin . Pharmacol . Ther. , 49:648-657). This is confirmed by measurements of apparent concentrations of IX when the ratio varied between 0-8.0 at the fixed IX concentration of 3xl0^~6 M (Table 4 below) . At 1U:1X ratios of 2.5 and 3.0, the apparent increases were 22% and 32%, respectively.

Table 4 The Effect of the Ratio 1U:1X on the Determination of IX Concentration by

ELISA at Fixed IX Concentration of 3xl0-⁶ M

1U:1X ratio [lX]χl0⁶ (M)

O0 3O0

0.50 2.75

1.00 3.25

1.50 3.25

2.00 3.60

2.50 3.65

3.00 3.95

4.00 4.20

5.00 4.30

6.00 4.50

8.00 4.30 The following observations attested to the validity of the competitive antigen ELISA for NAT2 phenotyping : 1) The ELISA assigned the correct phenotype in

29 of 30 individuals that have been phenotyped by capillary electrophoresis (CE) (Lloyd, D. et al . (1992) J. Chrom . , 578:283-291) . 2) In the CE method, the phenotype was determined using AFMU/1X peak height ratios rather than the AAMU/IX molar ratios used in the ELISA. When the molar ratios determined by ELISA and the peak height ratios determined by CE were correlated by regression analysis, the calculated regression equation was y=0.48+0.87x, with a correlation coefficient (r) of 0.84^'. Taking account that these two ratios are not exactly equal and that Kalow and Tang

(Kalow, W. et al . (1993) Clin . Pharmacol .

Ther. , 53:503-514) have pointed out that using AFMU rather than AAMU can lead to misclassification of NAT2 phenotypes, there is a remarkable agreement between the two methods .

3) The ELISA was used in determining the .NAT2 phenotype distribution within a group of 146 individuals. Fig. 16 illustrates a histogram of the NAT2 phenotypes of this group as determined by measuring the AAMU/lX ratio in urine samples by ELISA. Assuming an antimode of 1.80, the test population contained 60.4% slow acetylators and 39.6% fast acetylators. This is consistent with previously reported distributions (Kalow, W. et al . (1993) Clin . Pharmacol . Ther. , 53:503-514;

Kilbane, A.J. et al . (1990) Clin .

Pharmacol . Ther. , 47:470-477).

DETERMINATION OF 5-ACETAMINO-6-AMINO-1-METHYLURACYL (AAMU) AND 1-METHYL XANTHINE IN URINE SAMPLES WITH THE ELISA KIT

Table 5

Content of the ELISA Kit and Conditions of Stora ige

Item Unit State Amt Storage Conditions

Tween 20 1 vial Liquid 250 μL/vial 4°C

H₂O₂ 1 vial Liquid 250 μL/vial 4°C

AAMU-HRP 1 vial Liquid 250 μL/vial 4°C

IX-HRP 1 vial Liquid 250 μL/vial 4°C

Buffer A 4 vials Solid 0.8894 g/vial 4°C

Buffer B 6 vials Solid 1.234 g/vial 4°C

Buffer C 6 vials Solid 1.1170 g/vial 4°C

Buffer D 6 vials Solid 0.8082 g/vial 4°C

Plate(AAMU-Ab) 2 Solid - 4°C

Plate (IX-Ab) 2 Solid - 4°C

Buffer E 6 vials Solid 0.9567 g/vial -20°C

Standards 14 vials Liquid 200 μL -20°C

(AAMU)

Standards(lX) 14 vials Liquid 200 μL -20°C

INNaOH 1 bottle Liquid 15 mL 20°C

IN HCl 1 bottle Liquid 15 mL 20°C

CONVERSION OF AFMU TO AAMU

In order to determine the AAMU concentrations in urine samples by competitive antigen ELISA, a transformation of AFMU to AAMU is required. The contents of an ELISA kit for this assay are listed in Table 5. • Thaw and warm up to room temperature the urine sample .

• Suspend the sample thoroughly with the vortex before pipetting. • Add 100 μL of a urine sample in a '1.5 mL microtube .

• Add 100 μL of a IN NaOH solution.

• Leave at room temperature for 10 min.

• Neutralize with 100 μL IN HCl solution. • Add 700 μL of Buffer A (dissolve the powder of one vial A/50 mL) .

