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Definition The Role of Drug Metabolism Studies in

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					The Role of Drug Metabolism Studies in
     Optimizing Drug Candidates

           Kenneth Santone, PhD
            Bristol-Myers Squibb
     Metabolism and Pharmacokinetics /
    Pharmaceutical Candidate Optimization
     ALTERNATE TITLE:


Why All the Chemist's Wonderful
Compounds Don't Become Drugs!
Our Focus

   Unmet medical need
   First in class
   Best in class
   Need for efficiency and
    productivity enhancement
 What are we faced with?

 Industrialization of pharmaceutical research
  – Unprecedented increase in identification of targets
  – Corresponding increase in throughput of chemistry
  – Blurring of traditional discovery-development interface
      Focus and emphasis on “developability”
       (early go/no go decisions)
      Improve success rate
      Reduce development timeline
  – Necessity for increasing efficiency and productivity
Drug Discovery Paradigm Shift




            ‘Old’ Model                         ‘New’ Model
                                                                                       More informed
        of Drug Discovery                    of Drug Discovery                        decision making
                 Hits                              Validated Hits                       during Lead
                                                                                       Optimization,
                                                                                    through quicker and
                                                                                    earlier evaluation of
          Design          Efficacy &          Design                                   PAT attributes
        & Synthesis   Selectivity Testing               Efficacy &       PAT
                                            & Synthesis
                                                        Selectivity   Screening &
                                                         Testing      Predictions


             Lead Candidates

         Physicochemical, ADME                Detailed Physicochemical,
             & Tox Workup                       ADME & Tox Workup

             Development Compound                  Development Compound
The Hand-off from Drug Discovery to Development:
The Top Ten Quotations We All Know and Love*
10. “The molecular weight? 850. Why? Is that a problem?”
 9. “We’ll need eight different capsule strengths for Phase I.”
 8. “The compound is very potent in the in vitro screen but does not work well in the
     animal efficacy model.”
 7. “Now that you mention it, our solutions were a little cloudy.”
 6. “The compound is highly insoluble but Pharmaceutical Development will fix the
     problem.”
 5. “BMS-XXXXXX is a highly potent and selective inhibitor of (the target).
     In mouse models, the optimal dose was 200 mg/kg.”
 4. “Toxicity?! It’s not the drug; must be a metabolite unique to that animal species.”
 3. “Animal bioavailability ranged from 65% to <1%, depending on species.”
 2. “Gee, we didn’t have any problems when we gave it in DMSO.”
 1. “It’s a great compound, but it has formulation problems.”

   Partially adapted from R.A. Lipper   *why great compounds don’t always become drugs
         Critical Interfaces in Drug Discovery*
                       Chemistry


                         Biology


                         Activity


Safety        Metabolism & Pharmacokinetics   Pharmaceutics


                  Optimized Compound

    *Analytical Chemistry (Bioanalysis) involved
          in every one of these disciplines
              Role of ADME* Studies
 Selection of quality drug candidate for development
      – Developability
      – First-in-class vs. best-in-class
      – Crisp go/no go decisions

 Optimization of drug discovery and early development processes
     – Multi-tiered approach for ADME studies
     – Equal partnership with all functional areas
            Lead Discovery             Biology
            Chemistry                  Pharmaceutics
            Drug Safety                Analytical R&D
            Clinical Pharmacology Process Chemistry
 Blurring of traditional discovery-development interface
         * Absorption, Distribution, Metabolism, Excretion
Selection of Drug Candidates:
Focus on Developability
 Permeability           Protein binding
 Transport              Biopharmaceutics
 Metabolic stability    Active/reactive/
                          toxic metabolites
 P-450 mediated drug
  interactions           In vivo
                          PK/bioavailability
 PK/PD assessment        in animals
 Distribution           Prediction of PK
                          and efficacious
                          doses in humans
Tiered-Approach for ADME Studies

      Hits to Lead
      • In vitro Studies
          •Permeability
          •P450 inhibition
          •Metabolic Stability
      •In silico predictions

      •Objective
         •Develop SAR
         •Chemotype selection
Tiered-Approach for ADME Studies
       Lead Optimization
        In vitro Studies
            •Permeability/transport
            •P450 inhibition
            •Metabolic Stability
            •Reaction phenotyping
            •Protein binding
       •In vivo PK
            •Cassette dosing
            •Individual PK
       •Tissue penetration
       •Early biotransformation

