WO2017095921A1 - Compositions for cyp450 phenotyping using saliva samples - Google Patents

Compositions for cyp450 phenotyping using saliva samples Download PDF

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Publication number
WO2017095921A1
WO2017095921A1 PCT/US2016/064210 US2016064210W WO2017095921A1 WO 2017095921 A1 WO2017095921 A1 WO 2017095921A1 US 2016064210 W US2016064210 W US 2016064210W WO 2017095921 A1 WO2017095921 A1 WO 2017095921A1
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composition
substrate
unconverted
sub
eplerenone
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French (fr)
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Joseph MCGRAW
Armin Gerhardt
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Concordia University Inc
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Concordia University Inc
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Priority to JP2018547867A priority Critical patent/JP7086360B2/en
Priority to CA3006645A priority patent/CA3006645C/en
Priority to US15/779,788 priority patent/US10927398B2/en
Priority to AU2016365229A priority patent/AU2016365229B2/en
Priority to EP16871419.4A priority patent/EP3384040A4/en
Application filed by Concordia University Inc filed Critical Concordia University Inc
Publication of WO2017095921A1 publication Critical patent/WO2017095921A1/en
Anticipated expiration legal-status Critical
Priority to US17/153,333 priority patent/US20210139945A1/en
Priority to JP2022086475A priority patent/JP7529720B2/en
Priority to AU2023202583A priority patent/AU2023202583A1/en
Priority to US18/783,217 priority patent/US20250230481A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/485—Morphinan derivatives, e.g. morphine, codeine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0007—Effervescent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2054—Cellulose; Cellulose derivatives, e.g. hydroxypropyl methylcellulose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90241—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)

Definitions

  • the field of the invention relates to medical diagnostics. More particularly, the field of the invention relates to pharmacogenetic medical diagnostics. More particularly, the field of the invention relates to a compositions which may be utilized in methods for multiple simultaneous metabolic enzyme phenotyping using a saliva sample.
  • Cytochrome P450 (CYP450) enzymes are responsible for much of the variability in drug response and toxicity susceptibility.
  • the fields of pharmacogenetics and later pharmacogenomics began with observations that individuals from different racial/ethnic groups experienced different rates of response and toxicity to certain drugs.
  • scientists found significant differences in metabolic clearance of these 'highly variable' drugs on an individual and population level. They also found disproportionate frequencies of individuals with very low metabolic clearance amongst different racial/ethnic groups. Further investigations revealed genetic variants resulted in altered CYP450 enzyme activity and therefore differences in metabolic clearance of the drugs. After many years of investigation, CYP450 enzyme activity has remained the dominant determinant of metabolic clearance for many drugs.
  • the CYP450 enzyme family is the most studied enzyme family in the field of pharmacogenetics.
  • a drug When a drug is primarily metabolized via a specific CYP450 enzyme (i.e. responsible for 80% or more of drug clearance) it is known as a 'probe drug' .
  • alterations in CYP450 enzyme activity results in significant differences in drug clearance. Differences in metabolic enzyme activity are quantified by measuring the metabolic ratio i.e. the concentration or area ratio of a known enzyme substrate divided by the primary metabolite.
  • the metabolic ratio of the probe substrate is also called the metabolic phenotype although metabolic phenotype is often expressed as a categorical value such as poor metabolizer.
  • genetic assays which predict metabolic phenotype based on the presence or absence of genetic variants which result in altered metabolic clearance.
  • the genetic predicted metabolic phenotypes are categorical and labeled relative to an average individual being labeled a rapid metabolizer.
  • Other designations vary by CYP450 enzyme and particular study but common designations include: extensive metabolizer (EM) (i.e. the average wild type individual), poor metabolizer (PM) (i.e. individuals who have very poor metabolic clearance relative to the average wild type), intermediate metabolizer (IM) (i.e. individuals who have metabolic clearance between the average wild type and a poor metabolizer phenotype), rapid metabolizer (RM) (i.e. individuals who have higher metabolic clearance relative to the average wild type individual), and ultra-rapid metabolizers (UM) (i.e. individuals who have metabolic clearance significantly higher than rapid metabolizers).
  • EM extensive metabolizer
  • PM poor metabolizer
  • IM intermediate metabolizer
  • RM rapid metabolizer
  • UM ultra-rapid metabolizers
  • compositions, methods, and kit for the direct testing of metabolic phenotype for the major CYP450 enzymes using a melange of extremely safe phenotyping probes with a non-invasive saliva based testing assay This composition, methods, and kit allows for direct measurement of metabolic phenotype which can be converted into a traditional categorical phenotype or can be reported as a continuous variable metabolic phenotype.
  • P450 (CYP450) enzyme phenotyping The methods and compositions typically utilize a composition comprising one or more substrates for different CYP450 enzymes which may be administered orally to a patient. Subsequently, a metabolite of the one or more substrates may be detected in the patient' s saliva as well as any non-metabolized substrate to calculate a metabolic ratio for any given CYP450 enzyme in order to generate a phenytopic CYP450 enzyme profile for the patient.
  • the phenytopic CYP450 enzyme profile for the patient may be utilized in order to dose a drug for a patient and/or to assess hepatic function in the patient, for example, in a patient experiencing or at risk for developing hepatic failure.
  • Figure 1 Histogram of CYP1A2 Metabolic Phenotype in Healthy Adults.
  • Figure 2 Histogram of CYP2C19 Metabolic Phenotype in Healthy Adults.
  • Figure 3 Histogram of CYP2D6 Metabolic Phenotype in Healthy Adults.
  • a substrate and a metabolite should be interpreted to mean “one or more substrates” and “one or more metabolites,” respectively.
  • the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising” in that these latter terms are “open” transitional terms that do not limit claims only to the recited elements succeeding these transitional terms.
  • the term “consisting of,” while encompassed by the term “comprising,” should be interpreted as a “closed” transitional term that limits claims only to the recited elements succeeding this transitional term.
  • the term “consisting essentially of,” while encompassed by the term “comprising,” should be interpreted as a “partially closed” transitional term which permits additional elements succeeding this transitional term, but only if those additional elements do not materially affect the basic and novel characteristics of the claim.
  • a patient in need thereof may include a patient in need of phenotyping for cytochrome P450 (CYP450) enzymes.
  • CYP450 cytochrome P450
  • the term “patient” may be used interchangeably with the terms “subject” and “individual.”
  • a “patient” is intended to include human and non-human animals (e.g. , non-human primates, dogs, cats, horses, and the like).
  • a patient in need thereof may include a patient in need of drug dosing.
  • the disclosed methods may include methods for determining an appropropriate dose for a drug for a patient based on the patient's phenotype for one or more cytochrome P450 (CYP450) enzymes, which phenotype may be determined as disclosed herein.
  • CYP450 cytochrome P450
  • a patient in need thereof may include a patient experiencing or at risk for developing hepatic failure.
  • the disclosed methods may include methods of assessing liver function in a patient experiencing or at risk for developing hepatic failure based on the patient' s phenotype for one or more cytochrome P450 (CYP450) enzymes, which phenotype may be determined as disclosed herein.
  • CYP450 cytochrome P450
  • a patient in need thereof may include a patient enrolled in a drug study.
  • the disclosed methods may include methods of assessing liver function in a patient prior to participation in a drug study based on the patient's phenotype for one or more cytochrome P450 (CYP450) enzymes, which phenotype may be determined as disclosed herein.
  • CYP450 cytochrome P450
  • compositions disclosed herein typically include one or more substrates for one or more one or more isoforms of the cytochrome P450 (CYP450) enzymes.
  • the disclosed compositions may include a melange of substrates.
  • the term "melange” means a mixture, and the terms “melange” and “mixture” may be used interchangeably herein.
  • a melange may include a mixture of substrates for one or more enzymes.
  • a melange may include a mixture of substrates for one or more isoforms of the cytochrome P450 (CYP450) enzymes.
  • the substrates of the mixture may be individually formulated into multiple formulations which may be administered substantially concurrently and/or the substrates of the mixture may be formulated together into a single formulation.
  • a “substrate” refers to a chemical compound that is recognized by an enzyme and for which the enzyme catalyzes conversion of the substrate into a different chemical compound which may be referred to as a "metabolite.”
  • the liver contains enzymes that convert various drug substances (i.e. substrates) to metabolites, which are eliminated from the body in urine or excrement. This enzyme conversion process often determines the duration of action or intensity of drugs, which is why some drugs may be taken several times each day to treat diseases and produce desirable pharmacological effects.
  • Liver enzymes may include isoforms of cytochrome P450 (CYP450), N- acetyl transferases, UDP-glucuronosyltransferases, oxidases sulfotransferases and other enzymes.
  • CYP450 cytochrome P450
  • N- acetyl transferases N- acetyl transferases
  • UDP-glucuronosyltransferases oxidases sulfotransferases
  • oxidases sulfotransferases oxidases sulfotransferases
  • Each of these enzyme systems may be comprised of numerous isoforms, each of which is capable of metabolizing different substrates.
  • the CYP450 system in the human liver includes at least ten individual isoforms.
  • the CYP450 isoforms are often critical in determining the rate of elimination of drugs, and metabolism by CYP isoforms often represents the rate-limiting step in elimination of pharmaceutical
  • Prediction of metabolic phenotype based exclusively on genetic analysis, genetic markers, and/or genetic deficiencies may produce an imprecise result due to failure to include environmental factors, concomitant disease, levels of CYP450 isoform expression, translation and activity, and other factors.
  • Metabolic activity may be based on genetic markers including genetic deficiencies in a CYP450 isoform. As such, metabolic activity may be assessed by performing a genetic analysis.
  • the methods disclosed herein may include determining a patient' s methabolic phenotype and/or characterizing the patient's metabolic activity.
  • a patient' s metabolic activity may be referred to herein as a "metabolic phenotype.”
  • a patient may be characterized as a poor metabolizer (PM), and intermediate metabolizer (IM), an extensive metabolizer (EM), or an ultra-rapid metabolizer (UM).
  • PM poor metabolizer
  • IM intermediate metabolizer
  • EM extensive metabolizer
  • UM ultra-rapid metabolizer
  • metabolic phenotypes may be generated by administering a substrate for an enzyme to the patient. Subsequently, a sample may be taken from the patient and analyzed for the presence of a metabolite and any unconverted substrate to calculate a metabolic ratio, which can be used to characterize the patient's metabolic activity.
  • cytochrome P450 cytochrome P450
  • the methods and compositions may include or utilize one or more substrates for one or more different CYP450 enzymes (e.g., one or more substates for one or more of CYP1A2, CYP2C9, CYP2C19, CYP2E1, CYP2D6, CYP3A4, and CYP3A5) which may be administered orally to a patient.
  • the substrates of the disclosed compositions may be administered orally to a patient.
  • a saliva sample from the patient may be tested for a metabolite of one or more substrates of the composition that are metabolized by one or more CYP450 isoforms to generate the metabolite.
  • the saliva sample from the patient also may be tested for any non- metabolized substrate.
  • a ratio of the concentration of metabolite/non- metabolized substrate in saliva from the patient may be calculated to generate a metabolic phenotype for the patient for one or more CYP450 isoforms.
  • the disclosed methods include administering, typically orally, to a subject in need thereof a composition comprising one or more of: (i) a substrate for CYP1A2 (SUBCYPIA2), wherein CYP1A2 catalyzes conversion of SUBCYPIA2 to a metabolite (MET CY PIA2); ( ⁇ ) a substrate for CYP2C19 (SUB C YP2CI9), wherein CYP2C19 catalyzes conversion of SUBCYP2CI9 to a metabolite (METCYP2CI9); (hi) a substrate for CYP2D6 (SUBCYP2D6), wherein CYP2D6 catalyzes conversion of SUBCYP2D6 to a metabolite (METCYP2D6); and (iv) a substrate for CYP3A4 (SUBCYP3A4), wherein CYP3A4 catalyzes conversion of
  • the composition administered to the patient may comprise one or more tablet formulations of one or more substrates selected from SUBCYPIA2, SUBCYP2CI9, SUBCYP2D6, and SUB C Y3A4-
  • the composition includes multiple tablets, for example, one tablet each of SUBCYPIA2, SUBCYP2CI9, SUBCYP2D6, and SUBCY3A4-
  • the composition administered to the patient may comprise a single tablet formulation, the single table formulation including each of substrates including SUB C YPIA2 > SUB C YP2CI9, SUB C YP2D6, and SUBCY3A4-
  • Suitable tablet formulations may include immediate release tablet formulations, for example an immediate tablet release formulation for each of SUBCYPIA2, SUB C YP2ci9 > SUB C YP2D6 > and SUB C Y3A4-
  • the tablet formulations may include a non-substrate coating (
  • the composition administered in the disclosed methods is a buffered composition.
  • the composition administered in the disclosed methods may include a basic buffering agent, which may include but is not limited to sodium bicarbonate and/or calcium carbonate.
  • the composition administered in the disclosed methods has a basic pH when the composition is dissolved in water.
  • the composition administered in the disclosed methods may have a pH greater than about 7.5, 8.0, 8.5, or 9.0 when dissolved in water.
  • the metabolites and/or the unconverted substrates may be detected in saliva using any suitable procedure.
  • Suitable procedures may include but are not limited to procedures selected from the group consisting of Ultra High Pressure Liquid Chromatography (UHPLC), Mass Spectroscopy (MS), High Pressure Liquid Chromatography (HPLC), Ultraviolet Spectroscopy (UV), Gas Chromatography (GC), Electron Capture Detection (ECD), Flame Ionization Detection (FID), Raman Infrared (RI) Spectroscopy, Matrix- Assisted Laser Desorption/Ionization (MALDI), and combinations thereof.
  • UHPLC Ultra High Pressure Liquid Chromatography
  • MS Mass Spectroscopy
  • HPLC High Pressure Liquid Chromatography
  • UV Ultraviolet Spectroscopy
  • GC Gas Chromatography
  • ECD Electron Capture Detection
  • FID Flame Ionization Detection
  • RI Raman Infrared
  • MALDI Matr
  • the metabolites and/or the unconverted substrates may be detected in saliva using reagent composition that include one or more reagents for detecting the metabolites and/or the unconverted substrates via reacting the saliva with the reagent compositions.
  • the composition administered to the patient typically includes a substrate for one or more isoforms of CYP.
  • a substrate of the composition is a substrate for more than one isoform of CYP (e.g., a substrate for CYP1A2 may additionally be a substrate for another isoform of CYP such as any of CYP2C9, CYP2C19, CYP2E1, CYP2D6, CYP3A4, and CYP3A5).
  • a substrate of the composition is a substrate for one isoform of CYP and is not a substrate for another or any other isoform of CYP (e.g., a substrate for CYP3A4 may not be a substrate for another or any other of CYP1A2, CYP2C9, CYP2C19, CYP2E1, CYP2D6, and CYP3A5).
  • the substrates of the compositions that are administered in the disclosed methods may include "drugs" as known in the art.
  • the substrates (i.e. , drugs) of the compositions that are administered in the disclosed methods and the metabolites of the subtrates have suitable pharmaceutical characteristics such as oral bioavailability (e.g., greater than about 20%, 30%, 40% 50%, 60%, 70%, 80%, 90% or lower), and low or no plasma protein binding (e.g., less than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20%, or lower).
