Prediction of metabolites likely to be formed in vivo

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Prediction of metabolites likely to be formed in vivo Powered By Docstoc
					Role of Metabolite Elucidation in Support of
Drug Discovery and Development: Strategy
             and Techniques


                Ala F. Nassar
Today’s Topics

•   Introduction
•   Utilization of in-silico methods and automation
    techniques for rapid metabolite elucidation
•   Online H-D exchange method
•   Presentation of technique capable of detection
    down to 20 cpm 14C peaks
•   Using stable isotopes for metabolite elucidation
•   Case studies
Questions for Drug Metabolism Scientists
• What are the chemical reactions involved in the
    metabolism of foreign compounds?
•   Where do these reactions take place in the body?
•   Which enzyme systems catalyze the metabolism of
    foreign compounds?
•   What are the biochemical mechanisms of these
    processes?
•   What are the biological consequences of xenobiotic
    metabolism?

Outcome
Guiding drug candidate selection by taking advantage of
metabolic reactions to design more effective and safer drugs
      Typical Screening Cascade
                 In Vitro Enzyme Assay
                    Cell Based Assay
                       Selectivity

                   Exposure screening
In parallel         Microsomal stability
    or                 Metabolites ID
                     IV/PO PK studies
 In series        CYP inhibition screening
                   Caco-2 permeability
                         solubility
              Expanded PK (definitive, TK, formulations, etc.)
                Chronic efficacy model
              Acute in vivo efficacy model
                  Select Drug Candidate
     Techniques for ADME Studies
•   Microsomal Stability
•   Hepatocyte Stability
•   Caco-2 Permeability
•   Metabolic Profiling
•   Protein Binding
•   Whole Body Autoradiography
•   P450 Inhibition and Induction
•   Pharmacokinetic Analysis
•   Metabolite Elucidation
       Significance of Metabolite Elucidation
• Assistance with chemical synthesis efforts to block
    or enhance metabolism
• Prediction of metabolites likely to be formed in
    vivo
•   Determination of metabolic differences between
    species
•   Identification of potential pharmacologically-active
    or -toxic metabolites
•   Synthesis of metabolites for toxicology testing
•   Prediction of drug-drug interactions
Method for Characterization of Metabolites in Drug
Discovery Utilizing in-silico Prediction, GENESIS
           Workstation and QTOF-MS
The automated assay system consists of:
     Pallas MetabolExpert software to predict possible
                      metabolites

      Robotic liquid handler (Genesis workstation) to
               generate and process samples


     QTOF-MS coupled with liquid chromatography to
                  analyze samples


     MetaboLynx software to find potential metabolites

      Exact mass measurement to help for Met ID

     Advanced Chemistry Development/MS (ACD/MS)
    software to predict hypothetical metabolite chemical
                         structures
SELECTED COMPOUNDS TO EXAMINE NOVEL
      METHODS/TOOLS FOR MET ID
                O

                                                         OH
                                               N
                                               H

                                                   O
       N




  Dextromethorphan

                                           Propranolol




                     O                NH

                             OH


                         Alprenolol
Prediction of possible metabolites for propranolol using
          Pallas MetabolExpert 10.0 software

                             N-dealkylation
                                              Conjugation
                                  O
                         N



        N-oxidation          O
                                         O-dealkylaton


   Aromatic
   Oxidation or
   Epoxidation

                      Propranolol
Prediction of possible metabolites for propranolol using
          Pallas MetabolExpert 10.0 software
                              O                                                               O                                       O
                                                          O                       N
                      O                                                                                               N
          N
                                          O
                                                                                      O                                       O
                                              O
              O                                                                                           O



                                                                                          O
                                  N

                                                                      O


                                                                                                                              O
                                                                                                              N
                      O
      N
                                                                                                                  O

              O

                      O

                                                                          O
                                                          N                                       O

                                                                  O

          O
                                                                                                              N
                          O
          N
                                                                                                                  O
                  O
                                                  Propranolol                                                                 O
                      O