       •Objective
          •Identify a lead compound
          •Feedback to chemistry/biology
      Tiered-Approach for ADME Studies
Lead Selection
• Absolute bioavailability in pharmacology/toxicology models
• Dose dependency in PK
• Mechanism of absorption
• Assess potential for DDI
• Characterization of metabolites, routes of elimination
• Assess formation of active metabolites
• Interspecies differences in metabolism and in vitro-in vivo correlation
• Extrapolation of ADME properties to man from in vitro and in vivo
data
• Determination of PK/PD relationships; help selection of doses for First
in Human studies

•Objective
   •Characterize the lead compound
   •Identify risks/opportunities
   How In Vitro Metabolic Stability Relates to Clearance?
  TBC = CLhepatic + CLrenal + CLother

           CLhepatic = CLmetabolism + CLbiliary

           CLmetabolic = fB * CLintrinsic * Qh / fB * CLintrinsic + Qh
                   well stirred model of organ extraction



             Intrinsic Clearance (CLi) = Vmax / Km = vo / Cu
through rearrangement of the Michaelis-Menton eqn, assuming drug conc is < Km


        Depletion or Half-Life Method:
        CLi = (0.693 * liver wt) / (in vitro t1/2 * amount of liver)
Tools to Predict Metabolic Clearance
    In Vitro Systems
 Liver microsomes
    – high throughput and most common
    – mostly oxidative (CYP & FMO)
 S9 fraction                           In Vivo Animal Clearance
    – high throughput                           In Silico
    – Phase I & Phase II metabolism
 Hepatocytes
    –   low throughput
    –   cell membrane/transporters            In Vitro - In Vivo
                                                 Correlation
    –   intracellular concentration
    –   Phase I & Phase II metabolism
                            Metabolic Stability to Select Compounds with
                                    Potentially Longer Half-Life
                                                                                                                                                                                                                                                                                                                                                                                                                                                                               Human Metabolic Stability: Microsome vs Hepatocyte

                                                                                             Rate of Human Microsomal Metabolism                                                                                                                                                                                                                                                                                                                                    0.4




                                                                                                                                                                                                                                                                                                                                                                                                                                   Microsome Total Metabolic Rate
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         2
                     0.40                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              R = 0.8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                    0.3
                                                                                                                                                                                                                                                                                                Oxidation
                                                                                                                                                                                                                                                                                                Glucuronidation
                     0.30                                                                                                                                                                                                                                                                                                                                                                                                                                           0.2
Rate of metabolism




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               BMS:Y
                                                                                                                                                                                                                                                                                                                                                                                                                                                                    0.1
                     0.20
                                                                                                                                                                                                                                                                                                                                                                                                                                                                    0.0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                          -1          0         1         2          3           4   5
                     0.10                                                                                                                                                                                                                                                                                                                                                                                                                                                                      Hepatocyte Metabolic Rate




                     0.00
                                                                                                                                                                                                                                                                                                                                        BMS-428028



                                                                                                                                                                                                                                                                                                                                                                  BMS-250598



                                                                                                                                                                                                                                                                                                                                                                                            BMS-451491



                                                                                                                                                                                                                                                                                                                                                                                                                      BMS-231975
                            BMS-350869

                                         BMS-435689



                                                                   BMS-437134



                                                                                             BMS-227178



                                                                                                                       BMS-271494



                                                                                                                                                 BMS-201282



                                                                                                                                                                           BMS-214662



                                                                                                                                                                                                     BMS-214662



                                                                                                                                                                                                                               BMS-451503

                                                                                                                                                                                                                                            BMS-440883

                                                                                                                                                                                                                                                         BMS-437562

                                                                                                                                                                                                                                                                      BMS-212435

                                                                                                                                                                                                                                                                                   BMS-275816

                                                                                                                                                                                                                                                                                                 BMS-229983

                                                                                                                                                                                                                                                                                                              BMS-437917

                                                                                                                                                                                                                                                                                                                           BMS-437220



                                                                                                                                                                                                                                                                                                                                                     BMS-221970



                                                                                                                                                                                                                                                                                                                                                                               BMS-436039



                                                                                                                                                                                                                                                                                                                                                                                                         BMS-233406
                                                      BMS-227178



                                                                                BMS-434841



                                                                                                          BMS-338387



                                                                                                                                    BMS-225263



                                                                                                                                                              BMS-212347



                                                                                                                                                                                        BMS-437221



                                                                                                                                                                                                                  BMS-225975




                                                                                                          • Lead compound is primarily glucuronidated in humans
                                                                                                          • Human in vitro systems with combination of oxidation and
                                                                                                           glucuronidation employed for selection of back up
Major Reactions Involved in Drug Metabolism