  • the substrates and the metabolites of the substrates have a suitable half-life which, in some embodiments, may be a half-life of greater than about 0.5, 1.0, or 2.0 hours and less than about 8.0, 7.0, 6.0, 5.0, or 4.0 hours (e.g., a half life of 2.0 - 4.0).
  • Suitable substrates for the compositions administered in the disclosed methods may include one or more drugs listed in the following Table of Exemplary Drugs:
  • Prieriaeetia (analgesic) 179.216
  • Atorvasiaiin (Lipitor) 14% 98% 7-14 558.6
  • Bosentaa (Tradeer 50% ⁇ . ⁇ ., 5 551.6 2C9
  • Cilostazoi (Pletel) 87-100% 95-98% 11-13 369 5 2C19
  • Cisapride (Piopviisiii) 35-65% 9S% 6-12 465.9
  • Doxorubicin (Adriamycia.) 5% 74-76% 20-4S 579-99
  • Halofatmme 60-70% days 500.423
  • Imatinib (Gieevec) 9S% 95% is 5S9.7
  • Lovastatm (Mevacor; 5% >95% 2-5 404.5 3A, 2C8
  • Solifenacin (Vesicare) 90% 98% 45-68 352.5
  • Tacrolimus iPrograi 3 7-35% 99% 8.7-37.9 822.03
  • Testosterone Vanes 98% varies 2% i Tiagabine (GabifrU) 90% 7-9 412 3 A, 1A2. 2D6, 2C1
  • Verapamil iCalan 13-65% 36-94% 4-12 491.1 2C18 Vinblastine (Vel aije; 98-99.7% 24.8 909.06
  • the composition administered to the patient may include a substrate for CYP isoform 1A2 (SUBCYPIA2)-
  • the substrate for CYP1A2 (SUBCYPIA2) is also a substrate for additional enzymes such as additional CYP isoforms such as isoform 2A6 or other enzymes such as N-acetyl transferases (NATs) and/or xanthine oxidases (XOs).
  • the substrate for CYP isoform 1A2 is also a substrate for each of CYP1A2, CYP2A6, NAT, and XO.
  • Suitable substrates for CYP1A2 may include but are not limited to caffeine (e.g. , where the metabolite (METCYPIA2) is paraxanthine) and theophylline.
  • the composition administered to the patient may include a substrate for CYP isoform 2C19 (SUBCYP2CI9)-
  • the substrate for CYP2C19 also is a substrate for additional enzymes such as additional CYP isoforms such as isoform 3A4 (SUBCYP3A4)-
  • Suitable substrates for CYP2C19 may include but are not limited to omeprazole (e.g., where the metabolite (METCYP2CI9) is 50H-omeprazole), esomeprazole, mephenytoin, clopidogrel, and phenytoin.
  • the composition administered to the patient includes omeprazole and a basic buffering agent, which may include but is not limited to sodium bicarbonate and/or calcium carbonate.
  • the composition administered to the patient may include a substrate for CYP isoform 2D6 (SUBCYP2D6)- Suitable substrates for CYP2D6 may include but are not limited to dextromethorphan (e.g., where the metabolite (METCYP2D6) is dextrorphan), desipramine, and metoprolol.
  • a substrate for CYP isoform 2D6 e.g., where the metabolite (METCYP2D6) is dextrorphan
  • desipramine e.g., desipramine
  • metoprolol e.g., metoprolol
  • the composition administered to the patient may include a substrate for CYP isoform 3A4 (SUB CYP 3A 4 ), for example, wherein a metabolite (MET CYP 3A 4 ) is subsequently detected in saliva of the patient and any unmetabolized SUB CYP 3A 4 - Suitable substrates for CYP3A4 may include but are not limited to eplerenone (e.g.
  • metabolite is 6 -hydroxyeplerenone and/or 21- hydroxyeplerenone
  • midazolam simvastatin, alfentanil, dextrormethorphan, omeprazole, erythromycin, Cortisol, midazolam, quindine, and triazolam.
  • the composition administered to a patient may include a mixture of substrates comprising one or more of SUBC YPIA 2, SUBC YP 2C I9 , SUBC YP 2D6, and SUBC Y 3A 4 -
  • the composition administered to the patient further includes a substrate for an enzyme selected from one or more of CYP2C9, CYP2E1 , and CYP3A5 (i.e. , SUB CYP2A9 , SUB CYP2EI , and SUBC Y 3A5, respectively).
  • the composition administered to the patient include may include a substrate for CYP isoform 2C9 (SUB CYP 2C 9 ), for example, wherein a metabolite (MET CYP 2C 9 ) is subsequently detected in saliva of the patient and any unmetabolized SUB CYP 2C 9 - Suitable substrates for CYP2C9 may include but are not limited to warfarin, tolbutamide, diclofenac, flurbiprofen, celecoxib, lornoxicam, ibuprofen, naproxen, ketoprofen, piroxicam, meloxicam, suprofen, phenytoin, fluvastatin, glipizide, glibenclamide, glimepiride, glyburide, irbesartan, losartan, S-warfarin, sildenafil, terbinafine, amitriptyline, fluoxetine, nateglinide, ros
  • the composition administered to the patient include may include a substrate for CYP isoform 2E1 (SUB CYP 2 EI ), for example, wherein a metabolite (MET CYP 2 EI ) is subsequently detected in saliva of the patient and any unmetabolized SUB CYP 2 EI - Suitable substrates for CYP2E1 may include but are not limited to chlorzoxazone, desipramine, and metoprolol.
  • the composition administered to the patient include may include a substrate for CYP isoform 3A5 (SUB CYP 3A 5 ), for example, wherein a metabolite (MET CYP 3A 5 ) is subsequently detected in saliva of the patient and any unmetabolized SUBCYP3A5- Suitable substrates for CYP3A5 may include but are not limited to alprazolam (a-hydroxylation), Cortisol, alfentanil, midazolam, tacrolimus, and triazolam, and vincristine.
  • a substrate for CYP isoform 3A5 SUVB CYP 3A 5
  • MET CYP 3A 5 metabolite
  • any unmetabolized SUBCYP3A5- Suitable substrates for CYP3A5 may include but are not limited to alprazolam (a-hydroxylation), Cortisol, alfentanil, midazolam, tacrolimus, and triazolam, and vincristine.
  • the composition administered to the patient may include a substrate for additional enzymes which are not CYP isoforms.
  • a mixture of substrates administered to the patient may include a substrate for an enzyme selected from a group consisting of an N-acetyl transferase (NAT), a methyl transferase, a UDP glucuronosyl transferase (UGT), a sulfo transferases, and an oxidative enzyme, or a combination thereof.
  • NAT N-acetyl transferase
  • UDT UDP glucuronosyl transferase
  • sulfo transferases oxidative enzyme
  • a mixture of substrates administered to the patient may include a substrate for an isoform of UDP glucuronosyl transferase (UGT), which may include, but is not limited to a substrate for one or more of UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7.
  • UGT UDP glucuronosyl transferase
  • the composition administered to the patient may include as a substrate ketoprofen.
  • the method further may include detecting one or more metabolites selected from beta-estradio-3-glucuronide, trifluoperazine-N-glucuronide, 5-hydroxytryptophol-O-glucuronide, propofol-O- glucuronide, zidovudine-5 '-glucuronide, and combinations thereof.
  • the disclosed methods may include determining a metabolic ratio based on one or more metabolites detected in saliva versus one or more unconverted substrates detected in saliva (e.g., METCYP/SUBCYP detected in saliva after having administered SUBCYP and having waiting for a period of time such as 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 24.0 hours or more).
  • a metabolic ratio based on one or more metabolites detected in saliva versus one or more unconverted substrates detected in saliva (e.g., METCYP/SUBCYP detected in saliva after having administered SUBCYP and having waiting for a period of time such as 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 24.0 hours or more).
  • the disclosed methods may include determining a metabolic ratio for one or more of: (i) METCYPIA2 and unconverted SUBCYPIA2 (e.g., caffeine versus paraxanthine); (ii) METCYP2CI9 and unconverted SUB C YP2CI9 (e-g-, omeprazole versus 50H-omeprazole); (iii) MET C YP2D6 and unconverted SUBCYP2D6 (e.g., dextromethorphan versus dextrorphan); (iv) METCYP3A4 and unconverted SUBCYP3A4 (e.g., ⁇ -hydroxyeplerenone and/or 21 -hydroxy eplerenone versus eplerenone); (v) METCYP2C9 and unconverted SUBCYP2C9 ; (vi) METCYP2EI and unconverted SUBCYP2EI ; and (vii) METCYP3
  • compositions typically comprise one or more substrates for one or more CYP isoforms (e.g., one or more substates for one or more of CYP1A2, CYP2C9, CYP2C19, CYP2E1 , CYP2D6, CYP3A4, and CYP3A5).
  • CYP isoforms e.g., one or more substates for one or more of CYP1A2, CYP2C9, CYP2C19, CYP2E1 , CYP2D6, CYP3A4, and CYP3A5
  • the compositions may comprise one or more of: (i) a substrate for CYP1A2 (SUBCYPIA2), wherein CYP1A2 catalyzes conversion of SUB 1A2 to a metabolite ( ⁇ 2);( ⁇ ) a substrate for CYP2C19 (SUB C YP2CI9), wherein CYP2C19 catalyzes conversion of SUB 2C i9 to a metabolite (METCYP2CI9); (hi) a substrate for CYP2D6 (SUBCYP2D6), wherein CYP2D6 catalyzes conversion of SUBCYP2D6 to a metabolite (METCYP2D6); and (iv) a substrate for CYP3A4 (SUBCYP3A4), wherein CYP3A4 catalyzes conversion of SUBCYP3A4 to a metabolite (METCYP 3 A4)- Substrates for the disclosed
  • compositions may comprise one or more tablet formulations of one or more substrates selected from SUBCYPIA2, SUBCYP2CI9, SUBCYP2D6, and SUBCY 3 A4-
  • the composition includes multiple tablets, for example, one tablet each of SUBCYPIA2, SUBCYP2CI9, SUBCYP2D6, and SUBCY3A4-
  • the composition administered to the patient may comprise a single tablet formulation, the single table formulation including each of substrates including one or more of SUBCYPIA2, SUBCYP2CI9, SUBCYP2D6, and SUBCY3A4-
  • Suitable tablet formulations may include immediate release tablet formulations, for example an immediate table release formulation for one or more of SUBCYPIA2, SUBCYP2CI9, SUBCYP2D6, and SUB C Y 3 A4-
  • the tablet formulations may include a non-substrate coating (e.g., an enteric coating).
  • compositions typically include a substrate for one or more isoforms of CYP.
  • a substrate of the composition is a substrate for more than one isoform of CYP (e.g., a substrate for CYP1A2 may additionally be a substrate for another isoform of CYP such as any of CYP2C9, CYP2C19, CYP2E1 , CYP2D6, CYP3A4, and CYP3A5).
  • a substrate of the composition is a substrate for one isoform of CYP and is not a substrate for another or any other isoform of CYP (e.g., a substrate for CYP3A4 may not be a substrate for another or any other of CYP1A2, CYP2C9, CYP2C19, CYP2E1, CYP2D6, and CYP3A5).
  • compositions may comprise one or more of
  • SUBCYPIA2 may also be a substrate for other CYP isoforms or substrates for other enzymes.
  • SUBCYPIA2 also may be a substrate for an enzyme selected from the group consisting of CYP2A6, NAT2, XO, and combinations thereof.
  • SUBCYP2CI9 may also be a substrate for other CYP isoforms or substrates for other enzymes.
  • SUBCYP2CI9 may also be a substrate for CYP3A4.
  • Suitable substrates for the disclosed composition may include, but are not limited to, one or more of: (i) SUBCYPIA2 selected from caffeine and theophylline; (ii) SUBCYP2C I9 selected from omeprazole, esomeprazole, mephenytoin, clopidogrel, and phenytoin; (iii) SUBCYP2D6 selected from dextromethorphan, desipramine, and metoprolol; and (iv) SUBCYP3A4 selected from eplerenone, midazolam, simvastatin, alfentanil, dextrormethorphan, omeprazole, erythromycin, Cortisol, midazolam, quindine, and triazolam.
  • suitable substrates for the disclosed compositions may include one or more of caffeine, omeprazole, dextromethorphan, and eplerenone, and optionally a basic buffering agent, which may include but is not limited to sodium bicarbonate and/or calcium carbonate.
  • the disclosed mixtures may include a substrate for one or more of CYP2C9, CYP2E1 , and CYP3A5.
  • the disclosed compositions further may comprise a substrate for a non-CYP450 enzyme selected from an N-acetyl transferase (NAT), a methyl transferase, a UDP glucuronosyl transferase (UGT), a sulfo transferases, and an oxidative enzyme, or a combination thereof.
  • the compositions further comprises a substrate for an isoform of UDP glucuronosyl transferase (UGT) selected from the group consisting of UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7.
  • the disclosed compositions further may comprise ketoprofen.
  • the substrates utilized in the methods disclosed herein may be formulated as a pharmaceutical composition in solid dosage form, although any pharmaceutically acceptable dosage form can be utilized.
  • Exemplary solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage form can be, for example, an immediate release form.
  • the substrates may be formulated in the same dosage form (e.g., all tablet form). In other embodiments, the substrates may be formulated in different dosage forms (e.g., some in tablet form others in powder form).
  • the disclosed composition may include a buffer.
  • the composition may include a basic buffering agent, which may include but is not limited to sodium bicarbonate and/or calcium carbonate.
  • the disclosed composition may have s a basic pH when the composition is dissolved in water.
  • the composition may have a pH greater than about 7.5, 8.0, 8.5, or 9.0 when dissolved in water.
  • the substrates utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier.
  • the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.
  • the substrates utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents.
  • Filling agents may include lactose monohydrate, lactose anhydrous, and various starches;
  • binding agents are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCCTM).
  • Suitable lubricants may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel.
  • colloidal silicon dioxide such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel.
  • sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame.
  • sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame.
  • flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like.
  • preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.
  • the substrates utilized in the methods disclosed herein may be formulated as a pharmaceutical composition for delivery via any suitable route.
  • the pharmaceutical composition may be administered via oral routes, sublingual routes, or buccal routes.
  • Examples of pharmaceutical compositions for administration include capsules, syrups, concentrates, powders and granules.
  • Suitable capsules may include hard gelatin capsules or softgels (aka soft gelatin capsules).
  • the substrates utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.
  • kits may be utilized for testing metabolic activity in a patient and/or determining a phenotype of a patient.
  • the kits may be utilized for testing metabolic activity of one or more CYP450 isoforms and/or determining a phenotype of a patient in regarding to the activity of one or more CYP450 isoforms.
  • the disclosed kits may include as a component any of the compositions and/or substrates disclosed herein.
  • kits include one or more additional components selected from the group consisting of: (i) containers (e.g., salivettes) for collecting and transporting saliva samples; (ii) components and/or reagents for performing a UPLC-MS/MS assay; and/or (iii) components for generating a report of the results of the UPLC-MS/MS assay.