                                                                      O
                                                      N


                                                              O
                                                                                                      O                                   O
                      O                           O                                       N                               N
      N                               N

                                                                                              O                                   O
              O                           O
                                                                              O


                                                                                                          O

  O                                       O
TOF-MS/MS spectrum of (A) dextromethorphan and (B) dextromethorphan-M1
                                        -2H              -C3H7N
                                213              215                                                           -C3H7N
                                                                                                  -2H
                                                                                           199           201
                                                -C3H9N
                           -4H        198
                    147                                      O                                                       OH




                                            N                                                      N
                                                                   -CH2O
                                                             121           91                                        107
                                                                   -CH2                                                -CH2
                                                          173               159                                 159                 145
                                                             -H2                                               -H2         -C2H2

                                                          171                                                   157         133

                                                [M+H]+,   m/z 272                                          [M+H]+,     m/z 258




                                                                                                               TOF MSMS 272.00ES+
                                                                           171.1
         100                                              147.1


                    A                                                                            213.1


          %
                                                                   159.1                 198.1
                                       121.1
                                                                                                                                    272.2
                                                 135.1
                    91.1                                                        181.1
          0
                                                                                                               TOF MSMS 258.00ES+
                                                                 157.1
         100


                    B
                                                133.1                                    199.1
          %

                                                        145.1
                                                                                                                            258.2
                            107.1 121.1                                    171.1 185.1
          0                                                                                                                                 m/z
               80         100           120            140           160        180       200       220         240           260         280
TOF-MS/MS spectra of (A) propranolol and (B) propranolol-M1
             98
                                          218                          98
                                                                                                    234
                                       -H2O
               -H2O                             183
                                                                         -H2O                    -H2O
                                                                                                          199
                                              OH
             116
                              N                                                                         OH
                              H                                        116
                                                                                        N
                                                                                        H
                                   O
                                                                                             O

                                                         157
                                                           -2H                                                  173
                                              129                                                                 -2H
                                                                                                        145
                                                         155
                                  -C3H7                                                                   OH    171
                                                165                                         -C3H7
                                                                                                        181


                          [M+H]+, m/z 260                                             [M+H]+, m/z 276



                                                                                                        TOF MSMS 260.00ES+
                           116.1
  100
                                                      155.1
                                                                   183.1
              A
   %
                   98.1
                                                          165.1
                                    129.1                                                                        260.2
                                              141.1
         86.1                                                                           218.2
    0
                                                                                                        TOF MSMS 276.00ES+
                           116.1
  100                                                                        199.1
                                                               173.1

                   B
   %
                   98.1

                                                 145.1            181.1                                                  276.2
                                                         161.1
         86.1                      128.1                                                            234.2
    1                                                                                                                        m/z
        80         100        120             140        160      180           200         220           240    260       280
        ACD/MS software as efficient tool to help in
      assigning potential metabolite chemical structure
The ACD/MS software was used to help determine the chemical structures
for the metabolites. We performed the following steps to produce a potential
(hypothetical) metabolite chemical structure for each of our test compounds:

   Generate the MS/MS spectrum for each metabolite;
   Attach the corresponding potential (hypothetical) structure(s) to it;
   Run Autoassign;
   Use ACD/MS software to provide a score for percent of spectrum
     assignment;
   These scores then guided us to the most likely form for the potential
     (hypothetical) chemical metabolite structure.