OXIDATIVE REACTIONS (CYP, LM+NADPH)
•N-Dealkylation: erythromycin, morphine, caffeine
•O-Dealkylation: codeine, dextromethorphan
•Aliphatic Hydroxylation: tolbutamide, midazolam
•Aromatic Hydroxylation: phenytoin, amphetamine, warfarin
•N-Oxidation: chlorpheniramine, dapsone
•S-Oxidation: cimetidine, omeprazole
•Deamination: amphetamine
  Major Reactions Involved in Drug Metabolism

HYDROLYSIS REACTIONS (Esterase, ?LM+NADPH)
•Ester Hydrolysis: aspirin, cocaine
•Amide Hydrolysis: lidocaine, procainamide


CONJUGATION REACTIONS (Phase II, hepatocytes)
•Glucuronidation: morphine, ibuprofen
•Sulfation: acetaminophen
•Acetylation: sulfonamides, isoniazid
                Metabolic Stability Summary
•   Not all metabolism is hepatic.
•   Incubation concentration < Km balanced with assay sensitivity.
•   Need to correlate with in vivo model.
•   Fast in vitro clearance generally implies fast in vivo clearance, the reverse need not be
    true.
•   Confounding physical-chemical properties.
      solubility, stability, purity, non-specific binding

•   Real concentration at enzyme active site?
      protein binding, cell penetration, non-specific binding

•   In vitro systems generally underestimate CLi due to non-specific binding.
•   Can the stability be too good?   Yes, in certain situations.
•   Many unknown factors to can contribute to a poor in vitro - in vivo correlation or poor
    estimation of human metabolic stability.
      Nonetheless, in vitro methods are still the best method for predictions
            Drug-Drug Interaction Summary
• Major drug interactions are caused by either inhibition or induction of
  drug metabolizing enzymes.
• Semi-quantitative predictions of drug interactions
     many unknown factors
     human ADME properties in vivo

• Models provide numbers that must be placed in context with multiple
  factors:
     therapeutic area
     therapeutic index, route of administration
     market competition

• Animal models are not predictive of human interaction potential ???
• Static nature of in vitro systems compared to the dynamic in vivo system
• Mixtures of interaction mechanisms from the same compound are
  extremely difficult to predict:
     reversible + irreversible inhibition
     inhibition + induction
            Assessment of Active Metabolites
     Compound        Met Ratea        Cmaxb       AUC(0-8)b        IC50      Efficaceous
                                      (M)         (M.h)          (nM)         Dose
                                                                             (mole/kg)
        BMS-X           0.64           8.5             35               19       1.4
        BMS-Y           0.58           13.2           64.4              19       >60
    a: metabolism rate in nmol/min.mg protein in rat liver microsomes
    b: rat oral exposure studies at 0.1 mmol/kg




Issue
• Similar metabolism and in vitro activity profile but different in vivo
 activity profile
• Apparent PK/PD disconnect

Solution
• Rapid in vitro metabolism and biological activity assays
    Assessment of Active Metabolites
             In vitro Activity of Liver Microsomal
           Product in Cell Based Assay (IC50 (nM))       % parent
Compound
             Parent           0 min          30 min     remaining
                           incubation      incubation
 BMS-X         19               12             19          <1
 BMS-Y         19               60            490          20




Structural identification of active metabolites
• MS/MS indicated presence of monohydroxylation
• NMR showed site of hydroxylation

Subsequent steps
• Monohydroxylated metabolite synthesized
• Activity and PK properties confirmed
    Assessment of Reactive Metabolites

•A number of functional (chemical) elements have been
associated with problems in drug discovery leading to toxicity
   Metabolic activation to reactive intermediates
   Interference with metabolic processes

•Clinical manifestations include (preclinical measure)
   Cellular (hepatic) necrosis (animal toxicity)
   Idiosyncratic toxicity (glutathione adducts, protein
     covalent binding, immunogenic response)
   Drug-drug interactions (mechanism-dependent CYP
     inhibition)
      Examples of Reactive Metabolites
Furans
               O                          O

                                              O
                                                                             CYP3A4
       CYP3A4                  O      O           O   O                    (epoxidation)
                                                                     O
     (epoxidation)
                           O
                                                                                  OH
                                                              O
           O
                   O               OCH3                                                OH
                       Aflatoxin B                        6',7'-dihydroxybergamottin


Furan substructure is associated with toxicity (eg. aflatoxin) and
with CYP inhibition (eg. bergamottin)
      Examples of Reactive Metabolites
Thiophenes
                           O                  O
        S                  S                  S
                                                   Nu


                                                                 O       O
                                                  CYP2C9                       O
                                     O                                             S
                                                           H2N       N
        HO                                S
                 O
             O
                     Cl         Cl
                                                                         Cl
                          Tienilic acid                              Tenidap