DILUTIONS OF URINE SAMPLES FOR THE DETERMINATIONS OF [AAMU] AND [IX] BY ELISA The dilutions of urine samples required for determinations of AAMU and IX are a function of the sensitivity of the competitive antigen ELISA and AAMU and IX concentrations in urine samples. It is suggested to dilute the urine samples by a factor so that AAMU and IX concentrations are about 3x10-⁶ M in the well of the microtiter plate. Generally, dilution factors of 100-400 (Table 6) and 50-100 have been used for AAMU and IX, respectively. Table 6 Dilution Factors for Identifying AAMU and IX Concentrations

Microtube #

Dilution Factor 20x 40x 50x 80x lOOx 150x 200x 400x

Solution 1 2 3 4 5 6 7 8

Urine sample(mL)^a 500 250 200 125 100 66.7 50 25

10 x diluted

Buffer B (mL) 500 750 800 875 900 933.3 950 975 ^a. Vortex the microtubes containing the urine sample before pipetting.

Store the diluted urine samples at -20°C in a styrofoam box for microtubes .

Buffer B: dissolve the content of one vial B/100 mL.

DETERMINATION OF [AAMU] AND [IX] IN DILUTED URINE SAMPLES BY ELISA

Precautions :

The substrate is carcinogenic. Wear surgical gloves when handling Buffer E (Substrate buffer) . Each sample is determined in duplicate. An excellent pipetting technique is required. When this technique is mastered the absorbance values of duplicate should be within less than 5%. Buffers C, D and E are freshly prepared. Buffer E-H2O2 is prepared just prior pipetting in the microtiter ^' plate wells. Preparation of Samples:

Prepare Table 7 with a computer and print it. This table shows the content of each well of a 96-well microtiter plate. Enter the name of the urine sample (or number) at the corresponding well positions in Table 7. Select the dilution factor (D.F.) of each urine sample and enter at the corresponding position in Table 7. Enter the dilution of each urine sample with buffer B at the corresponding position in Table 7 : for example, for a D.F. of 100 (100 μL of lOx diluted urine sample + 900 μL buffer B) , enter 100/900. See "Dilutions of Urine Samples..." procedure described above for the preparation of the different dilutions. Prepare the different dilutions of the urine samples in 1.5 mL microtubes using a styrofoam support for 100 microtubes. Prepare Table 8 with a computer and print it. Using a styrofoam support (100 microtubes), prepare the following 48 microtubes in the order indicated in Table 8 :

Table 7 Positions of Blanks, Control and Urine Samples in a Microtiter Plate

Sample Well # D.F Dil. Sample Well # D.F Dil.

Blank 1-2 Control 49-50

Control 3-4 8 51-52

SI 5-6 9 53-54

S2 7-8 10 55-56

S3 9-10 11 57-58

S4 11-12 12 59-60

S5 13-14 13 61-62

S6 15-16 14 63-64

S7 17-18 15 65-66

S8 19-20 16 67-68

S9 21-22 17 69-70

S10 23-24 Control 71-72

Sl l 25-26 18 73-74

S12 27-28 19 75-76

S13 29-30 20 77-78

S14 31-32 21 79-80

S15 33-34 22 81-82

1 35-36 23 83-84

2 37-38 24 85-86

3 39-40 25 87-88

4 41-42 26 89-90

5 43-44 27 91-92

6 45-46 28 93-94

7 47-48 Blank 95-96 Table 8 Content of the Different Microtubes

Tube # Sample Content Tube # Sample Content

1 Blank Buffer B 25 7 Dil. Urine

2 Control Buffer B 26 8 Dil. Urine

3 SI AAMU or IX 27 9 Dil. Urine

4 S2 AAMU or IX 28 10 Dil. Urine

5 S3 AAMU or IX 29 11 Dil. Urine

6 S4 AAMU or IX 30 12 Dil. Urine

7 S5 AAMU or IX 31 13 Dil. Urine

8 S6 AAMU or IX 32 14 Dil. Urine

9 S7 AAMU or IX 33 15 Dil. Urine

10 S8 AAMU or IX 34 16 Dil. Urine

11 S9 AAMU or IX 35 17 Dil. Urine

12 S10 AAMU or IX 36 Control Buffer B

13 Sl l AAMU or IX 37 18 Dil. Urine

14 S12 AAMU or IX 38 19 Dil. Urine

15 S13 AAMU or IX 39 20 Dil. Urine

16 S14 . AAMU or IX 40 21 Dil. Urine

17 S15 AAMU or IX 41 22 Dil. Urine

18 1 Dil. Urine 42 23 Dil. Urine

19 2 Dil. Urine 43 24 Dil. Urine

20 3 Dil. Urine 44 25 Dil. Urine

21 4 Dil. Urine 45 26 Dil. Urine

22 5 Dil. Urine 46 27 Dil. Urine

23 6 Dil. Urine 47 28 Dil. Urine

24 Control Buffer B 48 Blank Buffer B SOLUTIONS :

Buffer A: Dissolve the content of one vial A/50 mL water.