  • containers e.g., salivettes
  • Example 1 CYP450 Phenotyping Using a Probe Melange and a Saliva
  • a saliva sample into a centrifuge tube (typically 500 mcL of liquid not including bubbles is required). Label this saliva sample "0". Place your initials on the tube, and place the tube in the freezer. Note: Do not try to express saliva (i.e., do not try to make more), just release what is already in your mouth.
  • OTC® (omeprazole 20 mg), and 1 caffeine tablet (caffeine 200 mg). Record the time in your lab notebook.
  • MS/MS instrument parameters for Method 1 are described in Table 2. Optimal voltage settings for each substrate and metabolite were determined via syringe pump direct infusion of each standard in 50% methanol/water solution. The optimized mass spectrometer parameters for Method 1 are listed in Table 3.
  • MS/MS instrument parameters for Method 2 are described in Table 5.
  • the optimized mass spectrometer parameters for Method 2 are listed in Table 6.
  • Standard curves were created using multiple injections at each data point for external standards from 0.06 to 1000 ng/ml.
  • Product quantification was performed using AB Sciex Multiquant software version 2.0.2.
  • Phenotyping results were generated for caffeine as a probe of CYP1A2, omeprazole as a probe for CYP2C19, and dextromethorphan as a probe for CYP2D6 in healthy adults.
  • L Caffeine probe of CYP1A2 phenotype Caffeine is commonly used as a probe in phenotyping studies and does not interact with the other probes used in our melange. Studies differ in their reports of CYP1A2 phenotype as determined by caffeine. Different researchers describe the histogram as unimodal, bimodal, or trimodal. (See Zhou et al. , Drug Metab. Rev. 41(2):89-295). In our cohort of primarily Caucasian subjects we observe a primarily unimodal distribution with outliers forming a small extreme mode. Our extreme mode corresponds to poor metabolizers and represents 7.8% of our subjects. This is in agreement with another study of Australian Caucasians which reported 5% poor metabolizers. (See Ilett et al, Clin. Pharmacol. Ther. 54(3):317-322).
  • Omeprazole has been used as a phenotyping probe for CYP2C19 in several studies in urine and plasma but never saliva. Omeprazole is commonly used as a probe in phenotyping studies and does not interact with the other probes used in our melange. Our saliva phenotyping results show a complex multimodal histogram consistent with other studies in other matrices. Frequencies of individuals within the different modes of the salivary CYP2C19 metabolic phenotype histogram corresponds very well with known frequencies of phenotypes reported based on known genetic polymorphisms.
  • Dextromethorphan is commonly used as a probe in phenotyping studies and does not interact with the other probes used in our melange.
  • the literature reported percentages of CYP2D6 metabolic phenotypes from a validation study based on genetic and phenotypic analysis were: Ultra-rapid (-1-2%), Extensive (-77-92%), Intermediate (-2-11%), and Poor (-5-10%). (See Gaedigk et al, Clin Pharma Ther 83(2): 234-242; Crews et al, Clin Pharma Ther 95(4): 376-382; and Fang et al. Pharmacogenomics J 14(6): 564-572). Literature reported percentages show close agreement with our findings: Ultra-rapid (2.3%), Extensive (83.9%), Intermediate (9.2%), and Poor (4.6%).
  • Salivary caffeine/paraxanthine metabolic ratio is an accepted measure of caffeine metabolic phenotype. Phenotypic examination using a substrate probe is necessary because genetics only explain approximately 42% of CYP1A2 catalytic activity and the common polymorphisms effect enzyme induction rather than enzyme activity (Klein, Winter et al. 2010).
  • Omeprazole is an accepted probe of CYP2C19 activity in urine and plasma.
  • omeprazole as a probe in saliva has not been previously reported. This is likely due to the low salivary concentrations of omeprazole and its metabolite 5- hydroxy omeprazole. However, we were able to develop a very sensitive assay which could simultaneously measure caffeine, dextromethorphan, omeprazole and their major metabolites in saliva.
  • UHPLC parameters are described in Table 7.
  • the MS/MS instrument parameters are described in Table 8.
  • the optimized mass spectrometer parameters are listed in Table 9.
  • the UHPLC parameters for Method 2 are described in Table 4.
  • the MS/MS instrument parameters for Method 2 are described in Table 5.
  • the optimized mass spectrometer parameters for Method 2 are listed in Table 6.
  • Product quantification was performed using AB Sciex Multiquant software version 2.0.2.
  • Table 10 shows the salivary logio molar metabolic ratios for eplerenone and its metabolite 6Beta-Hydroxy Eplerenone, which is formed via CYP3A4.
  • the metabolic ratios are fairly stable over different time points.
  • the 3 hour time point may be the best choice because the distribution phase will be over after 2 hours because eplerenone exhibits a T max of approximately 1.5 hours.
  • Five healthy volunteers aged 22-44 years old were given 50mg of commercially available generic eplerenone. As expected, the range of values is not very wide.
  • Constitutive CYP3A4 variability is estimated at about 5-fold; however, illness, inhibition, and induction-related interactions can enhance variability [1,2] with estimates ranging 40-50-fold in different populations [3].
  • a source of variability in omeprazole metabolic ratio measurements in saliva may be due to contamination of the mouth and saliva when the tablet is ingested.
  • contamination may occur due to residual powder from the tablet formulation contaminating the mouth and saliva.
  • formulations will be prepared with a film coating to impede powder contamination of the oral cavity while not disturbing rapid dissolution.

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Abstract

Disclosed are methods and compositions which may be used in human cytochrome P450 (CYP450) enzyme phenotyping. The methods and compositions typically utilize a melange of substrates for different CYP450 enzymes which may be administered orally to a patient. Subsequently, the metabolites of the substrates may be detected in the patient's saliva as well as any non-metabolized substrates to calculate a metabolic ratio for any given CYP450 enzyme in order to generate a phenytopic CYP450 enzyme profile for the patient.

Description

COMPOSITIONS FOR CYP450 PHENOTYPING USING SALIVA SAMPLES
CROSS-REFERENCED TO RELATED PATENT APPLICATIONS
[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to
U.S. Provisional Application No. 62/386,389, filed on November 30, 2015, the content of which is incorporated herein by reference in its entirety.
BACKGROUND
[0002] The field of the invention relates to medical diagnostics. More particularly, the field of the invention relates to pharmacogenetic medical diagnostics. More particularly, the field of the invention relates to a compositions which may be utilized in methods for multiple simultaneous metabolic enzyme phenotyping using a saliva sample.
[0003] Cytochrome P450 (CYP450) enzymes are responsible for much of the variability in drug response and toxicity susceptibility. The fields of pharmacogenetics and later pharmacogenomics began with observations that individuals from different racial/ethnic groups experienced different rates of response and toxicity to certain drugs. Scientists found significant differences in metabolic clearance of these 'highly variable' drugs on an individual and population level. They also found disproportionate frequencies of individuals with very low metabolic clearance amongst different racial/ethnic groups. Further investigations revealed genetic variants resulted in altered CYP450 enzyme activity and therefore differences in metabolic clearance of the drugs. After many years of investigation, CYP450 enzyme activity has remained the dominant determinant of metabolic clearance for many drugs. The CYP450 enzyme family is the most studied enzyme family in the field of pharmacogenetics.
[0004] When a drug is primarily metabolized via a specific CYP450 enzyme (i.e. responsible for 80% or more of drug clearance) it is known as a 'probe drug' . For these particular drugs, alterations in CYP450 enzyme activity results in significant differences in drug clearance. Differences in metabolic enzyme activity are quantified by measuring the metabolic ratio i.e. the concentration or area ratio of a known enzyme substrate divided by the primary metabolite. The metabolic ratio of the probe substrate is also called the metabolic phenotype although metabolic phenotype is often expressed as a categorical value such as poor metabolizer. Currently there are genetic assays which predict metabolic phenotype based on the presence or absence of genetic variants which result in altered metabolic clearance. The genetic predicted metabolic phenotypes are categorical and labeled relative to an average individual being labeled a rapid metabolizer. Other designations vary by CYP450 enzyme and particular study but common designations include: extensive metabolizer (EM) (i.e. the average wild type individual), poor metabolizer (PM) (i.e. individuals who have very poor metabolic clearance relative to the average wild type), intermediate metabolizer (IM) (i.e. individuals who have metabolic clearance between the average wild type and a poor metabolizer phenotype), rapid metabolizer (RM) (i.e. individuals who have higher metabolic clearance relative to the average wild type individual), and ultra-rapid metabolizers (UM) (i.e. individuals who have metabolic clearance significantly higher than rapid metabolizers).
[0005] Once the metabolic phenotype is determined, interventions such as lowering drug dosages in poor metabolizers can be performed. There is evidence showing benefits of genetic predicted phenotype guided drug dosing. However, there are some drawbacks to using genetic predicted phenotype. The genes encoding CYP450 enzymes do not change throughout a person's life but their level of expression, translation and activity do, thus the resulting metabolic phenotype is altered. A multitude of physiological and environmental factors such as alcohol ingestion, aging, diet, drug/pharmaceutical use producing enzyme induction or inhibition (drug-drug interactions), hepatic disease, renal disease, etc. impact the metabolic phenotype. In-vitro studies show a complex regulation of CYP450 activity including transcriptional regulation, translational regulation, post transcriptional modifications, and protein-protein interactions.
[0006] To avoid the pitfalls associated with genetic prediction of metabolic phenotype we have developed a composition, methods, and kit for the direct testing of metabolic phenotype for the major CYP450 enzymes using a melange of extremely safe phenotyping probes with a non-invasive saliva based testing assay. This composition, methods, and kit allows for direct measurement of metabolic phenotype which can be converted into a traditional categorical phenotype or can be reported as a continuous variable metabolic phenotype. SUMMARY
[0007] Disclosed are methods and compositions which may be used in cytochrome
P450 (CYP450) enzyme phenotyping. The methods and compositions typically utilize a composition comprising one or more substrates for different CYP450 enzymes which may be administered orally to a patient. Subsequently, a metabolite of the one or more substrates may be detected in the patient' s saliva as well as any non-metabolized substrate to calculate a metabolic ratio for any given CYP450 enzyme in order to generate a phenytopic CYP450 enzyme profile for the patient. The phenytopic CYP450 enzyme profile for the patient may be utilized in order to dose a drug for a patient and/or to assess hepatic function in the patient, for example, in a patient experiencing or at risk for developing hepatic failure.
BRIEF DESCRIPTION OF THE FIGURES
[0008] Figure 1. Histogram of CYP1A2 Metabolic Phenotype in Healthy Adults.
[0009] Figure 2. Histogram of CYP2C19 Metabolic Phenotype in Healthy Adults.
[0010] Figure 3. Histogram of CYP2D6 Metabolic Phenotype in Healthy Adults.
DETAILED DESCRIPTION
[0011] The present invention is described herein using several definitions, as set forth below and throughout the application.
[0012] Unless otherwise specified or indicated by context, the terms "a", "an", and
"the" mean "one or more." For example, "a substrate" and "a metabolite" should be interpreted to mean "one or more substrates" and "one or more metabolites," respectively.
[0013] As used herein, "about," "approximately," "substantially," and
"significantly" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of these terms which are not clear to persons of ordinary skill in the art given the context in which they are used, "about" and "approximately" will mean plus or minus <10% of the particular term and "substantially" and "significantly" will mean plus or minus >10% of the particular term.
[0014] As used herein, the terms "include" and "including" have the same meaning as the terms "comprise" and "comprising" in that these latter terms are "open" transitional terms that do not limit claims only to the recited elements succeeding these transitional terms. The term "consisting of," while encompassed by the term "comprising," should be interpreted as a "closed" transitional term that limits claims only to the recited elements succeeding this transitional term. The term "consisting essentially of," while encompassed by the term "comprising," should be interpreted as a "partially closed" transitional term which permits additional elements succeeding this transitional term, but only if those additional elements do not materially affect the basic and novel characteristics of the claim.
[0015] As used herein, "a patient in need thereof may include a patient in need of phenotyping for cytochrome P450 (CYP450) enzymes. The term "patient" may be used interchangeably with the terms "subject" and "individual." A "patient" is intended to include human and non-human animals (e.g. , non-human primates, dogs, cats, horses, and the like).
[0016] As used herein, "a patient in need thereof may include a patient in need of drug dosing. As such, the disclosed methods may include methods for determining an appropropriate dose for a drug for a patient based on the patient's phenotype for one or more cytochrome P450 (CYP450) enzymes, which phenotype may be determined as disclosed herein.
[0017] As used herein, "a patient in need thereof may include a patient experiencing or at risk for developing hepatic failure. As such, the disclosed methods may include methods of assessing liver function in a patient experiencing or at risk for developing hepatic failure based on the patient' s phenotype for one or more cytochrome P450 (CYP450) enzymes, which phenotype may be determined as disclosed herein.
[0018] As used herein, "a patient in need thereof may include a patient enrolled in a drug study. As such, the disclosed methods may include methods of assessing liver function in a patient prior to participation in a drug study based on the patient's phenotype for one or more cytochrome P450 (CYP450) enzymes, which phenotype may be determined as disclosed herein.
[0019] The compositions disclosed herein typically include one or more substrates for one or more one or more isoforms of the cytochrome P450 (CYP450) enzymes. The disclosed compositions may include a melange of substrates. As used herein, the term "melange" means a mixture, and the terms "melange" and "mixture" may be used interchangeably herein. A melange may include a mixture of substrates for one or more enzymes. In particular, a melange may include a mixture of substrates for one or more isoforms of the cytochrome P450 (CYP450) enzymes. The substrates of the mixture may be individually formulated into multiple formulations which may be administered substantially concurrently and/or the substrates of the mixture may be formulated together into a single formulation.
[0020] As used herein, a "substrate" refers to a chemical compound that is recognized by an enzyme and for which the enzyme catalyzes conversion of the substrate into a different chemical compound which may be referred to as a "metabolite." For example, the liver contains enzymes that convert various drug substances (i.e. substrates) to metabolites, which are eliminated from the body in urine or excrement. This enzyme conversion process often determines the duration of action or intensity of drugs, which is why some drugs may be taken several times each day to treat diseases and produce desirable pharmacological effects.
[0021] Liver enzymes may include isoforms of cytochrome P450 (CYP450), N- acetyl transferases, UDP-glucuronosyltransferases, oxidases sulfotransferases and other enzymes. Each of these enzyme systems may be comprised of numerous isoforms, each of which is capable of metabolizing different substrates. For example, the CYP450 system in the human liver includes at least ten individual isoforms. The CYP450 isoforms are often critical in determining the rate of elimination of drugs, and metabolism by CYP isoforms often represents the rate-limiting step in elimination of pharmaceuticals. Prediction of metabolic phenotype based exclusively on genetic analysis, genetic markers, and/or genetic deficiencies may produce an imprecise result due to failure to include environmental factors, concomitant disease, levels of CYP450 isoform expression, translation and activity, and other factors.
[0022] As such, a patient' s ability to metabolize a pharmaceutical is an important factor in determining a proper dose or dose regimen for the pharmaceutical. Metabolic activity may be based on genetic markers including genetic deficiencies in a CYP450 isoform. As such, metabolic activity may be assessed by performing a genetic analysis.