To speed up the process, we ran autoassign in batch mode on the various
metabolite possibilities for each particular molecule. We were able to increase
the intelligence of the process by using accurate mass data.
          Mass accuracy measurements of selected drugs and their metabolites,
           the exact mass difference between each metabolite and parent drug

    Substrate   Molecular         Mass, m/z             Mass        SD,      ppm     Exact Mass
                Formula                                 Error,      Da               Differencea,b
                             Theoretical   Measured
                                                         Da
                               Mass         Massa

    Dextrom1    C18H26NO     272.2014      272.2012     0.0002    0.0008      0.7          --
      M-1       C17H24NO     258.1858      258.1868     0.0010    0.0002      3.8       14.0146
      M-2       C16H22NO     244.1706      244.1707     0.0001    0.0010      0.4       28.0307
      M-3       C17H24NO     258.1858      258.1862     0.0004    0.0013      1.5       14.0152

Propranolol     C16H22NO2    260.1651      260.1649     0.0002    0.0005      0.7          --
   M-1          C16H22NO3    276.1600      276.1601     0.0001    0.0009      0.3       15.9950
   M-2          C16H22NO3    276.1600      276.1602     0.0002    0.0009      0.7       15.9951
   M-3          C16H22NO3    276.1600      276.1602     0.0002    0.0006      0.7       15.9951

Alprenolol      C15H24NO2    250.1807      250.1808     0.0001    0.0007      0.3          --
   M-1          C15H24NO3    266.1756      266.1763     0.0007    0.0015      2.6       15.9956
   M-2          C15H24NO3    266.1756      266.1752     0.0004    0.0003      1.5       15.9945
1
 Dextromethorphan
a
 Avg. of 5 injections.
b
 Exact mass difference between each metabolite and parent drug, theoretical mass of CH2 is 14.0157
  Da, C2H4 is 28.0313 Da, and oxygen is 15.9949 Da.
Pallas software was useful in predicting the possible
     metabolic fate of the compounds examined

Compound              Molecular    Pallas       ACD
                      Formula


Dextromethorphan       C18H26NO     --           --
Dextromethorphan-M1    C17H24NO    Yes           88
Dextromethorphan-M2    C16H22NO    Yes           98
Dextromethorphan-M3    C17H24NO    Yes           88
                                   n=7

Propranolol            C16H22NO2    --           --
Propranolol-M1         C16H22NO3   Yes           82
Propranolol-M2         C16H22NO3   Yes           86
Propranolol-M3         C16H22NO3   Yes           71
                                   n=15

Alprenolol             C15H24NO2    --           --
Alprenolol-M1          C15H24NO3   Yes           58
Alprenolol-M2          C15H24NO3   Yes           61
                                   n=11
Proposed metabolic pathways of dextromethorphan in hepatic
  microsomal incubations based on MS/MS and ACD data
                                             O




                                  N

                                                         O-demethylation
      N-demethylation
                                Dextromethorphan
                                     m/z 272                               OH


                   O




                                                               N


        N
        H              O-demethylation       N-demethylation       Dextrorphan, M-1
                                                                   [M+H]+ m/z 258
Methoxymorphinan, M-3
  [M+H]+ m/z 258                                 OH




                                      N
                                      H
                            Hydroxymorphinan, M-2
                              [M+H]+ m/z 244
Proposed metabolic pathways of propranolol in hepatic microsomal
          incubations based on MS/MS and ACD data

                                                       OH
                                             N
                                             H

                                                  O




                                         Propranolol
                                        [M+H]+ m/z 260
                         [O]                                       [O]
                                                 [O]

                                                                                      OH
                          OH                           OH                     N
               N                             N                                H
               H                             H
                                                                                  O
                   O                              O

                                   HO




                   OH                                                    OH
    Monohydroxy propranolol, M-1    Monohydroxy propranolol, M-2   Monohydroxy propranolol, M-3
         [M+H]+ m/z 276                  [M+H]+ m/z 276                 [M+H]+ m/z 276
 Proposed metabolic pathways of alprenolol in hepatic
microsomal incubations based on MS/MS and ACD data



                                O                    NH

                                          OH

                                      Alprenolol
                                    [M+H]+ m/z 250




                  [O]                                     [O]



            OH
                                               HO



        O               NH                                      O             NH

                 OH                                                   OH
  Monohydroxy-Alprenolol, M-1                             Monohydroxy-Alprenolol, M-2
       [M+H]+ m/z 266                                          [M+H]+ m/z 266
                       CONCLUSIONS
 Pallas is a useful software tool to predict possible metabolites