Buffer B: Dissolve the content of one vial B/100 mL water. Buffer C: Dissolve the content of one vial C/50 mL water. Add 25 mL of Tween™ 20. Buffer D: Dissolve the content of one vial D /25 mL water. Add 25 mL of Tween™ 20. 0.05 % Tween 20 Add 25 μL of Tween™ 20 in a 100 mL erlnemeyer flask containing 50 mL of water.

2.5 N HCl: 41.75 mL of 12 N HCl/200 mL water. Store in a 250 mL glass bottle.

AAMU-HRP Conjugate: Add 9 mL of Buffer C in a 15 mL glass test tube. Add 90 μL of AAMU- HRP stock solution.

IX-HRP Conjugate Add 9 mL of the 2 % BSA solution in a 15 mL glass test tube. Pipet 90 μL IX-HRP stock solution.

Buffer E-H₂θ2 : Dissolve the content of one vial

E- substrate/50 mL water. Pipet 25 μL of a 30% H2O2 solution (prepared just prior pipetting in the microtiter plate wells) . Table 9

Standard Solutions of AAMU and IX (Diluted with Buffer B)

AAMU IX

Standard [AAMU] Standard [IX]

1 1.12 x 10-4 M 1 2.00 x 10"⁴M

2 6.00 x 10-5 M 2 1.12 x 10"⁴M

3 3.56 x 10-5 M 3 6.00 x 10-⁵ M

4 2.00 x 10^"5 M 4 3.56 x lO-⁵ M

5 6.00 x 10^"6M 5 2.00 x lO-⁵ M

6 3.56 l0"⁶M 6 1.12 10-5 M

7 2.00 10-6 M 7 6.00 x 10^"6M

8 1.12 x 10-6 M 8 3.56 x 10^"6M

9 6.00 10"⁷M 9 2.00 x 10"⁶M

10 3.56xl0^"7M 10 1.12 x 10^"6M

11 2.00 10^~7M 11 6.00 x 10"⁷M

12 1.12 10-7 M 12 3.56 x 10^"7M

13 6.00 x 10"⁸ M 13 2.00 x 10"⁷M

14 3.56 lO^"8M 14 1.12 x 10"⁷M

15 2.00 x 10"⁸ M 15 6.00 x 10^"8M

CONDITIONS OF THE ELISA

Add 50 μL/well of AAMU-HRP (or IX-HRP) conjugate solution, starting from the last row. Add 50 μL/well of diluted urine samples in duplicate, standards (see Table 9) , blank with a micropipet (0-200 μL) , starting from well #96 (see Table 7) . Cover the plate and mix gently by vortexing for several seconds. Leave the plate at room temperature for 3 h. Wash 3 times with 100 μL/well with buffer C, using a microtiter plate washer. Wash 3 times with 100 μL/well with the 0.05%

Tween 20 solution. Add 150 μL/well of Buffer E-H₂0₂

(prepared just prior pipetting in the microtiter plate wells) . Shake 20-30 min at room temperature with an orbital shaker. Add 50 μL/well of a 2.5 N HCl solution. Shake 3 min with the orbital shaker at room temperature. Read the absorbance of the wells with microtiter plate reader at 490 nm. Print the sheet of data and properly identify the data sheet.

CALCULATION OF THE [AAMU] AND [IX] IN URINE SAMPLES FROM THE DATA

Draw a Table 10 with a computer. Using the data sheet of the microtiter plate reader, enter the average absorbance values of blanks, controls (no free hapten present), standards and samples in Table 10. Draw the calibration curve on a semi-logarithmic plot

(absorbance at 490 nm as a function of the standard concentrations) using sigma plot (or other plot software) . Find the [AAMU] (or [IX] ) in the microtiter well of the unknown from the calibration curve and enter the data in Table 11. Multiply the [AAMU] (or

[IX] ) of the unknown by the dilution factor and enter the result in the corresponding case of Table 11.