[0023] However, an understanding of the patient's actual metabolic activity is the most important factor for determining a proper dose or dose regimen for the pharmaceutical. The methods disclosed herein may include determining a patient' s methabolic phenotype and/or characterizing the patient's metabolic activity. A patient' s metabolic activity may be referred to herein as a "metabolic phenotype." Based on a metabolic phenotype, a patient may be characterized as a poor metabolizer (PM), and intermediate metabolizer (IM), an extensive metabolizer (EM), or an ultra-rapid metabolizer (UM). For example, metabolic phenotypes may be generated by administering a substrate for an enzyme to the patient. Subsequently, a sample may be taken from the patient and analyzed for the presence of a metabolite and any unconverted substrate to calculate a metabolic ratio, which can be used to characterize the patient's metabolic activity.
[0024] Probe Melange for Simultaneous CYP450 Phenotyping Using Saliva
Samples
[0025] As such, disclosed herein are methods and compositions including mixtures which may be used in human cytochrome P450 (CYP450) enzyme phenotyping. The methods and compositions may include or utilize one or more substrates for one or more different CYP450 enzymes (e.g., one or more substates for one or more of CYP1A2, CYP2C9, CYP2C19, CYP2E1, CYP2D6, CYP3A4, and CYP3A5) which may be administered orally to a patient. The substrates of the disclosed compositions may be administered orally to a patient. Subsequently, for example, after 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 24.0 hours or later, a saliva sample from the patient may be tested for a metabolite of one or more substrates of the composition that are metabolized by one or more CYP450 isoforms to generate the metabolite. The saliva sample from the patient also may be tested for any non- metabolized substrate. As such, a ratio of the concentration of metabolite/non- metabolized substrate in saliva from the patient may be calculated to generate a metabolic phenotype for the patient for one or more CYP450 isoforms.
[0026] In some embodiments, the disclosed methods include administering, typically orally, to a subject in need thereof a composition comprising one or more of: (i) a substrate for CYP1A2 (SUBCYPIA2), wherein CYP1A2 catalyzes conversion of SUBCYPIA2 to a metabolite (METCYPIA2); (ϋ) a substrate for CYP2C19 (SUBCYP2CI9), wherein CYP2C19 catalyzes conversion of SUBCYP2CI9 to a metabolite (METCYP2CI9); (hi) a substrate for CYP2D6 (SUBCYP2D6), wherein CYP2D6 catalyzes conversion of SUBCYP2D6 to a metabolite (METCYP2D6); and (iv) a substrate for CYP3A4 (SUBCYP3A4), wherein CYP3A4 catalyzes conversion of SUBCYP3A4 to a metabolite (METCYP3A4)- Subsequently, the methods may include (b) detecting in a saliva sample from the subject one or more of: (i) METCYPIA2 and unconverted SUBCYPIA2; (ϋ) METCYP2CI9 and unconverted SUBCYP2CI9; (iii) METCYP2D6 and unconverted SUBCYP2D6; and (iv) METCYP3A4 and unconverted
SUBcYP3A4-
[0027] In the disclosed methods, the composition administered to the patient may comprise one or more tablet formulations of one or more substrates selected from SUBCYPIA2, SUBCYP2CI9, SUBCYP2D6, and SUBCY3A4- In some embodiments, the composition includes multiple tablets, for example, one tablet each of SUBCYPIA2, SUBCYP2CI9, SUBCYP2D6, and SUBCY3A4- In other embodiments, the composition administered to the patient may comprise a single tablet formulation, the single table formulation including each of substrates including SUBCYPIA2> SUBCYP2CI9, SUBCYP2D6, and SUBCY3A4- Suitable tablet formulations may include immediate release tablet formulations, for example an immediate tablet release formulation for each of SUBCYPIA2, SUBCYP2ci9> SUBCYP2D6> and SUBCY3A4- In some embodiments, the tablet formulations may include a non-substrate coating (e.g. , an enteric coating or other type of coating).
[0028] In some embodiments, the composition administered in the disclosed methods is a buffered composition. For example, the composition administered in the disclosed methods may include a basic buffering agent, which may include but is not limited to sodium bicarbonate and/or calcium carbonate.
[0029] In some embodiments, the composition administered in the disclosed methods has a basic pH when the composition is dissolved in water. For example, the composition administered in the disclosed methods may have a pH greater than about 7.5, 8.0, 8.5, or 9.0 when dissolved in water.
[0030] In the disclosed methods, the metabolites and/or the unconverted substrates may be detected in saliva using any suitable procedure. Suitable procedures may include but are not limited to procedures selected from the group consisting of Ultra High Pressure Liquid Chromatography (UHPLC), Mass Spectroscopy (MS), High Pressure Liquid Chromatography (HPLC), Ultraviolet Spectroscopy (UV), Gas Chromatography (GC), Electron Capture Detection (ECD), Flame Ionization Detection (FID), Raman Infrared (RI) Spectroscopy, Matrix- Assisted Laser Desorption/Ionization (MALDI), and combinations thereof. In addition, in the disclosed methods the metabolites and/or the unconverted substrates may be detected in saliva using reagent composition that include one or more reagents for detecting the metabolites and/or the unconverted substrates via reacting the saliva with the reagent compositions.
[0031] In the disclosed methods, the composition administered to the patient typically includes a substrate for one or more isoforms of CYP. In some embodiments, a substrate of the composition is a substrate for more than one isoform of CYP (e.g., a substrate for CYP1A2 may additionally be a substrate for another isoform of CYP such as any of CYP2C9, CYP2C19, CYP2E1, CYP2D6, CYP3A4, and CYP3A5). In other embodiments, a substrate of the composition is a substrate for one isoform of CYP and is not a substrate for another or any other isoform of CYP (e.g., a substrate for CYP3A4 may not be a substrate for another or any other of CYP1A2, CYP2C9, CYP2C19, CYP2E1, CYP2D6, and CYP3A5).
[0032] The substrates of the compositions that are administered in the disclosed methods may include "drugs" as known in the art. Preferably, the substrates (i.e. , drugs) of the compositions that are administered in the disclosed methods and the metabolites of the subtrates have suitable pharmaceutical characteristics such as oral bioavailability (e.g., greater than about 20%, 30%, 40% 50%, 60%, 70%, 80%, 90% or lower), and low or no plasma protein binding (e.g., less than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20%, or lower). Preferably, the substrates and the metabolites of the substrates have a suitable half-life which, in some embodiments, may be a half-life of greater than about 0.5, 1.0, or 2.0 hours and less than about 8.0, 7.0, 6.0, 5.0, or 4.0 hours (e.g., a half life of 2.0 - 4.0).
[0033] Suitable substrates for the compositions administered in the disclosed methods may include one or more drugs listed in the following Table of Exemplary Drugs:
[0034] Table of Exemplary Drugs for Assessing Metabolism by CYPl A2.
PLASMA HALF-
ORAL PROTEIN LIFE MOLECULAR
RRtTG BIOAVAILABILITY BINDING (hs-sj WEIGHT METABOLISM
AmiSiipx iiiie -43-46% ->99% -43-36 277.4 2D6
Clomipramine 20-78% 97% 19-37 314. SS 2C19, 3A4
Iniiprajiiins 22-77% 60-96% 8-21 280.41 2D6
Agumelaiine 5%. 95% 1-2 243.3
Clozapine 12-81% 97% 4-66 326.82 2D6, 3A4
Olanzapine 87% 93% 21 -54 312.43 2D6
S8.4-
Haioperidoi 60% 14-37 375. S6 1A2. 2DS
Caffeine 17-36% 5 194.19
Ropivaeaiise 87-98% - IV 94% 5-7 274.4
Tiieoph iiiae - 100% 40% 1.5-9.S 180.16 2.E1
Zohintripmi! -40% 25% 3 2S7.36
35-50
Melatonin 3-75% min 52 :·>. 1AI, 2C19
-5-7 2C9, 3A4, 3A5, 2C19, 2D6, i.
Tauioxifer: 9% d3y» 371.51 3A7, 2B6, 231
Erio!ifiib 60-100% 93% 36.2 393.44 3A4, iAl, 1C
Cyctobeiizapriiie 33-55% 93% g-37 275.39 2Ό6., 3A4
Esrradioi 43% >95% 6 hours 272.38 3A4
FiavoxaoM&e S3-S4% -77-S0¾ -14- IS 318.33
Mexiletme 90% 50-60% 10-12 179.26 2D6
Naproxen 95% 99% S-21 230.26 2C9. 2C8 ftidavisefioii 100% 70-76% 3-6 293.36 2D6, 3A4
Piienacetm 179.22
Paracetamol 85-98% !0-25% 5-4 151.16 2D6. 2E1
Propranolol 100% 9&% 4 259.34 2D6
Ri!uzole -60% 96% 12 234.2
Ta iine 2.4-36% 55% 2-4 198.26
Tizsnidme 40% 30% 2.5 253.71
Verapamil 20-35% 90% 2.8-7.4 454,6 2CS, 2C1S, 2C9, 3A4, 3A5
2C9, 2C8, 2CIS, 2C1S. 1A1
Warfarin 100% 99% 20-60 308 33 3A4,
ZiSeu!cn 93% 2.5-3.2 236.29 2C9. 3A4
[0035] Table of Exemplary Drugs for Assessing I Metabolism by CYP2C9.
PLASMA HALF-
ORAL PROTEIN LIFE MOLECULAR
DRUG BIOAVAILABILITY BINDING (los) WEIGHT METABOLISM
Cetecoxib 97% 1 1 3SL3S
Loiaosicaai Evi: S7% 99.7% 4 371.8
Diclofenac 50% >99% 1.9-2.2 296.15
I iiprofen «7-100% 99% 2.44 206.3
Naproxen 95% >99% 12-17 230.26
Ketoprofen 90% >99% 2.1 254.29
Pifoxicam 99% 50 331.348
Meloxkam B9% 99.4% 5-20 351.4
Siiprofen 92% y&% 2-4 260.3
P eiiy!oin 20-90% SS-93% 4-2:2 274.25 2C9, 2C1
Ffevnstatiii 24¾ 9s% 2.5 411 5 2C9, 2CS, 3A4
Glipizide 90-100% 98-99% 2-5 445.55
Gii eiicl-!iiiide 99% 10 493.99
Gumepintfe im¾ 5-9.2 490.62
Tolbutamide 80-99% 4.5-6.5 270.35
Glyburide 99% iO 493.99
Irbesartan 60-80% 90% 11-1.5 428.5
Lt¾arr.m 33% 13% 46 i 2C9. 3A4
S-warlan 100% 99% 40 330.31 2C9. 2C19, 2C18. ΪΑ2, 3A4
Sildeiiafii 41% 96% 4 666.7 3A4, 2C
Ter iinrfiiffi 40% >99% 22-26 327.9 2C9. 1A2. 3A4. 2CS, 2C19
Aasiuiptylifie 30-60% 90% 1 313.S7 3A4, 2C9. 206. 1A2. C19
4-6
Fiaoxetiae 60-80% 94.5%. days 309.3 2D6. 2C9
Nai. slinide 73% 9S¾ 1 5 317.423 2C9. A4
Rosigiiazcne 99% 99.8 3-4 357.4 2CS, 2C9
5-7
Tamoxifen 99% days 371.5 3 A, 2C9. 2D6
Torasemide S0% 2.2-3. S 34S.43
eiaiaixie JM: 90-93%. PO: 16% 47% 2-3 274.19
THC 10-35% 97-99% 1.6-59 314.469 2C9, 2i ;v. A4
Limoneiie 136.24 2( -.·. 2C19
Tapeis!adol 32% 20% 4-5 257.S 2C9, 2D6
Poiyutisatarated FAs
ontefakast 64-73% 99% 2.7-5.5 60S. IS 2C¾, 2C9, 3A4 [0036] Table of Exemplary Drugs for Assessing Metabolism by CYP2C19.
PLASMA HALF- ORAL PROTEIN LIFE MOLECULAR
DRUG BIOAVAILABILITY BINDING (his) WEIGHT METABOLISM
3A4, 2C . 2EX>, 1A2,
Ami !iipi ime 30-60% --90% 15 3 S3 87 2C19
Cfoimpiamiiie 20-78% 97% 32 351.3
Ii i i iiiiiiie 94-96% 89% 6-1 S 316.9 :·.< ; ·,· 2D 6
Crialoprarn 80% S0¾ 35 324.4 3A4
Moclobemkie 55-95% 50%
Bupropion 84% 19-21.3 276-320 2B6
Diazepam 9S% 95-99 3% Up to 48 284.75 3A4
Mephen toiii "Well-absorbed" 59.5% 17 218 25
ordazepairi 36-200 270 7
Pheir toin 20-90% SS-93% 14-22 274.25 2C9
Pfaetiob¾bitai 80-100% 20-60% 36-120 232.24
Primidone 90-109% 20-30% .3 ,3-7 218 25
Hexobarbiral 25% 236 3 2C9
Methylpheaobarbitai 50% 70-76% 1 1-67 246.3
Llansoprazcie 81-91% 97-99% 0.9-1.5 369.37 1A2
Omeprazole 30-40% 95-9(5% 0.5-1 345.42
FairtopiHzt'fc 77% 1 405-432 3A4. 2D6, 2C9
Rabeprazok 52% 96.3% 1-2 381.43 3A
E;;Mneprazo5e 90% 7% 1.5 367.4 3A4
Ckipidogies 50%. 98% 6 419 9 3A. 236. 1A2
Proguanii 75% 12-1S .253.7
Propranolol 30-70% 93% 3-6 295.S
Liuionsrie 12-24 136.26
Giiciazide 80% 85-99% S-12 323 4
Cariioprodoi 60% 8 260.3
Chloramphenicol 90-100% 50-80% 1.6-3.3 323.13
cl hos hamide 75 ■-•60% 3-12 279 1
Indoiitemaciii 100% 99% 4.5 357 8
Neifkiavir 9S% 3.5-5 567.7S 3 A. 2C19 Nflutamide SO-84% 38-59.1 317.2
?V: 5-
Progesterone !0-i5% 96-99% 20mii 314.5
Teiiiposide 99% 5 656,6
2C9. 2C19. 2C1S, 1.
Waiferiii 100% 99% 40 330.31 3A4
Tspentadol 32% 20% 4-5 257. S 2C9. 2C1 [0037] Table of Exemplary Drugs for Assessing Metabolism by CYP2D6.
PLASMA HALF
ORAL PROTEIN LIFE MOLECULAR
DRUG BIOAVAILABILITY BINDING (tit s) WEIGHT METABOLISM
Iinip3"3niii: 94-96% 89% 6- IS 31 .9 2C19. 2D6
Ainitriptyhse -43-46% >90% ~! 3-36 277.4 JA4, 2C9. 2D6. 1A2, 2
4-S
Fluoxetine 60-S0¾ 94.5% days 309.3 2D6. 2C9
Paroxetine 93-95% 15-25 329.3
Ftav Ki iine 53% S0% 15.6 318.3 1 A2, 2C .. 19. 2DS.