 The QTOF mass spectrometer generates accurate MS data
  (high resolution) which can rapidly identify metabolites
  when combined with MetaboLynx software

 MetaboLynx   software is useful to automatically find
  potential metabolites, avoiding time-consuming data analysis

 ACD software is useful to assist in the mass spectral data
  interpretation, leading to tentative structures for the
  proposed metabolites in drug discovery

 The major metabolites were found in rat and human liver
 microsomal preparations and were in good agreement with
 previously published results
 Hydrogen-deuterium Exchange and
QTOF-MS for Metabolite Elucidation in
        Drug Metabolism
H-D exchange methods are useful for determination of:

• N- or S-oxide formation and mono-
  hydroxylation

• Conjugation such as glucuronidation

• Dehydrogenation or dealkylation
     Prediction of possible metabolites for
      Nimodipine using Pallas software

                                                                      9
1.   Cleavage
                                                      O+
2.   Cleavage                                         N
                                                        O         7
3.   Cleavage                    3
4.   Hydroxylation                       O            O
5.   Hydroxylation       O                        8
6.   Cleavage                        O                    O
7.   Cleavage                                N
8.   Hydroxylation           2               H*                           6
9.   Cleavage        1
                                 4                            5
    Exchange of labile hydrogens in nimodipine and
             metabolites formed in-vitro

Compound             Labile          Mass         Mass [MD2+D]+, m/z
                    Hydrogen        1
                                 [MH +H]+, m/z   predicted measured
Nimodipine              1            419           421       421
M-1                     1            359           361       361
M-2                     2            435           438       438
M-3                     1            403           405       405
M-4                     2            405           408       408
M-5                     0            417           418       418
1
MH=the molecular weight in H2O
2
MD=the molecular weight in D2O
Proposed metabolic pathways of nimodipine in hepatic microsomal incubations in H2O
                                                                                       O
                                                                                           +
                                                                                       N
                                                                                               O

                                                                       O               O
                                                         O
                                                                   O                           O

                                                                                   N       CH2OH
                                                                                   H
                                                                       [MH+H]+ m/z 435
                                                                            M-2
                                         O                                                                              O
                                             +                                                                              +
                                         N                                                                              N
                                                 O                                                                              O

                         O               O                                                          O                   O
        O                                                                      HO
                     O                           O                                         O                                    O

                             N                                                                              N
                             H                                         O
                                                                           +                                H
                     [MH+H]+ m/z 419                                   N
                                                                               O               [MH+H]+ m/z 405
                      Nimopdipine
                                                                                                    M-4
                                                         O             O
                                         HO          O                         O

                                                              N        C
                                                                   H           H
                             O                                                                                  O
                                 +                       [MH+H]+m/z 359                                         N
                                                                                                                    +
                             N
                                     O                        M-1                                                       O

                 O           O                                                                  O               O
   O
            O                        O
                                                                           HO          O                                O

                         N                                                                              N

                [MH+H]+ m/z 417                                                                [MH+H]+ m/z 403
                     M-5                                                                            M-3
Proposed metabolic pathways of nimodipine in hepatic microsomal incubations in D2O
                                                                                         O
                                                                                             +
                                                                                         N
                                                                                                 O

                                                                        O                O
                                                          O
                                                                   O                             O

                                                                                     N   CH2OD
                                                                                     D
                                                                       [MD+D]+ m/z 438
                                                                            M-2
                                         O                                                                                O
                                             +                                                                                +
                                         N                                                                                N
                                                 O                                                                                O

                         O               O                                                            O                   O
        O                                                                        DO
                     O                           O                                           O                                    O

                             N                                                                                N
                              D                                         O                                 D
                                                                            +
                           + m/z 421
                     [MD+D]                                             N                        [MD+D]+ m/z 408
                                                                                O
                      Nimopdipine                                                                     M-4
                                                          O             O
                                                 DO
                                                      O                          O