The compositions of the buffers used in the ELISA kit are shown in Table 12. Table 10

Average Absorbance values of Samples in the Microtiter Plate

Sample Well # A490 Sample Well # ^A490

Blank 1-2 Control 49-50

Control 3-4 8 51-52

SI 5-6 9 53-54

S2 7-8 10 55-56

S3 9-10 11 57-58

S4 11-12 12 59-60

S5 13-14 13 61-62

S6 15-16 14 63-64

S7 17-18 15 65-66

S8 19-20 16 67-68

S9 21-22 17 69-70

S10 23-24 Control 71-72

Sll 25-26 18 73-74

S12 27-28 19 75-76

S13 29-30 20 77-78

S14 31-32 21 79-80

S15 33-34 22 81-82

1 35-36 23 83-84

2 37-38 24 85-86

3 39-40 25 87-88

4 41-42 26 89-90

5 43-44 27 91-92

6 45-46 28 93-94

7 47-48 Blank 95-96 Table 11 AAMU (or IX) Concentrations in Urine Samples

Sample D.F. [AAMU] [AAMU] x D.F.

1

2

3

4

^• 5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29 Table 12

Compositions of the different buffers

Buffer pH Composition Concen. [P] (mM) (mM)

A 7.50 0.15629 g/100 mL NaH2PO₄ 11.325

1.622 g/100 mL Na₂HPO₄.7 H₂O 60.099

1.778 g/100 mL (total weight) 71.424

B 7.50 0.1210191 g /100 mL NaH₂PO4 8.769

1.11309 g /100 mL ofNa2HPO₄.7H₂O 41.23 1.2341 g/100 mL (total weight) 49.999

C 7.50 1 g/100 mL of BSA

0.1210191 g /100 mL of NaH₂PO₄ 8.769

1.11309 g /100 mL of Na₂HPO₄.7H₂O 41.23

2.2341 g/100 mL (total weight) 49.999

D 7.50 2 g/100 mL of BSA

0.1210191 g 00 mL of NaH₂PO₄ 8.769

1.11309 g /100 mL of Na₂HPO₄.7H₂O 41.23

3.2341 g/100 mL (total weight) 49.999

E 5.00 0.52508 g/100 mL of citric acid 25

1.34848 g/100 mL of Na₂HPO₄.7H₂O 50

40 mg/100 mL of o-phenylenediamine hydrochloride

1.913567 g/100 mL (total weight)

In accordance with one embodiment of the present invention, the ELISA protocol outlined hereinabove, is adapted to provide an ELISA specific to at least one metabolic factor identified to influence the metabolism of a given drug. An example of a metabolic factor of interest may be CYP3A4 or CYP2D6. Similarly, according to another embodiment of the present invention, a genomic assay specific to at least one metabolic factor identified to influence the metabolism of a given drug or class of drugs is provided for quickly and accurately characterizing a metabolic profile of an individual for use in the individualization of therapy and/or drug dosing. For example, a metabolic profile according to a preferred embodiment of the present invention may be based on at least an individual's enzyme-specific genotype.

In accordance with another embodiment of the present invention the ELISA protocol and/or genomic assay is also adapted to provide a multi-determinant assay for providing metabolic determinants for' a plurality of metabolic factors of interest. In accordance with this embodiment, the metabolic factors may be enzymes identified to influence the metabolism of a preferred drug, and may include, but are not limited to cytochrome P450 enzymes and N-acetylation enzymes. In the case of CYP3A4 , a CYP3A4-specific ELISA is provided for rapidly and accurately identifying CYP3A4 phenotypic determinants of an individual for use in treating - that individual with a dosage of an antihistamine, for example, that is specific to at least their CYP3A4 phenotype. Likewise, in the case of CYP2D6, a CYP2D6-specific ELISA may be provided for rapidly and accurately identifying CYP2D6 phenotypic determinants of an individual for use in treating that individual with a dosage of an antihistamine that is specific to at least their CYP3A4-specific phenotype and/or genotype. Furthermore, in the case of a multi- determinant ELISA according to an aspect of the present invention metabolic determinants for at least CYP3A4 and CYP2D6 may be determined to identify an individual's capacity for the metabolism of an antihistamine. Alternatively, a genomic assay specific to these and/or other metabolic factors may be provided.

Fig. 17 exemplifies a multi-determinant assay according to an embodiment of the present invention. A multi-determinant assay of the present invention may provide more than one 6 X 6 array, as illustrated in Fig. 18, in each well of a standard microplate. Preferably, each well will be provided with 4 6 x 6 arrays according to this aspect of the present invention. Furthermore, the microplate of Fig. 18 may be adapted to provide the ELISA or genomic assay of the present invention.