Veaiafasiae 12.6% 27-30% 5 277
Diiioseiiae 30-80% •>90% i2 333.88 1A2. 2D6
Miaaserin 20-30% 90% 1.4 264.4
Mirtazapine 50% :- '·· .. 26-37 265.36 2D6, 1A2. 3A4
Codeine '.·' "' .. 7-25% 2.5-3 299.36 2Ό6, Ϊ.Α4
Tiamadoi 75% 20% 5,6-6.7 299.8 2D6, 3A4
O-desmethyfcamaite: 9 249.349 Ml
N-desmei vteaiaadoi
[ioactive]
Oxycodone 60-87% 45% 5.S 315.37 3A4, 2D6
Hydroeodone 25% •••••15·';. 7-9 494.5 3A4. 2D6. 2C1 . 2B
TapeiiWd l 32% 20% 4-5 25 · x 2C9. 2D5
SS.4-
Hsioperidol 60% 92.5% 14-37 375.86 1A2, 2D6
Risperidone 70% 90% 3-20 410.49
Perphenazine 20% 9-12 403.97
Tliioridezine 21-24 370.577
Zi-dijpenih oi 49% 98% 20 400.965 2D6. 3A4
Ilopeiidone 96% 9S% 18-33 426.48 5A4, 2D6
Aiipiprszele 87%, >99% 75 448.38 2D6, 3A4
ChiorpiOinazine 32% 90-99% 6 318.86
Levoaiepromaane 50-60% 15 32S.5
Remoxipride 96% 5-6%. 3-6 371.27
Minaprine 2-2.5 298.38
Tamoxifen 99% days 371.5 3A, 2CS, 2D6 etopraioi 77% 12% 3-7 652.8
Timolol 90% ·. ! <■% 4 31S.4
Aiprerio!ot 20% S0% 2-3 249.34
Carveiiiiol 25-35% 95-98% 6-10 406.5 2D6. 2C9„ 3A4. 2C19. Bufiiraiei 297.S ebivoiol 12-96% 9S% 12-19 441.9
Pt'opraiioioi 30-70% 93% 3-6 295. S
Debrisoquiue 448.5
Fkcftinide 70-95% 40% 20 474.4
Propafenone 3.4-10.6% 95% 2-10 377 92 2D6, 3A4. 1A2
I .5- Eacaitiide 25-90% 70.5-7S I I .3 352.47
Mexfe!ae 80-90% 50-70% 6-17 179.259 2! 16. 1A2 Lidocaine
(msmly by 3A4) 35% 60-80% 1.5-2 2.34.34 1A2, 3A4, 2D6
Sparteine 234.38
56% 70-76% 3-6.2 293.4
Doaepezil 100% 96% 70 379.5 2D6, 3A4
P aifonaki 40-60% 12-20% 4-13 205.26
Tropisetroii 60-100% 71% 5.6-8.6 2S4.4
11-
Amphetamine lOCH-% 20% 12.36 135.21
MetiiOKyamphetsmiiie 165.232
DexriometiianiphetsaiHie 12 149.24
Aioriioxetme 63% 9S% 5.2 291.82
CMoiphenauii&e 25-50% 72% 20 274.8
Des feriilva'amine 6S% 36% 17-20 231.257
BeXtiOHi6(ilOipll3i! 11% 1 4-3.9 370.53 2D6. 3A4, 3A5 etodopraaiide S0% 30% 5-6 354.3
Peihexfline 12-18 277.488
Prieriaeetia (analgesic) 179.216
ProHietiaziae 88% 93% 16-19 2S4.42
[0038] Table of Exemplary Drugs for Assessing Metabolism by CYP3A4.
PLASMA HALF-
ORAL PROTEIN LIFE MOLECULAR
DRUG BIOAVAILABILITY BINDING (In-*) WEIGHT METABOLISM
Alfeiitanii 'Alfenta) 92% 1.5 452.9S
Alfnzosin (Uroxatral) 49% 86% 10 425 9
Ahnoiriptan (Axeit) 70% 35% 3.5 469.56 2D6, MAO
Alprazolam (Xanax) 90% S0% 11 308.7S
Aiiiiodai'one (Cea darone) s 96% >9days 681.78 2C8
Amlodipine iTfcrvasc) (· Ι 93% 30-60 567.1
Aprepiiaiit (Eniead) 60-65% 95% 9-13 534.4 1A2. 2C19, 2C9
Atazanavir Reyataz) 60-68% S6¾ 704.9
Atorvasiaiin (Lipitor) 14% 98% 7-14 558.6
Bepridil (Vascor) 601· a 99-100% 42 366.5
Bexarotei e (Targretb.) 99% 3 ;>■ ··
Bosentaa (Tradeer 50% <·.ν·., 5 551.6 2C9
Bromociipriiie (Parlodel) 65-95% 90-96% 6-20 654.6
Budesoinde t'Entocon) 9-21% 85-90% 2-3.6 430.5
Boprencrpiiine (Subvnex) 46-65% 96% 24-4S 467 6
Bupropion (Zybaa, Weillnitna, Voxra) S4% 19-21.3 239.7 2B
Carbaisaz-pine (ee. Tea'etol S9% 76% 12-17 236.3
Cevimeiiii* (Evoxac) <20¾ 4-6 199.3 2D6, 3A3
Cilostazoi (Pletel) 87-100% 95-98% 11-13 369 5 2C19
Cisapride (Piopviisiii) 35-65% 9S% 6-12 465.9
daiitoomycia iBiaxia) 55% Low 4 747.9
Clonazepam ί'Κίοηοριη) 90% 85% 30-40 315.7
Clopjtlogrei ί'ΡΙΐΓ,άχ) >5C¾ 94-98% 6 321 S 2C1S, 3 A, 2B6, 1A2
26.S-
Cokhicine 45% 39% 31.2 399.4
Cv¾lophos»haiiaidfi. (C'vtoxani 75% >60% 3-12 261.1
Cyciosporiiie ( eorai) 30% 90% 19 1202.6
Dapsone (Avlosui&ii) 86-104»'» 70-90% 10-50 24S
Darunavir (Prezista) 37% 95% 15 593.73
Dasnt ib (Sprycef) 96% 3-5 488.01
Delavirdme (Rescriptor.i 85% 98% 5.S 552.6S 3A: 2D6
1.8S-
Dexamethasone (Decadtoa) 86.10% 77¼ 2.23 392.47
Di!iydroergoraiiiiiie 32% 93% 9-10 679.S
Diltiazetn (Cardizera) --40% 70-80% 3-6 450.99
E»isopyrarmde (Norpaee 80% 50-65% 6.7 437.47
Docetaxei (Taxoiere) S 94-97% 11.1 8 1.9
Doiiepezil (Arscepi > 100% 96% 70 379.5 3A4. 2D6
Doxorubicin (Adriamycia.) 5% 74-76% 20-4S 579-99
Drop ridol 379 43 Duiasteride (Avodait) 60% 99% 5 weeks 52g.5
Ebastine (Kestise) "High" 98% 24 8 469.658
99,5-
Efavaeaz {Sustivs} 40-45% 99,75% 40-55 315.675 3 A, 2B6
Eietnptan (Relpax) 50% 85% 4 463.4
EpSerenone (Inspia) 69% 50% 3-6 414.5
Ergotamine (Ergomar) <5% i.S-2.5 SSI .66
Eiiotinib (Tarceva) 60% 93% 36.2 393.436 3A4, 1A2
Ei iiii' niy iii 30-65% 90% 1-1.5 744.94
Eitazolani ProSoin) 93% 93% 10-24 294 74
Eszopiclone (Ltmesta) 52-59% 6 3S8.80S A4, 2ΕΪ
Ethinyl Estradiol 38-4S% 97-9S¾ 7-36 296,403
Ethosuximide (Zaiontm.i 93% 25-60 I4L168 3A4, 2E1
Etoposide (Vepesid) 50% 97% 7 588.57
E :€ine ta n e (Aro inasi ii} 42% 90% 24 296 41
Felodipine (Pieadil) i3-20% 99% 33,2 384,259
Fentan i (Sublimaze) 76-92% 80-86% 3-27 336.5
Finasteride (Proscar) 65% 90% 4.5 372.55
Fluiazepam i'Dakuane) ;·.-.-;.. 97.2% 2 3 3S7 9
Fosamprenavir Lexiva j 90% 7 7 5S5.60S
Gatantaiiiine (Ss-minyl) 90% 1S% 7 368.27 3A4. 2D6
Gefi!iaib (Ire&sa) 60% 90% 48 446.9 3A4, 2D6
Graaiseton i yirii) 60% 65% 3-24 312.4 lAi. A4
6-10
Halofatmme (Halfan) 60-70% days 500.423
Ifosfamide ,'Ifex) 92-100% 20% 261.1 3 A, 2B1. 2B
Imatinib (Gieevec) 9S% 95% is 5S9.7
Indinavir i'Qixi an ; 30% 60% 1.8 71 1.&S
Irii!oiecan (Camptosar) 30-6S¾ 6-12 677 1
IR: 90-95%;
Isradipine (BynaCirc) <. XV ϊί-24% 95% S 371.39
Itraconazole (Sporanox) 55% 99.8% 35-64 705.64
Ixabepilone (Txernpra) 67-77% 52 506.7
Ketocoiiazoie (Nizoiai) 75% 99% 2-8 5 1.43
Lapatiaib (Tykerb) >99% 24 581.1 3A4, 3A5
Levomeiiiadvi Orlaam) 80% 35-60 353.5
Loperamide (Imodium) G.3 % 97% 9-14 477
Lopia-vvir (Kaietra.) 98-99% 5-6 628.8
Loraiadine (Claritin 100% 97% 8 382.9 2D6. 3A4
Lovastatm (Mevacor; 5% >95% 2-5 404.5 3A, 2C8
Maraviroc (Selzentry) 23-33% 76% 14-18 513.67
Mefloquine (Lariam) S5¾ 98% 3 weeks 414 78
Me tbylprednisolone 78% 2-3 374.5
Midazolam (Versed) PO: 36% 97% 3-6 362.25 Mifepristone (Mifeprex) 99.2% 20-85 429.6
ModaSml (Provigift 60% 7.5-15 273.35
erazodone 20% >99% 2-4 506.5
Nevirapine (Viranajiie) SO-94 60% 25-45 266.3 3A4. 2B6 i ai dipiiie (Cat dene) 35% 95% 8.6- Ϊ4.4 515.99 3A4, 2C8, 2D6
Nifedipine (Adalat) 45-56% 92-98% i.. 346.3
Niiuotu'pine iNimoiop) 13% 95% 8-9 418.4
Nisoidipsne (Su!ar) 5% 99% 13.7 3SS.4
Nitrendipine (Bsypres ) 25% 97-99% 2-24 360.4
Oxybufyiiir: i Dinopao) 91-93% 7-30 357.5
Oxycodone i'Pereodan) 60-87% 45% 5.6 3 : 5 " 3A4. 2D6
Paditaxei (Taxol) 6.50% 89-98% 52.7 853.9 2CS, 3A4
Paricalcitol (Zeaiplar) 72% 99 8% 5-7 416.6
Pimozide (Grap) 50% 55 461 .56 3A4, !A2, 2D6
Pioglrfazone 50% >99%. 3-7 392.9 3A4, 2CS
Praziquantel (BilBicide) 80% 80% 0.8-3 312.4
Prednisolone -"/.-:■··;.5% 70-90% 2-4 360.4
Pfeil soiie 92% 70% 2-3 358,43
Propoxyphene (Darvon) 40% 78% 6-12 339.5
Q'.iazepani (Dorai; :··.·'- ·: .. 95% 25-41 3S6.S
Quefiapme ( Sei ¾uei) 100% 6-7 ■·■■■■ :. 5
Qoiuaernie SO-9 % 5 days 399.96
Quinidme 70-80% 50-83% 6-8 324.43
Quinine 7(5-88% 69-92% 9.7-20 7S2.96
Raae!aziae (Raaess) 55% 62% 7-8,9 4 7,54 3A, 2D6
Repagliuide (Prand ) 56% 1 452.6 3A4. 2CS
Rifabutin (Riicacrane) 53% 85% 36-45 847.02
Ritonavir {Norvk) ·%- 3-5 720.95 3A4, 2D6
Saquinavir {Invirase} 9S% 13 670.86
Sib tramiae {Meiidia} 77% 97% 1.1 334.33
Sildenafil (Viagra) 41%. 96% 4 666.7 3A4, 2 9
Simvastatin (Zccor) 5% 95% 2.S-3.2S 41S.57
61.3-
Sirelimvis (Rapaniune) 14-27% 92% 72.3 91 .2
Solifenacin (Vesicare) 90% 98% 45-68 352.5
.Sufentanil (Suienta) 93% 2.5 S.68
Staiianib (Sutent) 95% 40-60 532.6
Tacrolimus iPrograi) 3 7-35% 99% 8.7-37.9 822.03
Tadaiafii (Craiis) 94% 15-35 3S9.4!
Tamoxifen (Nolvadex) 99% 5-7days 371.5 3A4, 2C9. 2D6
Tamstilosit; (Fioioax) 94-99% 9-15 444.9S 3A4, 2D6
Teiiipo.¾de (Yumon) 99% 5 656.7
Testosterone Vanes 98% varies 2% i Tiagabine (GabifrU) 90% 7-9 412 3 A, 1A2. 2D6, 2C1
11.1- Tmisazoie (Tiudaiuax) •00% 12% 14.7 247 3
Tipinnavir ('Aptivnsj 307% 99.9% 5.5-6 602.7
Topirsiaa te (Topsmsx) 80% 15-41% 21 339.3S
343.21
Vardenafil (Levitia) 15-44% 95% 4-5 57S 1 3A4. 3A5, 2C
3A4. 1A2. .· ;· . 2i39.
Verapamil iCalan) 13-65% 36-94% 4-12 491.1 2C18 Vinblastine (Vel aije; 98-99.7% 24.8 909.06
Vincristine (Oncovin) 85 923 04
Zipraskfaie (Geodon) 60% ¾ 467.42 3A4. 1A2 Zolpidem (Ambieu) 70% 92.5% 2,5-2. S 764.9
Zoaisaaiide (ZonegKm) 40-60% 63 212.23
Zopicicns (Imovaiie) so% 45% 3.5- j,5 SS 8
[0039] In the disclosed methods, the composition administered to the patient may include a substrate for CYP isoform 1A2 (SUBCYPIA2)- In some embodiments, the substrate for CYP1A2 (SUBCYPIA2) is also a substrate for additional enzymes such as additional CYP isoforms such as isoform 2A6 or other enzymes such as N-acetyl transferases (NATs) and/or xanthine oxidases (XOs). In some embodiments, the substrate for CYP isoform 1A2 is also a substrate for each of CYP1A2, CYP2A6, NAT, and XO. Suitable substrates for CYP1A2 may include but are not limited to caffeine (e.g. , where the metabolite (METCYPIA2) is paraxanthine) and theophylline.