Thiophene substructure has been associated with several types of
toxicity (predominately hepatotoxicity). Other thiophene
containing drugs: ticlopidine, clopidigrel, raloxifene.
        Examples of Reactive Metabolites
Anilines, Nitroaromatics
                                                       OH                O
                 NO2               NH2            HN                N




                                                       O       O
                       O                                   S
                               O
           H2N             S
                               N       O
                               H   N       H2N                     NH2

                    Sulfamethoxazole                   Dapsone

Anilines are associated with a number of types of toxicity (eg. methemoglobinemia, skin
rashes, etc.). Nitroaromatics are primarily activated by initial reduction, often in the
gut, followed by N-oxidation.
Anilines of polycyclic aromatic systems are often potent mutagens and carcinogens (eg.,
naphthylamine, aminofluorene) through conjugation of the hydroxylamine and
subsequent loss of the conjugate to leave a nitrenium ion.
       Examples of Reactive Metabolites
                                                            O
Amines, alkylamines          N                              N

                                                        O


                                    S
                                                O
                                    N
                                            O       O

                                        N


                                   Diltiazem
The metabolism of amines or alkylamines is generally related to time-
dependent inhibition of CYP enzymes, with the nitroso species forming a tight
complex with the heme iron, known as a MI complex. Other compounds that
undergo this type of transformation and inhibit CYPs are TAO, erythromycin
and verapamil
      Examples of Reactive Metabolites
Quinone, Quinoid
                     O                  O


                                            X = O, N, C

                     X                  X
                         O

                    HN                                             O
                                            O
                                                      O        S
                                                                   NH
                                   HO
                                                                   O
                     OH
                   Acetaminophen                Troglitazone


Quinone-like compounds can exert their effects through direct
alkylation of nucleophiles or through redox cycling between their
oxidized and reduced forms
    Examples of Reactive Metabolites

Acetylenes
                              O


                                  N
                         OH                      OH




     O                            O

             Gestodene                Mifepristone (RU 486)



Acetylenes have been found to be time-dependent inhibitors of
CYP enzymes.
      Examples of Reactive Metabolites
Acyl glucuronidation formation


                                 Direct reaction with nucleophiles
       O                O

       OH               OGluc
                                 Amidori rearrangement, then reaction with nucleophiles
                                       O
            O
                                             N           OH
                  N         OH
                                                     O
                        O
 Cl

            Zomipirac                 Tolmentin




Acyl glucuronides have been implicated in both direct hepatic
damage and idiosyncratic toxicities
Challenges and Opportunities
  HTS screens for prediction of permeability, metabolic stability, metabolic
   reactivity and DDI
    – How are we using these data?
    – Retrospective analysis on return of investment
    – The numbers in gray zone!
    – Secondary assays for better predictability

  Application of animal PK/bioavailability data for lead optimization
    – Adequacy of permeability and metabolic stability data
    – Animals vs. humans: quantitative and qualitative differences in ADME
      properties

  Informed decision based on drug metabolism and pharmacokinetic data
    – Low bioavailability vs. oral efficacy
    – Role of metabolite(s), reactivity of metabolite(s)
    – Protein binding
    – In vitro- in vivo correlation in animals and extrapolation to humans

  Issue of enzyme induction in humans
    – In-vitro models and predictability
    – False and real alarm from in-vivo animal data
Challenges and Opportunities
 Use of biomarkers
  – In-vivo biology, animals vs. humans
  – Development and validation of assays
  – Transfer from preclinical to clinical laboratories
  – Biomarkers = Surrogate marker = Efficacy/Toxicity
  – A balancing act of emerging science

 The feedback loops
  – To and from chemistry
  – To and from biology
  – To and from drug safety
  – To and from pharmaceutics
  – To and from clinical pharmacology

 Volume of data
  – Conversion of information into knowledge
  – Timing and availability
A Focused Application of ADME Studies
• Active involvement earlier in the Discovery Process

• Timely guidance to Chemistry to select chemotypes with
desirable ADME properties

• Maximize informed decision making during Lead
Optimization

• Improved ability to predict human metabolism and
pharmacokinetics

• Stronger partnerships with Drug Discovery and all areas
of Pharmaceutical Development
 Our Mission


To ensure that no development candidate
       fails in the clinic due to an
        unforeseen metabolic or
       pharmacokinetic property
Acknowledgements


      David Rodrigues and Griff Humphreys

    Saeho Chong, Punit Marathe, Wen Chyi Shyu
                  and Mike Sinz


                   And finally ….

				
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