The single or multi-determinant ELISA system of the present invention include (s) metabolite-specific binding agents for the detection of drug-specific metabolites in a biological sample. Such binding agents are preferably antibodies and the assay system is preferably an ELISA, as exemplified in the cases of NAT2 discussed herein above. A detection method according to an embodiment of the present invention is exemplified in Fig. 19. An assay system of the present invention is exemplified in Fig. 20 and provides means to detect metabolites specific to the metabolic pathway (s) used to metabolize a given drug or class- of drugs. In the case of a genomic assay according to the present invention, the binding agents are preferably genetic probes specific to predetermined allelic variations corresponding to a metabolic factor of interest. According to one embodiment each well of a microplate as illustrated in Fig. 18 may contain at least one genetic probe specific to a metabolic factor of interest. Examples of such probes are described in accordance with the genotypic determinants of those enzymes discussed herein above.

The present invention provides a convenient and effective tool for use in both a clinical and laboratory environment. The present invention is particularly suited for use by a physician in a clinic, whereby metabolic determinants corresponding to drug- specific metabolic factors, can be quickly and easily obtained. According to an embodiment of the present invention, a ready-to-use kit is provided for fast and accurate determination of at least one metabolic determinant for a metabolic factor specific for the metabolism of a given drug. The kit of the present invention may be tailored for phenotypic and/or genotypic screening. According to one embodiment, the ELISA assay system and kit preferably employ antibodies specific to a plurality of metabolites on a suitable substrate allowing for detection of the preferred metabolites in a biological sample of an individual after consumption of a corresponding probe substrate.

The assay systems of the present invention may be provided in a plurality of forms including but not limited to a high-throughput assay system or a dipstick based assay. EXAMPLE II

Determining Genotypic Determinants

Individuals with extreme metabolic phenotypes or genotypes are often at high risk for either toxicity or inefficacy of particular drug treatments. In some instances, these ultraextensive or extremely poor metabolizers can be identified by genotyping. For several metabolic enzymes genetic polymorphisms exist which result in an enzyme deficiency or the production enzyme with null activity. These individuals will not be affected by enzyme inducers or inhibitors and will consistently be extremely poor metabolizers . Identifying those individuals who carry these genetic polymorphisms allows physicians to avoid prescribing a drug metabolized by the enzyme in question. Conversely, several genetic polymorphisms have been identified that result in high levels of enzyme and/or increased enzyme activity. In addition, some individuals have been identified with multiple copies of the gene containing the polymorphism. As for the extremely poor metabolizers, these individuals may be identified as "high risk" candidates and subsequently excluded from certain treatment regimes.

According to an embodiment of the present invention genotypic determinants may be employed to individualize an effective dosage regime for an individual. In doing so, an individual having a specific allelic variation corresponding to an enzyme specific inefficiency in metabolism can be identified by genotyping and a corresponding dosage regime determined that will be safe for that individual . According to this example, an allelic variation may be identified as a metabolic factor of interest and metabolic determinants for that metabolic factor may be characterized to provide a metabolic profile. Based on an individual's metabolic profile, a corresponding individualized dosage may be determined.

According to an alternate embodiment of the present invention, a genomic assay is provided to characterize genotypic determinants for use in the individualization of therapy.

A genomic assay of the present invention may include a means for identifying a genetic marker such as a metabolic factor corresponding to an individual's capacity for the metabolism of a given drug or class of drugs, for example. This genetic marker may be- quantified in accordance with the genomic assay to provide an indicator of metabolic capacity. This indicator of metabolic capacity may be a genotypic determinant. This genotypic determinant may be employed to subsequently characterize a metabolic profile specific to an individual's metabolic capacity for the drug or class of drugs of interest. The genomic assay of the present invention may further include means for correlating said indicator with a therapeutically- effective dosage of the drug or class of drugs of interest for the individual .

According to an embodiment of the present invention a genetic probe specific to an allelic polymorphism of interest may be provided for identifying said genetic marker. Preferably, the allelic polymorphism is specific to a gene associated with metabolism, such as a gene known to influence the activity of at least one of the following enzymes : NAT1, NAT2, CYP2A6, CYP2D6, CYP3A4 , CYP2E1, CYP2C9, CYP1A2 and CYP2C19, for example. The genomic assay of the present invention may be specific to one or more genotypic determinants for the individualization of therapy and/or treatment with a drug or class of drugs. Typically, without limitation, the genomic assay will provide for the genotyping a biological sample obtained from an individual to • identify one or more genotypic determinants corresponding to metabolic factor (s) of interest. Based on the one or more genotypic determinants a metabolic profile may be characterized. A metabolic profile may correspond to a rate of drug metabolism of an individual. The metabolic profile may be employed ' in the determination of an individual dosage of a drug of interest .