[0040] In the disclosed methods, the composition administered to the patient may include a substrate for CYP isoform 2C19 (SUBCYP2CI9)- In some embodiments, the substrate for CYP2C19 also is a substrate for additional enzymes such as additional CYP isoforms such as isoform 3A4 (SUBCYP3A4)- Suitable substrates for CYP2C19 may include but are not limited to omeprazole (e.g., where the metabolite (METCYP2CI9) is 50H-omeprazole), esomeprazole, mephenytoin, clopidogrel, and phenytoin. In some embodiments, the composition administered to the patient includes omeprazole and a basic buffering agent, which may include but is not limited to sodium bicarbonate and/or calcium carbonate.
[0041] In the disclosed methods, the composition administered to the patient may include a substrate for CYP isoform 2D6 (SUBCYP2D6)- Suitable substrates for CYP2D6 may include but are not limited to dextromethorphan (e.g., where the metabolite (METCYP2D6) is dextrorphan), desipramine, and metoprolol. [0042] In the disclosed methods, the composition administered to the patient may include a substrate for CYP isoform 3A4 (SUBCYP3A4), for example, wherein a metabolite (METCYP3A4) is subsequently detected in saliva of the patient and any unmetabolized SUBCYP3A4- Suitable substrates for CYP3A4 may include but are not limited to eplerenone (e.g. , where the metabolite (METCYP33A4) is 6 -hydroxyeplerenone and/or 21- hydroxyeplerenone), midazolam, simvastatin, alfentanil, dextrormethorphan, omeprazole, erythromycin, Cortisol, midazolam, quindine, and triazolam.
[0043] In the disclosed methods, the composition administered to a patient may include a mixture of substrates comprising one or more of SUBCYPIA2, SUBCYP2CI9, SUBCYP2D6, and SUBCY3A4- In some embodiments of the disclosed methods, the composition administered to the patient further includes a substrate for an enzyme selected from one or more of CYP2C9, CYP2E1 , and CYP3A5 (i.e. , SUBCYP2A9, SUBCYP2EI , and SUBCY3A5, respectively).
[0044] In the disclosed methods, the composition administered to the patient include may include a substrate for CYP isoform 2C9 (SUBCYP2C9), for example, wherein a metabolite (METCYP2C9) is subsequently detected in saliva of the patient and any unmetabolized SUBCYP2C9- Suitable substrates for CYP2C9 may include but are not limited to warfarin, tolbutamide, diclofenac, flurbiprofen, celecoxib, lornoxicam, ibuprofen, naproxen, ketoprofen, piroxicam, meloxicam, suprofen, phenytoin, fluvastatin, glipizide, glibenclamide, glimepiride, glyburide, irbesartan, losartan, S-warfarin, sildenafil, terbinafine, amitriptyline, fluoxetine, nateglinide, rosiglitazone, tamoxifen, torasemide, ketamine, THC, JWH-018, AM-2201, and limonene.
[0045] In the disclosed methods, the composition administered to the patient include may include a substrate for CYP isoform 2E1 (SUBCYP2EI), for example, wherein a metabolite (METCYP2EI) is subsequently detected in saliva of the patient and any unmetabolized SUBCYP2EI- Suitable substrates for CYP2E1 may include but are not limited to chlorzoxazone, desipramine, and metoprolol.
[0046] In the disclosed methods, the composition administered to the patient include may include a substrate for CYP isoform 3A5 (SUBCYP3A5), for example, wherein a metabolite (METCYP3A5) is subsequently detected in saliva of the patient and any unmetabolized SUBCYP3A5- Suitable substrates for CYP3A5 may include but are not limited to alprazolam (a-hydroxylation), Cortisol, alfentanil, midazolam, tacrolimus, and triazolam, and vincristine.
[0047] In the disclosed methods, the composition administered to the patient may include a substrate for additional enzymes which are not CYP isoforms. In some embodiments of the disclosed methods a mixture of substrates administered to the patient may include a substrate for an enzyme selected from a group consisting of an N-acetyl transferase (NAT), a methyl transferase, a UDP glucuronosyl transferase (UGT), a sulfo transferases, and an oxidative enzyme, or a combination thereof. In some embodiments of the disclosed methods, a mixture of substrates administered to the patient may include a substrate for an isoform of UDP glucuronosyl transferase (UGT), which may include, but is not limited to a substrate for one or more of UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7.
[0048] In some embodiments of the disclosed methods, the composition administered to the patient may include as a substrate ketoprofen. The method further may include detecting one or more metabolites selected from beta-estradio-3-glucuronide, trifluoperazine-N-glucuronide, 5-hydroxytryptophol-O-glucuronide, propofol-O- glucuronide, zidovudine-5 '-glucuronide, and combinations thereof.
[0049] The disclosed methods may include determining a metabolic ratio based on one or more metabolites detected in saliva versus one or more unconverted substrates detected in saliva (e.g., METCYP/SUBCYP detected in saliva after having administered SUBCYP and having waiting for a period of time such as 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 24.0 hours or more). The disclosed methods may include determining a metabolic ratio for one or more of: (i) METCYPIA2 and unconverted SUBCYPIA2 (e.g., caffeine versus paraxanthine); (ii) METCYP2CI9 and unconverted SUBCYP2CI9 (e-g-, omeprazole versus 50H-omeprazole); (iii) METCYP2D6 and unconverted SUBCYP2D6 (e.g., dextromethorphan versus dextrorphan); (iv) METCYP3A4 and unconverted SUBCYP3A4 (e.g., όβ-hydroxyeplerenone and/or 21 -hydroxy eplerenone versus eplerenone); (v) METCYP2C9 and unconverted SUBCYP2C9 ; (vi) METCYP2EI and unconverted SUBCYP2EI ; and (vii) METCYP3A5 and unconverted SUBCYP3A5- [0050] Also disclosed herein are compositions suitable for use in the disclosed methods. The disclosed compositions typically comprise one or more substrates for one or more CYP isoforms (e.g., one or more substates for one or more of CYP1A2, CYP2C9, CYP2C19, CYP2E1 , CYP2D6, CYP3A4, and CYP3A5). In some embodiments, the compositions may comprise one or more of: (i) a substrate for CYP1A2 (SUBCYPIA2), wherein CYP1A2 catalyzes conversion of SUB 1A2 to a metabolite (ΜΕΤογρΐΑ2);(ϋ) a substrate for CYP2C19 (SUBCYP2CI9), wherein CYP2C19 catalyzes conversion of SUB2Ci9 to a metabolite (METCYP2CI9); (hi) a substrate for CYP2D6 (SUBCYP2D6), wherein CYP2D6 catalyzes conversion of SUBCYP2D6 to a metabolite (METCYP2D6); and (iv) a substrate for CYP3A4 (SUBCYP3A4), wherein CYP3A4 catalyzes conversion of SUBCYP3A4 to a metabolite (METCYP3A4)- Substrates for the disclosed compositions may include one or more "drugs" as known in the art and as provided above in the "Table of Exemplary Drugs."
[0051] In the disclosed compositions may comprise one or more tablet formulations of one or more substrates selected from SUBCYPIA2, SUBCYP2CI9, SUBCYP2D6, and SUBCY3A4- In some embodiments, the composition includes multiple tablets, for example, one tablet each of SUBCYPIA2, SUBCYP2CI9, SUBCYP2D6, and SUBCY3A4- In other embodiments, the composition administered to the patient may comprise a single tablet formulation, the single table formulation including each of substrates including one or more of SUBCYPIA2, SUBCYP2CI9, SUBCYP2D6, and SUBCY3A4- Suitable tablet formulations may include immediate release tablet formulations, for example an immediate table release formulation for one or more of SUBCYPIA2, SUBCYP2CI9, SUBCYP2D6, and SUBCY3A4- In some embodiments, the tablet formulations may include a non-substrate coating (e.g., an enteric coating).
[0052] The disclosed compositions typically include a substrate for one or more isoforms of CYP. In some embodiments, a substrate of the composition is a substrate for more than one isoform of CYP (e.g., a substrate for CYP1A2 may additionally be a substrate for another isoform of CYP such as any of CYP2C9, CYP2C19, CYP2E1 , CYP2D6, CYP3A4, and CYP3A5). In other embodiments, a substrate of the composition is a substrate for one isoform of CYP and is not a substrate for another or any other isoform of CYP (e.g., a substrate for CYP3A4 may not be a substrate for another or any other of CYP1A2, CYP2C9, CYP2C19, CYP2E1, CYP2D6, and CYP3A5).
[0053] As such, the disclosed compositions may comprise one or more of
SUBCYPIA2, SUBCYP2CI9, SUBCYP2D6, and SUBCYP3A4- In some embodiments of the disclosed compositions, SUBCYPIA2 may also be a substrate for other CYP isoforms or substrates for other enzymes. For example, SUBCYPIA2 also may be a substrate for an enzyme selected from the group consisting of CYP2A6, NAT2, XO, and combinations thereof. In some embodiments, of the disclosed compositions, SUBCYP2CI9 may also be a substrate for other CYP isoforms or substrates for other enzymes. For example SUBCYP2CI9 may also be a substrate for CYP3A4. Suitable substrates for the disclosed composition may include, but are not limited to, one or more of: (i) SUBCYPIA2 selected from caffeine and theophylline; (ii) SUBCYP2C I9 selected from omeprazole, esomeprazole, mephenytoin, clopidogrel, and phenytoin; (iii) SUBCYP2D6 selected from dextromethorphan, desipramine, and metoprolol; and (iv) SUBCYP3A4 selected from eplerenone, midazolam, simvastatin, alfentanil, dextrormethorphan, omeprazole, erythromycin, Cortisol, midazolam, quindine, and triazolam. In particular, suitable substrates for the disclosed compositions may include one or more of caffeine, omeprazole, dextromethorphan, and eplerenone, and optionally a basic buffering agent, which may include but is not limited to sodium bicarbonate and/or calcium carbonate. Optionally, the disclosed mixtures may include a substrate for one or more of CYP2C9, CYP2E1 , and CYP3A5.
[0054] Optionally, the disclosed compositions further may comprise a substrate for a non-CYP450 enzyme selected from an N-acetyl transferase (NAT), a methyl transferase, a UDP glucuronosyl transferase (UGT), a sulfo transferases, and an oxidative enzyme, or a combination thereof. Optionally, the compositions further comprises a substrate for an isoform of UDP glucuronosyl transferase (UGT) selected from the group consisting of UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7. Optionally, the disclosed compositions further may comprise ketoprofen.
[0055] The substrates utilized in the methods disclosed herein may be formulated as a pharmaceutical composition in solid dosage form, although any pharmaceutically acceptable dosage form can be utilized. Exemplary solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage form can be, for example, an immediate release form. In some embodiment, the substrates may be formulated in the same dosage form (e.g., all tablet form). In other embodiments, the substrates may be formulated in different dosage forms (e.g., some in tablet form others in powder form).
[0056] In some embodiments, the disclosed composition may include a buffer.
For example, the composition may include a basic buffering agent, which may include but is not limited to sodium bicarbonate and/or calcium carbonate.
[0057] In some embodiments, the disclosed composition may have s a basic pH when the composition is dissolved in water. For example, the composition may have a pH greater than about 7.5, 8.0, 8.5, or 9.0 when dissolved in water.
[0058] The substrates utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier. For example, the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.
[0059] The substrates utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Filling agents may include lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. Examples of flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.
[0060] The substrates utilized in the methods disclosed herein may be formulated as a pharmaceutical composition for delivery via any suitable route. For example, the pharmaceutical composition may be administered via oral routes, sublingual routes, or buccal routes. Examples of pharmaceutical compositions for administration include capsules, syrups, concentrates, powders and granules. Suitable capsules may include hard gelatin capsules or softgels (aka soft gelatin capsules).
[0061] The substrates utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.
[0062] Also disclosed herein are kits. The disclosed kits may be utilized for testing metabolic activity in a patient and/or determining a phenotype of a patient. For example, the kits may be utilized for testing metabolic activity of one or more CYP450 isoforms and/or determining a phenotype of a patient in regarding to the activity of one or more CYP450 isoforms. The disclosed kits may include as a component any of the compositions and/or substrates disclosed herein. In some embodiments, the disclosed kits include one or more additional components selected from the group consisting of: (i) containers (e.g., salivettes) for collecting and transporting saliva samples; (ii) components and/or reagents for performing a UPLC-MS/MS assay; and/or (iii) components for generating a report of the results of the UPLC-MS/MS assay.
EXAMPLES
[0063] The following Examples are illustrative and are not intended to limit the scope of the claimed subject matter. [0064] Example 1. CYP450 Phenotyping Using a Probe Melange and a Saliva
Sample
[0065] A. Materials and Methods
[0066] L Cohort Characteristics
[0067] Healthy adult, primarily Caucasian college students and faculty aged 18-65 were enrolled in the study from February 2013 to April 2015. Exclusion criteria included allergy to caffeine, dextromethorphan, or omeprazole. Other exclusion criteria were pregnancy, possible pregnancy, or chronic illness.
[0068] IL Saliva Collection Protocol yl.
[0069] The patient is instructed as follows:
[0070] 1. Do not drink or eat anything with caffeine or related products (ex. tea, coffee, chocolate, guarana extract) after 9 pm.
[0071] 2. Drink a glass of water after waking. Wait 1 hour, then spit at least
0.5 ml of a saliva sample into a centrifuge tube (typically 500 mcL of liquid not including bubbles is required). Label this saliva sample "0". Place your initials on the tube, and place the tube in the freezer. Note: Do not try to express saliva (i.e., do not try to make more), just release what is already in your mouth.
[0072] 3. Take 2 Robitussin® tablets (dextromethorphan 30 mg), 1 Prilosec
OTC® (omeprazole 20 mg), and 1 caffeine tablet (caffeine 200 mg). Record the time in your lab notebook.
[0073] 4. Drink a glass of water every couple hours.
[0074] 5. Four, Six, and Eight hours after taking the pills, spit out at least 0.5 ml of a saliva sample into a centrifuge tube and label the tubes "4, 6, and 8" respectively. Initial the tubes and place the tubes in the freezer. Record the time.
[0075] ILL Saliva Extraction a.k.a. Oral Fluid Extraction [0076] Method A
[0077] 1. Withdraw 500 mcL saliva and place into a new 1.5 mL centrifuge tube, also labeled with identifier and the sample number 0, 4, 6, or 8. Repeat with the other three saliva samples. Spike all four samples with 10 mcL internal standard solution (i.e., for effective quantitation in the final instrumental analysis), add 490 mcL of methanol, and mix by vortexing.
[0078] 2. Freeze the samples in a -80°C freezer (in the research lab in
Heidelberg or Barth).
[0079] 3. Remove the samples from the freezer once frozen (which typically takes -60-90 minutes) and thaw.
[0080] 4. Centrifuge the samples in the Eppendorf centrifuge at 13, 100 RPM for 3 minutes.
[0081] 5. Transfer 500 mcL of supernatant to a 3 kDa molecular weight cutoff centrifugal filter (Amicon Ultra 0.5 mL, or VWR 0.5 mL) nested in a new 1.5 mL centrifuge tube labeled with subject number and sample number, 0, 4, 6, or 8.
[0082] 6. Centrifuge the tube and sieve at 13, 100 RPM for 30 minutes.
[0083] 7. Remove 350 mcL of supernatant (flow through) from the bottom of the centrifuge tube from step 6 and mix it with 150 mcL of deionized ¾0 in a HPLC vial labeled with your study number and 0, 4, 6, or 8 to identify the sample.