                                                               N        C
                                                                    H           H
                             O                                                                                    O
                                 +                        [MD+D]+ m/z 361                                         N
                                                                                                                      +
                             N
                                     O                         M-1                                                        O

                 O           O                                                                    O               O
   O                                                                            DO
            O                        O                                                   O                                O

                         N                                                                                N

                [MD+D]+ m/z 418                                                                  [MD+D]+ m/z 405
                     M-5                                                                              M-3
                      CONCLUSIONS
•   One advantage of the H-D exchange method is that, with
    LC-MS/MS, it offers an easy estimation of the number of
    labile hydrogen atoms in such groups as -OH, -NH, -NH2
    and -COOH

•   This number is useful in comparing the metabolite structure
    with that of the parent drug to determine the presence or
    absence of the above groups

•   H-D exchange experiments have facilitated structural
    elucidation and interpretation of fragmentation processes
    as well

•   Our results indicated that this method should be
    particularly desirable for identification of metabolites
    produced by dehydrogenation, oxidation, and dealkylation
 NOVEL APPROACH TO PERFORMING
METABOLITE IDENTIFICATION IN DRUG
          METABOLISM
 AIMS

• Validate capability of on-line radioactivity
  detector coupled with MS to measure
  radiolabeled compounds

• Enhance the sensitivity of radioisotope
  measurement for metabolite identification
                LC-ARC-MS-FC System

                                                           PC
         Sampler




                Column
Agilent 1100
   Pump                  StopFlow
                         Controller
                                       Packard 500    Waste
                                        Detector


                                      LCQ MS

                         Cocktail

                                          Fraction Collector


    Laptop PC

    ARC Data System                                            ARC Control Lines

                                                               LCQ Control Lines
                              Linearity of DynamicFlow Radioactivity Detection


                   700                                                           y = 1.1213x - 50.702
                                                                                       2
                                                                                     R = 0.996
                   600
      Peak (DPM)




                   500
Peak Area Area




                   400
                                                                                 Detected DPM
                                                                                 Linear (Detected DPM)
                   300

                   200

                   100

                     0
                         0   200          400           600           800

                                   Applied DPM
                                    Applied DPM
HPLC-MS chromatograms of [14C]dextromethorphan following incubation with
HLM in the presence of NADPH showing M-1, M-2, M-3 and dextromethorphan
   RT: 0.00 - 32.00 SM: 15G
                                                                                                          20.52                                                  NL: 1.10E6
      100
                                                                                                                                                                 m/z= 273.00-275.00 F:
                                                                                                                                                                 ITMS + c ESI Full ms [
                                                                                                                                                                 200.00-300.00] MS
       50                                                                                                                                                        HLM-C14-60min

                0.44       3.16 3.86             7.16        9.77 12.29 14.05 15.82 16.71             19.48       21.03 22.17 24.58 26.46 28.23          31.59
        0
                                                                      12.40                                                                                      NL: 4.28E5
      100
                                                                                                                                                                 m/z= 257.95-258.52 F:
                                                                    12.34                                                                                        ITMS + c ESI Full ms [
                                                                            12.50
                                                                                                                                                                 200.00-300.00] MS
       50                                                                                                                                                        HLM-C14-60min

                0.16       3.01 4.02 5.95 7.45              9.31    12.22   12.58 15.28           19.09 21.76 22.03 22.26 25.08 26.97 28.38              31.58
        0
                                                                            13.35                                                                                NL: 4.54E4
      100
                                                                                                                                                                 m/z= 243.00-245.00 F:
                                                                                                                                                                 ITMS + c ESI Full ms [
                                                                                                                                                                 200.00-300.00] MS
       50                                                                                                                                                        HLM-C14-60min