EXAMPLE III

DETERMINING INDIVIDUALIZED TREATMENT REGIMES AND/OR DRUG DOSAGES

The exposure of an individual to a drug is described by the concept of area-under-the curve (commonly referred to as AUC) . AUC is related to clearance by the following equation:

AUC=dose/clearance Thus, if an individual's rate of drug clearance is known, the dose can be individualized to achieve a desired AUC by the equation:

Dose = desired AUC x clearance

An individual's rate of drug clearance is important as it determines the circulating drug concentrations. Both efficacy and toxicity are determined, in part, by the circulating concentrations of the drug .

Therefore, to individualize therapy a model is developed encompassing a plurality of factors, which could possibly play a role in an individual ' s clearance value for a particular medication (s) and hence predict a dose with maximal efficacy and minimal toxicity. As drug metabolism is the principal determinant of circulating drug concentrations, determining an individual's rate of drug metabolism is an important factor for the development of a successful model for the individualization of therapy. The model of the present invention will account for at least one metabolic factor contributing to an individual's rate of metabolism for a given drug. Metabolic determinant (s) corresponding to said at least one metabolic factor may be used in determining a specific dose of the given drug for that individual .

Other factors can alter drug clearance, such as body surface area, hepatic enzyme levels (e.g. serum alanine aminotransferases (ALT) , albumin, alkaline phosphatases and serum α-1-acidicglycoprotein (AAG) ) , and drug transport proteins (e.g. P-glycoprotein (pgp) ) ■

Other individual specific characteristics may play a role in determining individual dose-limiting toxicity. According to another aspect of the present invention, other influencing factors may be accounted for, in addition to the rate of metabolism, in the model for the individualization of therapy. For example, in the case of many chemotherapeutic drugs, myelosuppression is the dose-limiting toxicity, and hence an individual's pretreatment white blood cell

(WBC) count could be an important factor in predicting toxicity. Using multivariate analysis such individual factors are examined for correlation to efficacy and toxicity. In accordance with one embodiment of the present invention, factors identified as having a significant correlation to either efficacy or toxicity are included in the model along with drug metabolism.

The importance of drug metabolism in determining an individual ' s rate of drug clearance renders it as a key factor in determining the efficacy and toxicity of many drugs. Some of the metabolic enzymes mentioned in the context of this invention have a clear bimodal distribution of metabolism, allowing the separation of the population into poor and extensive metabolizers. However, within each phenotypic and genotypic group there is a wide variation in metabolic capacity. It may be naϊve to regard all individuals with metabolic ratios greater than a predetermined cut off value as being equivalent . This attempt to classify the population in two or three phenotypic or genotypic groups is even more difficult for enzymes without a bimodal distribution. The segregation of individuals into these limited classifications may not allow for the complete exploitation of an individual ' s pattern of metabolism. In some cases this simple classification is sufficient. For example, some individuals may have an enzyme specific deficiency, such as CYP2D6 and as a result are at risk for severe complications if high doses of a particular drug, such as Prozac™ are prescribed. However, this simple classification does not allow for differential dosing of the extensive metabolizers as a function of the molar ratio calculated during determination of phenotype. If the simple classification of extensive CYP2D6 metabolizers was used, all individuals with a molar ratio of >0.3

(dextromethorphan as probe substrate) would receive the same dose . Alternatively, the present invention provides a dosing scale that would produce an increasing dose with increasing molar ratio, as exemplified in Fig. 21. If only the bimodal distribution is considered, only two possible doses can be prescribed. Accordingly, the use of metabolic determinants in the individualization of therapy and/or drug dosing is proposed in accordance with the present invention. According to the present invention, current non-individualized or categorical treatment may be replaced with individualization of treatment whereby the metabolic phenotype or genotype of each individual is assessed on an individual basis and a corresponding individual dosage is determined. In accordance with this embodiment, an assessment of an individual's metabolic phenotype and/or genotype is employed to correlate an individual's specific rate of metabolism for an agent or drug of interest . In this manner, drugs may be prescribed on an individual basis in dosages corresponding to at least an individual's phenotypic or genotypic ability for metabolism.