[0084] Method B
[0085] 1. Freeze the samples in a -80°C freezer to denature salivary proteins
(See Francis et al, Arch. Oral. Biol. 45(7):601-606).
[0086] 2. Remove the samples from the freezer once frozen (takes -60-90 minutes) and thaw.
[0087] 3. Centrifuge the samples in an Eppendorf centrifuge at 13,100 RPM for 5 minutes to precipitate mucous and denatured (from freezing) salivary protein. [0088] 4. Decant saliva supernate into a new 1.5 mL centrifuge tube.
Withdraw 250 mcL saliva and place into another new 1.5 mL centrifuge tube. Also labeled with identifier and the sample number 0, 4, 6, or 8. Repeat with the other three saliva samples. Spike all four samples with 50 mcL internal standard solutions (for effective quantitation in the final instrumental analysis), add 200 mcL of methanol, and mix by vortexing.
[0089] 5. Centrifuge the samples in the Eppendorf centrifuge at 13, 100 RPM for 5 minutes to precipitate residual protein and lipid from saliva.
[0090] 6. Transfer supernate to a 3 kDa molecular weight cutoff centrifugal filter (Amicon Ultra 0.5 mL, or VWR 0.5 mL) nested in a new 1.5 mL centrifuge tube labeled with subject number and sample number, 0, 4, 6, or 8.
[0091] 7. Centrifuge the samples at 13,100 RPM for 30 minutes.
[0092] 8. Remove supernate (flow through) from the bottom of the centrifuge tube from step 7 and place in an 300mcl HPLC vial insert.
[0093] IV. UHPLC - MS/MS Method
[0094] Two UHPLC MS/MS methods were developed for initial data collection
(Method 1 and Method 2).
[0095] The UHPLC parameters for Method 1 are described in Table 1. The
MS/MS instrument parameters for Method 1 are described in Table 2. Optimal voltage settings for each substrate and metabolite were determined via syringe pump direct infusion of each standard in 50% methanol/water solution. The optimized mass spectrometer parameters for Method 1 are listed in Table 3.
[0096] The UHPLC parameters for Method 2 are described in Table 4. The
MS/MS instrument parameters for Method 2 are described in Table 5. The optimized mass spectrometer parameters for Method 2 are listed in Table 6. [0097] Standard curves were created using multiple injections at each data point for external standards from 0.06 to 1000 ng/ml. Product quantification was performed using AB Sciex Multiquant software version 2.0.2.
Table 1. Method 1 UHPLC Parameters
Figure imgf000029_0001
-acetont e, -tetra ycro ran, A- rmc ac , o n-souton, n. -necton
Table 2. Method 1 MS/MS Parameters
Figure imgf000029_0002
-temperamre, -eectrospray ionzaton
Table 3. Method 1 Optimized Mass Spectrometer Parameters
Figure imgf000029_0003
Table 4, Method 2 UHPLC Parameters
Figure imgf000030_0001
ACN-aceienitrile, FA-formic acid, SoPn-solution, Inj. -injection
Table 5. Method 2 MS/MS Parameters
Instrument Information Mlli Source Parameters
Instrument Sciex 4000 Qtrap IHHI Curtain Gas 20 PSI
Source Turbo V Collision Gas High
Probe ESI Ion Spray Voltage 4500 V
Software Analyst, v. 1.5.1 Source T 550aC
Scan time 50ms HHHl Gas 1 45 PSI
Mode Positive Bill Gas 2 60 PSI
T-temperature, ESI-electrospray ionization
Table 6. Method 2 Optimized MS/MS Parameters
Figure imgf000030_0002
[0098] FL Results
[0099] Phenotyping results were generated for caffeine as a probe of CYP1A2, omeprazole as a probe for CYP2C19, and dextromethorphan as a probe for CYP2D6 in healthy adults.
[00100] L Caffeine probe of CYP1A2 phenotype [00101] Caffeine is commonly used as a probe in phenotyping studies and does not interact with the other probes used in our melange. Studies differ in their reports of CYP1A2 phenotype as determined by caffeine. Different researchers describe the histogram as unimodal, bimodal, or trimodal. (See Zhou et al. , Drug Metab. Rev. 41(2):89-295). In our cohort of primarily Caucasian subjects we observe a primarily unimodal distribution with outliers forming a small extreme mode. Our extreme mode corresponds to poor metabolizers and represents 7.8% of our subjects. This is in agreement with another study of Australian Caucasians which reported 5% poor metabolizers. (See Ilett et al, Clin. Pharmacol. Ther. 54(3):317-322).
[00102] IL Omeprazole probe of CYP2C19 phenotype
[00103] Omeprazole has been used as a phenotyping probe for CYP2C19 in several studies in urine and plasma but never saliva. Omeprazole is commonly used as a probe in phenotyping studies and does not interact with the other probes used in our melange. Our saliva phenotyping results show a complex multimodal histogram consistent with other studies in other matrices. Frequencies of individuals within the different modes of the salivary CYP2C19 metabolic phenotype histogram corresponds very well with known frequencies of phenotypes reported based on known genetic polymorphisms. The literature reports ranges of metabolic phenotypes as percentages: Ultra-rapid (2+4), Rapid (18+17), Extensive (51+15), Intermediate (34+17), Poor (7+8) (McGraw 2014). Literature reported percentages show close agreement with our findings: Ultra-rapid (2.4), Rapid (9.6), Extensive (49.4), Intermediate (31.3), and Poor (7.2).
[00104] We experienced problems with quantification of omeprazole in some saliva samples due to very low concentrations of both omeprazole and 50H-omeprazole. These problems have been overcome by creating an immediate release formulation with increased dosage of omeprazole and added buffer.
[00105] IIL Dextromethorphan probe of CYP2D6 phenotype
[00106] Dextromethorphan is commonly used as a probe in phenotyping studies and does not interact with the other probes used in our melange. The literature reported percentages of CYP2D6 metabolic phenotypes from a validation study based on genetic and phenotypic analysis were: Ultra-rapid (-1-2%), Extensive (-77-92%), Intermediate (-2-11%), and Poor (-5-10%). (See Gaedigk et al, Clin Pharma Ther 83(2): 234-242; Crews et al, Clin Pharma Ther 95(4): 376-382; and Fang et al. Pharmacogenomics J 14(6): 564-572). Literature reported percentages show close agreement with our findings: Ultra-rapid (2.3%), Extensive (83.9%), Intermediate (9.2%), and Poor (4.6%).
[00107] Discussion
[00108] The oral melange of caffeine, dextromethorphan, and omeprazole is the first to simultaneously probe CYP1A2, CYP2D6, and CYP2C19 using saliva as a matrix. The results show good agreement with known rates of metabolic phenotypes in other primarily Caucasian cohorts.
[00109] Salivary caffeine/paraxanthine metabolic ratio is an accepted measure of caffeine metabolic phenotype. Phenotypic examination using a substrate probe is necessary because genetics only explain approximately 42% of CYP1A2 catalytic activity and the common polymorphisms effect enzyme induction rather than enzyme activity (Klein, Winter et al. 2010).
[00110] Omeprazole is an accepted probe of CYP2C19 activity in urine and plasma.
However, the utility of omeprazole as a probe in saliva has not been previously reported. This is likely due to the low salivary concentrations of omeprazole and its metabolite 5- hydroxy omeprazole. However, we were able to develop a very sensitive assay which could simultaneously measure caffeine, dextromethorphan, omeprazole and their major metabolites in saliva.
[00111] An early paper by Hou et al. determined that saliva was an appropriate matrix for CYP2D6 phenotyping in a Chinese cohort. (See Hou et al. , Clin. Pharmacol. Ther. 59(4):411-417). They described an antimode (MR=14) in healthy subjects determined 3 hours after a 50 mg immediate release (drug powder in capsule) marking the beginning of poor metabolizer MRs. In another study of patients with renal disease they used a lower 30mg immediate release dose (drug powder in capsule) and the antimode at 3 hours was higher (MR=33). (See Hou et al, Clin Pharmacol Ther 59(4):411-417). Our antimode for dextromethorphan 6 hours after a 30mg delayed release dose is MR=27.5. It is not surprising that the MR is similar amongst studies since Yeh et al. showed a stable MR in plasma whether an immediate release (Medicon ® 15mg) or extended release formulation (Detusiv ® 60mg) was given and the MR was maintained over several hours. (See Yeh et al, J. Biomed. Sci. 10(5):552-564). In a study by Hu et al, significant variability was found for repeated measures of dextromethorphan MRs from saliva samples taken 3 and 5 hours after a single 15mg dose of Medicon while the 4 hour sample was borderline significant and the 6 hour sample was acceptable. (See Hu et al, J. Pharmacol. Exp. Ther. 285(3):955-960). These data provide external validation of our data.
[00112] Future work is aimed at expanding the study populations in which the probe melange is used and expanding the probe set. Our preliminary studies have investigated CYP3A4 probes to add to the melange for salivary analysis. Probe substrates were chosen based on safety considerations and previous reports of utility as CYP3A4 probes. Substrates we have tested include atorvastatin, Cortisol, eplerenone, erythromycin, dextromethorphan, and omeprazole. Salivary concentrations represent free drug and metabolite concentrations. Therefore, metabolite production and diffusion ability must be high with low plasma protein binding for sufficient parent drug and metabolite to be present in saliva. Our preliminary results show erythromycin is the most promising candidate for a salivary CYP3A4 probe. Since genetics only explain a small fraction of CYP3A4 activity, the need for a rapid, inexpensive method to probe CYP3A4 activity is urgently needed.
[00113] References
[00114] Crews, K. R., A. Gaedigk, H. M. Dunnenberger, J. S. Leeder, T. E. Klein,
K. E. Caudle, C. E. Haidar, D. D. Shen, J. T. Callaghan, S. Sadhasivam, C. A. Prows, E. D. Kharasch, T. C. Skaar and C. P. I. Consortium (2014). "Clinical Pharmacogenetics Implementation Consortium guidelines for cytochrome P450 2D6 genotype and codeine therapy: 2014 update." Clin Pharmacol Ther 95(4): 376-382.
[00115] Crouch, D.J., Day, J., Baudys, Jakub, and Fatah, A.A., "Evaluation of
Saliva/Oral Fluid as an Alternate Drug Testing Specimen," NIJ Report 605-03, Document No. 203569, February 2005. [00116] Donzelli, M., Derungs, A., Serratore, M.-G., Noppen, C, Nezic, L.,
Krahenbuhl, S., and Haschke, M., "The Basel Cocktail for Simultaneous Phenotyping of Human Cytochrome P450 Isoforms in Plasma, Saliva and Dried Blood Spots, Clin Pharmacokinet (2014) 53:271-282.
[00117] Fang, H., X. Liu, J. Ramirez, N. Choudhury, M. Kubo, H. K. Im, A.
Konkashbaev, N. J. Cox, M. J. Ratain, Y. Nakamura and P. H. O'Donnell (2014). "Establishment of CYP2D6 reference samples by multiple validated genotyping platforms." Pharmacogenomics J 14(6): 564-572.
[00118] Gaedigk, A., S. D. Simon, R. E. Pearce, L. D. Bradford, M. J. Kennedy and
J. S. Leeder (2008). "The CYP2D6 activity score: translating genotype information into a qualitative measure of phenotype." Clin Pharmacol Ther 83(2): 234-242.
[00119] Hou, Z. Y., C. P. Chen, W. C. Yang, M. D. Lai, E. T. Buchert, H. M.
Chung, L. W. Pickle and R. L. Woosley (1996). "Determination of dextromethorphan metabolic phenotype by salivary analysis with a reference to genotype in Chinese patients receiving renal hemodialysis." Clin Pharmacol Ther 59(4): 411-417.
[00120] Hou, Z. Y., L. W. Pickle, P. S. Meyer and R. L. Woosley (1991). "Salivary analysis for determination of dextromethorphan metabolic phenotype." Clin Pharmacol Ther 49(4): 410-419.
[00121] Hu, O. Y., H. S. Tang, H. Y. Lane, W. H. Chang and T. M. Hu (1998).
"Novel single-point plasma or saliva dextromethorphan method for determining CYP2D6 activity." J Pharmacol Exp Ther 285(3): 955-960.
[00122] Ilett, K. F., W. M. Castleden, Y. K. Vandongen, M. C. Stacey, M. A. Butler and F. F. Kadlubar (1993). "Acetylation phenotype and cytochrome P450IA2 phenotype are unlikely to be associated with peripheral arterial disease." Clin Pharmacol Ther 54(3): 317-322.
[00123] Klein, K., S. Winter, M. Turpeinen, M. Schwab and U. M. Zanger (2010).
"Pathway-Targeted Pharmacogenomics of CYP1A2 in Human Liver." Front Pharmacol 1 : 129. [00124] McGraw, J. (2014). Chapter 16 - CYP450 and Ethnicity A2 -
Padmanabhan, Sandosh. Handbook of Pharmacogenomics and Stratified Medicine. San Diego, Academic Press: 323-340.
[00125] Yeh, G. C, P. L. Tao, H. O. Ho, Y. J. Lee, J. Y. Chen and M. T. Sheu
(2003). "Analysis of pharmacokinetic parameters for assessment of dextromethorphan metabolic phenotypes." J Biomed Sci 10(5): 552-564.
[00126] Zhou, S. F., J. P. Liu and B. Chowbay (2009). "Polymorphism of human cytochrome P450 enzymes and its clinical impact." Drug Metab Rev 41(2): 89-295.
[00127] US Patent No. 8,315,815.
[00128] US Publication No. 20010036440 Al, published on November 1, 2001.
[00129] US Publication No. 20030104453 Al, June 5, 2003.
[00130] US Publication No. 20050032070 Al, February 10, 2005.
[00131] EP Publication No. 0 921 396 A2, published on June 9, 1999.
[00132] EP Publication No. 1 138 778 A2, published on March 14, 2001.
[00133] PCT Publication No. WO 2001/092596, published on August 4, 2011.\
[00134] Example 2 - Eplerenone Probe of CYP3A4 Phenotype
[00135] An extensive search was conducted for CYP3A4 probe substrates and their metabolites, which could be identified in saliva. Only safe probes that would have minimal to no side effects from a one-time dose were considered. Candidates were also selected based on optimal PK parameters such as low to no protein binding and significant lipophilicity, so that they could pass through biological membranes such as salivary glands, and be present in saliva. Although we could identify most probe parent drugs in saliva, metabolites were often too low to quantify and/or very short lived in saliva. However, unlike the other CYP3A4 probes tested, eplerenone and its primary metabolite, 6Beta-Hydroxy Eplerenone, were both found in sufficiently high concentrations in saliva.
[00136] UHPLC MS/MS methods were developed for initial data collection. The
UHPLC parameters are described in Table 7. The MS/MS instrument parameters are described in Table 8. The optimized mass spectrometer parameters are listed in Table 9. The UHPLC parameters for Method 2 are described in Table 4. The MS/MS instrument parameters for Method 2 are described in Table 5. The optimized mass spectrometer parameters for Method 2 are listed in Table 6. Product quantification was performed using AB Sciex Multiquant software version 2.0.2.