                   3.03    3.13       3.35       7.41 9.40 11.24 12.78              13.88   17.76 18.60        21.26 22.15 23.85 25.54 27.85             31.58
        0
                                                                                                                 21.89                                           NL: 5.91E5
      100
                                                                                                                                                                 m/z= 260.00-262.00 F:
                                                                                                                                                                 ITMS + c ESI Full ms [
                                                                                                                                                                 200.00-300.00] MS
       50                                                                                                                                                        HLM-C14-60min
                                                                                                                        22.26
                0.44       3.01 3.71 5.31         8.33 9.74 12.15 12.41         15.34 16.79 18.91 19.85                         23.39 25.83 28.03        31.58
        0
            0          2          4          6          8      10      12      14        16      18       20       22       24       26     28      30
                                                                                      Time (min)
                LC-MS spectra of [M+H]+ m/z 274, nonmetabolized [14C]dextromethorphan, (A) MS2, (B) MS3
                         and (C) MS4 following incubation of [14C]dextromethorphan with HLM
                   dex274 #237-272 RT: 0.63-0.72 AV: 36 NL: 1.58E6
                   F: ITMS + c ESI Full ms2 274.00@50.00 [ 75.00-300.00]
                                                                                                                                                       217.12


                        1400000

                        1200000

                        1000000
    Intensity




                         800000

                         600000

                         400000                                                                                                                  215.14

                                                                                                  149.03
                         200000
                                                                             122.97                        161.06    175.07                                               243.12
                                        83.00 90.99             109.05                      148.26                                     201.11 214.32             231.16             256.23       274.22
                                    0
                                         80               100             120               140            160             180             200            220             240             260        280      300
                                                                                                                                 m/z
                   dex274 #383-419 RT: 1.09-1.22 AV: 37 NL: 5.88E5
                   F: ITMS + c ESI Full ms3 274.00@50.00 217.00@50.00 [ 55.00-300.00]
                                                                                   148.98

                       550000
                       500000
                       450000
                       400000

                       350000
  Intensity




                       300000
                                                                                                                             175.02
                       250000
                       200000
                                                                                                                  161.00
                       150000
                       100000
                                                                                      122.94
                         50000                                                                    136.99                                189.04
                                             68.92   80.93      92.91    106.97                                       163.01                     198.13
                                0
                                        60            80            100               120            140            160           180            200            220         240            260        280     300
                                                                                                                                 m/z
                   dex274 #701-736 RT: 2.55-2.70 AV: 36 NL: 3.56E4
                   F: ITMS + c ESI Full ms4 274.00@50.00 217.00@50.00 149.00@50.00 [ 50.00-150.00]
                                                                                    90.88
                        35000


                       30000


                       25000
Intensity




                       20000


                       15000


                       10000                                                                                                                                      120.97
                                                                                                                                                                                     131.98
                        5000
                                                                                                                  92.90                                     118.94               130.97
                                                                                                                                          106.91                                           133.99
                                        54.88                                                                        94.99                             114.97                   129.82                      149.04
                            0
                                50                   60                 70                  80               90               100                110              120               130             140       150
                                                                                                                              m/z
      -CH2         -2H          -C3H7N
201          215          217


                                         *
                                  O      CH3




                                                              121
                   N
                                         -*CH2O                 -2H
                                  123                 91

                                175             149           123
                                        -C2H2         -C2H2

                         [M+H]+, m/z 274
                   [14C]dextromethorphan
                            *=C14
Proposed metabolic pathways of dextromethorphan in hepatic
                 microsomal incubations
                                                       O    *
                                                            CH3




                                       N


           N-demethylation           Dextromethorphan               O-demethylation
                                      [M+H]+ m/z 274
                                                                                     OH
                          O                    *=C14
                              *
                              CH3



                                                                      N
            N                                                               Dextrorphan
            H
                                                                          [M+H]+ m/z 258
       Methoxymorphinan
       [M+H]+ m/z 260

                O-demethylation                            OH     N-demethylation




                                           N
                                           H

                                    Hydroxymorphinan
                                     [M+H]+ m/z 244
                         CONCLUSIONS
   This system detects 14C peaks down to 6 cpm, about 20 times more
    sensitively than commercially available flow-through radioactivity
    detectors