In some cases multiple enzymes play key roles in determining the rate of drug metabolism. Therefore, the monitoring of only one metabolic enzyme in such cases may not provide complete information for individualizing therapy. The use of a multi-determinant assay allows for the examination of multiple metabolic factors to provide additional metabolism-related information and thereby providing a more accurate model for individualizing therapy. As one drug or drug metabolite can be acted on by several enzymes the use of a multi-determinant assay, which accounts for multiple enzymes associated metabolism, may, in some cases, provide a more accurate model. A multi- determinant assay of the present invention may employ genotyping and/or phenotyping to characterize a metabolic profile of an individual . The knowledge of an individual's (multiple) metabolic profile of metabolic determinant (s) allows physicians, without limitation, to: determine if the individual has a phenotype and/or genotype that allows for the safe prescription of a drug or class of drugs; determine which drug of a plurality of drugs used for treating an individual's pathology or disease is the optimal drug in terms of drug efficiency and drug safety for that individual; and determine the optimal drug dose in terms of drug efficiency and drug safety for an individual . The knowledge of an individual's metabolic profile for one or more enzymes provides for the identification of drug(s) that could cause significant side effects or be ineffective in treating that individual. In addition, a metabolic profile as provided by the present invention provides for the development of an individualized dosing scheme where a dosage corresponds to a level of enzymatic activity or rate of metabolism expressed by an individual . The

- implementation of a metabolic phenotyping and/or. genotyping in the individualization of treatment and/or individualization of dosing determination in accordance with the present invention will lead to a marked decrease in side effects and increase in therapeutic efficiency. The capacity of an individual to metabolize a drug is a critical factor in determining the efficacy of that drug for treating that particular individual. Accordingly, the ability to be able to individualize treatment, as provided by the present invention, based on an individual's phenotypic and/or genotypic capacity with respect to a metabolic factor of interest would provide an enormous benefit. The methodology of the present invention can be used to guide dosing of a plurality of drugs to maximize their efficacy. The present invention provides for an individualization model. According to one embodiment of the present invention, the individualization model is based upon at least one of a an individual's phenotype or genotype for metabolism for use in the individualization of therapy. This proactive procedure will identify starting doses much more accurately than the standard methods, and will result in much less post-administration "fine-tuning" of the dose.

In accordance with one embodiment of the present invention, prior to beginning a treatment, regime individuals are administered a predetermined dose of a probe substrate specific to an enzyme and/or metabolic pathway of interest, such as CYP3A4 and/or CYP2D6, for example. A biological sample is collected (e.g. urine) after the probe substrate is consumed. The concentrations of the probe substrate and metabolite (s) are determined and a molar ratio calculated. This molar ratio is specific to the individual's level of CYP3A4 and/or CYP2D6 activity.

The levels of activity of at least one enzyme and/or metabolic pathway as determined by genotyping and/or phenotyping are incorporated into an individualization of therapy model in accordance with one aspect of the present invention to determine an individualized treatment dosage of a drug that correlates with an individual's capacity to metabolize the same. An ELISA system as exemplified above may be employed to detect phenotypic determinants specific to the metabolism of a drug or class of drugs of interest for determining an individual's capacity to metabolize a candidate drug for treatment therewith.

Alternatively, a genomic assay may be employed to detect genotypic ^"determinants specific for the metabolism of a given drug or class of drugs. These genotypic determinants may be subsequently employed for determining an individual's capacity to metabolize a given drug. The present invention also provides for an individualization model based upon at least an individual's specific phenotype and/or genotype for at least one metabolic factor for use in the individualization of therapy an/or drug dosing. The_. individualization model of the present invention may further include other enzyme-specific determinants as well as other factors, which have a significant contribution to the clearance of a drug in the body or a significant contribution to ^' toxicity (e.g. pretreatment renal function) .

In accordance with an embodiment of the present invention, an assay system is provided that can be used in a clinical environment, whereby metabolic determinants can be quantified from a biological sample, such as urine or blood, for example, and applied to an individualization model to determine a dosage of a drug for treating an individual which corresponds to the individual's capacity metabolism of the drug. As a result, physicians will be provided with a tool for the individualization of therapy providing an alternative to the arbitrary selection of medications based on prognosis and categorical dosing.

While the invention has been described in connection with specifid embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from. the present disclosure as come within known or customary practice within the art to which the invention, pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims .

Claims

WHAT IS CLAIMED IS:

1. A method of individualizing drug treatment for an individual, wherein an individualized dosage of a drug selected from a drug or class of drugs known for treating a condition is determined for said individual, said method comprising: a) determining a metabolic profile of said individual corresponding to at least one

-metabolic factor known to influence the metabolism of said class of drugs; and b) calculating said individualized dosage of said drug according to metabolic determinants specific for said at least one metabolic factor; wherein said metabolic determinants are correlated to a rate of drug metabolism specific of said individual and said individualized dosage is calculated therefrom.