Table 7. Method 3 UHPLC Parameters
Figure imgf000036_0001
Table 8. Method 3 MS/MS Parameters
Figure imgf000036_0002
T-temperature, ESI-electrospray ionization, *APCI used for eplerenone and metabolite. ESI for all others
Table 9. Method 3 Optimized MS /MS Parameters
Figure imgf000037_0001
[00137] Table 10. shows the salivary logio molar metabolic ratios for eplerenone and its metabolite 6Beta-Hydroxy Eplerenone, which is formed via CYP3A4.
Table 10, Log10 Eplerenone / 6Beta-Hydroxy Eplerenone Metabolic Ratios
Figure imgf000037_0002
[00138] As indicated in Table 10, the metabolic ratios are fairly stable over different time points. The 3 hour time point may be the best choice because the distribution phase will be over after 2 hours because eplerenone exhibits a Tmax of approximately 1.5 hours. Five healthy volunteers aged 22-44 years old were given 50mg of commercially available generic eplerenone. As expected, the range of values is not very wide. Constitutive CYP3A4 variability is estimated at about 5-fold; however, illness, inhibition, and induction-related interactions can enhance variability [1,2] with estimates ranging 40-50-fold in different populations [3].
[00139] References [00140] [1] Wilkinson GR. Drug metabolism and variability among patients in drug response. N Engl J Med 2005;352(21):2211-21.
[00141] [2] Ingelman-Sundberg M. Human drug metabolising cytochrome P450 enzymes: properties and polymorphisms. Naunyn Schmiedebergs Arch Pharmacol 2004;369(1):89-104.
[00142] [3] Galetin A, Brown C, Hallifax D, Ito K, Houston JB. Utility of recombinant enzyme kinetics in prediction of human clearance: impact of variability, CYP3A5, and CYP2C19 on CYP3A4 probe substrates. Drug Metab Dispos 2004;32(12): 1411-20.
[00143] Example 3 - Optimized Formulations for Detection of Metabolites in
Saliva
[00144] In our initial studies with over the counter formulations of caffeine, dextromethorphan, and omeprazole we identified several problems. Caffeine doses of 200 mg were very high for some individuals and they complained of nervousness and anxiety. Dextrorphan, the dextromethorphan metabolite, was sometimes found in very low concentrations in saliva of individuals dosed with over the counter formulations. In addition, 5 -hydroxy omeprazole and omeprazole were also found in low concentrations in saliva of some individuals. To overcome these issues we began formulating immediate release tablets containing caffeine, dextromethorphan, and omeprazole with sodium or calcium bicarbonate. The bicarbonate salts were added to enhance stability of omeprazole as a dosage form as well as stability in the stomach. Without an enteric coating or base protection, omeprazole rapidly degrades in acidic gastric fluids. Table 11 lists several formulations that were tested for melange prototypes. Formulation #4 was utilized for repeat metabolic ratio measurements. Table 11, Formulations Tested for Parent and Metabolites in Saliva
Figure imgf000039_0001
[00145] Table 12. below shows that metabolic ratio measurements may be unstable during the absorption phase but become stable during the elimination phase.
Table 12. Logio Omeprazole 5-Hydroxy Omeprazole Metabolic Ratio Measurements after Repeat Doses of Formulation
Figure imgf000040_0001
Bold italicized values were signiiicantly different via two tailed paired t-test.
X-tnissing data due to inadequate sample.
[00146] All individuals showed maximal omeprazole concentrations in saliva at 0.5 or 1 hour. Time points after 4 hours were not reported as parent and metabolite concentrations approach the limit of detection after 4 hours. The data indicate that 3 hours (+/- 0.5 hours) may be an optimal time point for reproducible metabolic ratio measurements of immediate release omeprazole.
[00147] A source of variability in omeprazole metabolic ratio measurements in saliva may be due to contamination of the mouth and saliva when the tablet is ingested. For example, contamination may occur due to residual powder from the tablet formulation contaminating the mouth and saliva. As such, formulations will be prepared with a film coating to impede powder contamination of the oral cavity while not disturbing rapid dissolution. [0001] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
[0002] Citations to a number of patent and non-patent references are made herein.
The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.

Claims

CLAIMS We claim:
1. A method comprising:
(a) administering to a subject in need thereof a composition comprising:
(i) a substrate for CYP1A2 (SUBCYPIA2), wherein CYP1A2 catalyzes conversion of SUBCYPIA2 to a metabolite (METCYPIA2);
(ii) a substrate for CYP2C19 (SUBCYP2CI9), wherein CYP2C19 catalyzes conversion of SUBCYP2CI9 to a metabolite (METCYP2CI9); and
(iii) a substrate for CYP2D6 (SUBCYP2D6), wherein CYP2D6 catalyzes conversion of SUBCYP2D6 to a metabolite (METCYP2D6); and
(b) quantifying in a saliva sample from the subject:
(i) METCYPIA2 and unconverted SUBCYPIA2;
(ii) METCYP2CI9 and unconverted SUBCYP2CI9; and
(iii) METCYP2D6 and unconverted SUBCYP2D6-
2. The method of claim 1 , wherein the composition comprises a tablet formulation of each of SUBCYPIA2, SUBCYP2CI9, and SUBCYP2D6, which optionally includes a coating.
3. The method of claim 2, wherein the tablet formulation is an immediate release tablet formulation of each of SUBCYPIA2> SUBCYP2CI9> and SUBCYP2D6> which optionally includes a coating.
4. The method of claim 1, wherein one or more of METCYPIA2 and unconverted SUBCYPIA2> METCYP2CI9 and unconverted SUBCYP2CI9> and METCYP2D6 and unconverted SUBCYP2D6 are quantified using one or more procedures selected from the group consisting of Ultra High Pressure Liquid Chromatography (UHPLC), Mass Spectroscopy (MS), High Pressure Liquid Chromatography (HPLC), Ultraviolet Spectroscopy (UV), Gas Chromatography (GC), Electron Capture Detection (ECD), Flame Ionization Detection (FID), Raman Infrared (RI) Spectroscopy, Matrix- Assisted Laser Desorption/Ionization (MALDI), and combinations thereof.
5. The method of claim 1, wherein one or more of METCYPIA2 and unconverted SUBCYPIA2, METCYP2CI9 and unconverted SUBCYP2CI9, and METCYP2D6 and unconverted SUBCYP2D6 are quantified by reacting the saliva sample with one or more reagent compositions comprising reagents for quantifying one or more of METCYPIA2, METCYP2CI9, and METCYp2D6-
6. The method of claim 1, wherein SUBCYPIA also is a substrate for an enzyme selected from the group consisting of CYP2A6, NAT2, XO, and combinations thereof.
7. The method of claim 6, wherein SUBCYPIA2 also is a substrate for each of CYP2A6, NAT2, and XO.
8. The method of claim 1, wherein SUBCYPIA2 is caffeine or theophylline.
9. The method of claim 1, wherein SUBCYPIA2 is caffeine and METCYPIA2 is paraxanthine.
10. The method of claim 1, wherein SUBCYP2CI9 also is a substrate for CYP3A4 (SUBCYP3A4), wherein CYP3A4 catalyzes conversion of SUBCYP3A4 to a metabolite (METCYP3A4)-
11. The method of claim 1, wherein SUBCYP2CI9 is selected from the group consisting of omeprazole, esomeprazole, mephenytoin, clopidogrel, and phenytoin.
12. The method of claim 1, wherein SUBCYP2CI9 is omeprazole and METCYP2C19 is 50H-omeprazole, and the composition administered in the method optionally includes a basic buffering agent.
13. The method of claim 1, wherein SUBCYP2D6 is selected from the group consisting of dextromethorphan, desipramine, and metoprolol.
14. The method of claim 1, wherein SUBCYP2D6 is dextromethorphan and METCYP2D6 is dextrorphan.
15. The method of claim 1, wherein the composition further comprises a substrate for an enzyme selected from CYP2C9, CYP2E1, CYP3A4, and CYP3A5.
16. The method of claim 1, wherein the composition further comprises a substrate for CYP3A4 (SUBCYP3A4), wherein CYP3A4 catalyzes conversion of SUBCYP3A4 to a metabolite (METCYP3A4)-
17. The method of claim 16, wherein SUBCYP3A4 is eplerenone and METCYP3A4 is 6 -hydroxy eplerenone and/or 21 -hydroxy eplerenone.
18. The method of claim 1, wherein the composition further comprises a substrate for an enzyme selected from a group consisting of an N-acetyl transferase (NAT), a methyl transferase, a UDP glucuronosyl transferase (UGT), a sulfo transferases, and an oxidative enzyme, or a combination thereof.
19. The method of claim 1, wherein the composition further comprises a substrate for a UDP glucuronosyl transferase (UGT) selected from the group consisting of UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7.
20. The method of claim 1, further comprising administering ketoprofen to the subject, and the method further comprising quantifying in a biological sample from the subject metabolites selected from the group consisting of beta-estradio-3- glucuronide, trifluoperazine-N-glucuronide, 5-hydroxytryptophol-O-glucuronide, propofol-O-glucuronide, zidovudine-5'-glucuronide, and combinations thereof.
21. The method of claim 1, further comprising determining a metabolic ratio for one or more of: (i) METCYPIA2 and unconverted SUBCYPIA2 ; (ϋ) METCYP2CI9 and unconverted SUBCYP2CI9; and (iii) METCYP2D6 and unconverted SUBCYP2D6-
22. The method of claim 1, wherein the method further includes administering a dose of a drug, optionally by determining a metabolic ratio for one or more of: (i) METCYPIA2 and unconverted SUBCYPIA2; (ϋ) METCYP2CI9 and unconverted SUBCYP2CI9; and (iii) METCYP2D6 and unconverted SUBCYP2D6-
23. The method of claim 1, wherein the subject is experiencing or at risk for developing hepatic failure and optionally the method includes assessing hepatic function in the patient, optionally by determining a metabolic ratio for one or more of: (i) METCYPIA2 and unconverted SUBCYPIA2; (ϋ) METCYP2CI9 and unconverted SUBCYP2CI9; and (iii) METCYP2D6 and unconverted SUBCYP2D6-
24. The method of claim 1, wherein the method is performed in order to assess the subject's suitability for a drug study prior to the subject participating in the drug study, optionally by determining a metabolic ratio for one or more of: (i) METCYPIA2 and unconverted SUBCYPIA2; (ϋ) METCYP2CI9 and unconverted SUBCYP2CI9; and (iii) METCYP2D6 and unconverted SUBCYP2D6-
25. A composition comprising:
(i) a substrate for CYP1A2 (SUBCYPIA2), wherein CYP1A2 catalyzes conversion of SUBCYPIA2 to a metabolite (METCYPIA2);
(ii) a substrate for CYP2C19 (SUBCYP2ci9), wherein CYP2C19 catalyzes conversion of SUBCYP2C I9 to a metabolite (METCYp2ci9); and
(iii) a substrate for CYP2D6 (SUBCYP2D6), wherein CYP2D6 catalyzes conversion of SUBCYP2D6 to a metabolite
(METcYP2D6)-
26. The composition of claim 25, wherein the composition comprises a tablet formulation of each of SUBCYPIA2, SUBCYP2CI9, and SUBCYP2D6, which optionally includes a coating.
27. The composition of claim 26, wherein the tablet formulation is an immediate release tablet formulation of each of SUBCYPIA2, SUBCYP2CI9, and SUBCYP2D6, which optionally includes a coating.
28. The composition of claim 24, wherein SUBCYPIA2 also is a substrate for an enzyme selected from the group consisting of CYP2A6, NAT2, XO, and combinations thereof.
29. The composition of claim 25, wherein SUBCYPIA2 also is a substrate for each of CYP2A6, NAT2, and XO.
30. The composition of claim 25, wherein SUBCYPIA2 is caffeine or theophylline.
31. The composition of claim 25, wherein SUBCYPIA2 is caffeine.
32. The composition of claim 25, wherein SUBCYP2CI9 also is a substrate for CYP3A4 (SUBCYP3A4), wherein CYP3A4 catalyzes conversion of SUBCYP3A4 to a metabolite (METCYP3A4)-
33. The composition of claim 25, wherein SUBCYP2CI9 is selected from the group consisting of omeprazole, esomeprazole, mephenytoin, clopidogrel, and phenytoin.
34. The composition of claim 25, wherein SUBCYP2CI9 is omeprazole and the composition optionally includes a basic buffering agent.
35. The composition of claim 25, wherein SUBCYP2D6 is selected from the group consisting of dextromethorphan, desipramine, and metoprolol.
36. The composition of claim 25, wherein the composition further comprises a substrate for an enzyme selected from CYP2C9, CYP2E1, CYP3A4, and CYP3A5.
37. The composition of claim 25, wherein the composition further comprises a substrate for CYP3A4 (SUBCYP3A4)-
38. The composition of claim 37, wherein SUBCYP3A4 is eplerenone.
39. The composition of claim 25, wherein the composition further comprises a substrate for an enzyme selected from a group consisting of an N-acetyl transferase (NAT), a methyl transferase, a UDP glucuronosyl transferase (UGT), a sulfo transferases, and an oxidative enzyme, or a combination thereof.
40. The composition of claim 25, wherein the composition further comprises a substrate for a UDP glucuronosyl transferase (UGT) selected from the group consisting of UGT1A1 , UGT1A4, UGT1A6, UGT1A9, and UGT2B7.
41. The composition of claim 25, wherein the composition further comprises ketoprofen.
42. A kit for testing metabolic activity in a patient and/or determining a phenotype of a patient, the kit comprising any of the compositions of claims 25-41.
43. The kit of claim 42 further comprising one or more components selected from the group consisting of: (i) containers (e.g., salivettes) for collecting and transporting saliva samples; (ii) components and/or reagents for performing a UPLC- MS/MS assay; and/or (iii) components for generating a report of the results of the UPLC- MS/MS assay.
44. A method comprising:
(a) administering to a subject in need thereof a composition comprising eplerenone; and
(b) quantifying in a saliva sample from the subject 6β- hydroxy eplerenone and/or 21 -hydroxy eplerenone.
45. The method of claim 44, further comprising determining a metabolic ratio for όβ-hydroxy eplerenone and/or 21 -hydroxy eplerenone versus unconverted eplerenone.
46. The method of claim 44, wherein the method further includes administering a dose of a drug, optionally by determining a metabolic ratio for 6β- hydroxy eplerenone and/or 21 -hydroxy eplerenone versus unconverted eplerenone.
47. The method of claim 44, wherein the subject is experiencing or at risk for developing hepatic failure and optionally the method includes assessing hepatic function in the patient, optionally by determining a metabolic ratio for 6β- hydroxy eplerenone and/or 21 -hydroxy eplerenone versus unconverted eplerenone.
48. The method of claim 44, wherein the method is performed in order to assess the subject's suitability for a drug study prior to the subject participating in the drug study, optionally by determining a metabolic ratio for 6 -hydroxyeplerenone and/or 21 -hydroxy eplerenone versus unconverted eplerenone.
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WO2019032589A1 (en) 2017-08-07 2019-02-14 Concordia University, Inc. Eplerenone for phenotyping of cyp3a5 enzyme activity in saliva samples
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