2. The method of claim 1, wherein said step of determining a metabolic profile includes genotyping.

3. The method of claim 2, wherein said metabolic determinants are genotypic determinants.

4. The method of claim 1, wherein said step of determining a metabolic profile includes phenotyping and/or genotyping.

5. The method of claim 1, wherein said at least one metabolic factor is specific the an activity level of an enzyme activity known to influence the metabolism of said drug or class of drugs.

6. The method of claim 5, wherein said activity level of said enzyme is specific for at least one cytochrome P450 or N-acetyltransferase enzyme.

7. The method of claim 1, wherein said at least one metabolic factor is an activity level of a metabolic pathway known to influence the metabolism of said drug or class of drugs.

8. The method of claim 7, wherein said activity level of said metabolic pathway is specific for at least one cytochrome P450 or N-acetyltransferase metabolic pathway.

9. The method of claim 2, wherein genotyping is performed by a genomic assay.

10. The method of claim 5, wherein said at least one enzyme is at least one of the following enzymes: NAT1, NAT2, CYP2A6, CYP2D6, CYP3A4 , CYP2E1, CYP2C9, CYP1A2 and CYP2C19.

11. The method of claim 1, further including: c) measuring at least one determinant for drug clearance known to affect the toxicity or efficacy of said drug or class of drugs; wherein said at least one determinant for drug clearance is factored together with at least said rate of drug metabolism to determine a therapeutically- effective amount of said drug or class of drugs to be prescribed to said individual.

12. The method of claim 11, wherein said at least one determinant for drug clearance is based on at least one of body surface area and hepatic enzyme levels of said individual .

13. Use of genotyping for the individualization of therapy and/or treatment, wherein an individual is genotyped for a specific metabolic factor and a corresponding genotypic determinant is characterized.

14. The use according to claim 13, wherein said genotypic determinant is used to quantify an individualized dosage regime of a drug.

15. The use according to claim 13, wherein said genotypic determinant is used to identify an individual having a metabolic incompatibility with a selected therapy and/or treatment .

16. The use according to claim 13, wherein said metabolic factor is at least one metabolism-specific allelic polymorphism.

17. The use according to claim 16, wherein said allelic polymorphism in a gene for one of the following enzymes: NAT1, NAT2 , CYP2A6, CYP2D6, CYP3A4, CYP2E1, CYP2C9, CYP1A2 and CYP2C19.

18. A genomic assay for use in the individualization of therapy and/or treatment, said assay comprising: a) a means for identifying a genetic marker corresponding to an individual's capacity for the metabolism of a given drug or class of drugs ; b) a means for quantifying said genetic marker to provide an indicator of metabolic capacity specific for said drug or class of drugs ; and c) a means for correlating said indicator with a therapeutically-effective dosage of said drug or class of drugs for said individual .

19. The genomic assay of claim 18, wherein said means for identifying said genetic marker includes a genetic probe specific to an allelic polymorphism of interest .

20. The genomic assay of claim 19, wherein said allelic polymorphism is specific to a gene associated with metabolism.

21. The genomic assay of claim 20, wherein said allelic polymorphism is specific to a gene know to influence the activity of at least one of the following enzymes: NAT1 , NAT2 , CYP2A6, CYP2D6, CYP3A4 , CYP2E1, CYP2C9, CYP1A2 and CYP2C19.

22. A method of using a genomic assay specific to a plurality of genootypic determinants for the individualization of therapy and/or treatment with a drug or class of drugs, said method comprising: a) genotyping a biological sample obtained from an individual to identify said plurality of genotypic determinants corresponding to metabolic factors of interest; b) calculating a rate of drug metabolism according to said plurality of genotypic determinants; and c) determining an individual dosage of said drug or class of drugs corresponding to said rate of drug metabolism; wherein said rate of drug metabolism is indicative of the rate of metabolism of said drug or class of drugs in said individual .

23. A method of selectively treating an individual with a drug or class of drugs; said method comprising: a) genotyping an individual to identify at least one allelic polymorphism known to influence the metabolism of said drug or class of drugs; b) phenotyping said individual to confirm their phenotypic capacity to metabolize said at drug of class of drugs; . c) calculating a therapeutically-effective amount of said drug or class of drugs specific for said individual based on said genotyping and phenotyping; and d) selectively treating said individual with the same .

24. The method of claim 23 wherein an allelic polymorphism known to adversely influence the metabolism of a drug or class of drugs is identified by genotyping, an individual is identified as high risk and treatment with said drug or class of drugs is avoided.