   The LC-ARC on-line stop-flow method detects 3H peaks up to 10
    times more sensitively than commercially available flow-through
    radioactivity detectors

   On-line mass spectrometer data were acquired

   Using this method, it is possible to generate high resolution
    radiochromatograms, accurately measure volatile metabolites which
    fraction-collection methods are not even able to detect, and acquire
    mass spectra on-line

   An important safety benefit is that, by using this method, we reduced
    sample injection size, thus reducing radioactivity exposure and wastes
Stable Isotope Approach for Metabolite
    Elucidation in Drug Metabolism
Case Studies
            Considerations to Enhance Metabolic Stability.
 One of the most important keys to successful drug design and development is a
process of finding the right combination of multiple properties such as activity,
toxicity and exposure. Optimize these three properties for drug candidates, and thus
their suitability for advancement to development.

 The responsibility of the drug metabolism scientist is to optimize plasma T1/2
(clearance compound), drug/metabolic clearance, metabolic stability, and the ratio
of metabolic to renal clearance.

 Another concern is to minimize or eliminate the following:
• gut/hepatic-first-pass metabolism,
• inhibition/induction of drug-metabolizing enzymes by metabolites,
• biologically active metabolites,
• metabolism by polymorphically expressed drug-metabolizing enzymes
• formation of reactive metabolites.
         Advantages of Enhancing Metabolic Stability

 Increased bioavailability and longer half-life, which in turn should allow lower
and less frequent dosing thus promoting better patient compliance.

 Better congruence between dose and plasma concentration, thus reducing or
even eliminating the need for expensive therapeutic monitoring.

 Reduction in metabolic turnover rates from different species which, in turn,
may permit better extrapolation of animal data to humans.

 Lower patient-to-patient and intra-patient variability in drug levels, since this is
largely based on differences in drug metabolic capacity.

 Diminishing the number and significance of active metabolites and thus
lessening the need for further studies on drug metabolites in both animals and
man.
             Strategies to Enhance Metabolic Stability
 In general, metabolism can be reduced by incorporation of stable functions
(blocking groups) at metabolically vulnerable sites. Substrate structure activity
relationships of metabolizing enzymes have to be accommodated within the
structure activity relationships of the actual pharmacological target.

  The following strategies have been used:

• Deactivating aromatic rings towards oxidation by substituting them with strongly
electron withdrawing groups (e.g., CF3, SO2NH2, SO3-).
• Introducing an N-t-butyl group to prevent N-dealkylation.
• Replacing a labile ester linkage with an amide group.
• Constraining the molecule in a conformation which is unfavorable to the metabolic
pathway, more generally, protecting the labile moiety by steric shielding
• The phenolic function has consistently been shown to be rapidly glucuronidated.
Thus, avoidance of this moiety in a sterically unhindered position is advised in any
compound intended for oral use.
      Strategies to Enhance Metabolic Stability (cont’d)
• Avoidance of other conjugation reactions as primary clearance pathways, would
also be advised in the design stage in any drug destined for oral usage.

• Sometimes the best strategy is to anticipate a likely route of metabolism and
prepare the expected metabolite if it has adequate intrinsic activity. For example,
often N-oxides are just as active as the parent amine, but won't undergo further N-
oxidation.
Examples from literature to enhance metabolic stability in
                  the molecular design

  Reduce the overall lipophilicity (logP, logD) of the structure
                                                                H
                                                    O           N

                O                           O
                                N                                       O
                    N                           N
                    H                           H                   O
     O N                    O                                                  EC50 = 0.078mM, clogP = 2.07, C7hr = 0.012mM

                                                        F
                                            F




                                                H
                                        O       N

                                                                             EC50 = 0.058mM, clogP = 0.18, C7hr = 0.057mM
        O                       O
                        N                                   O
            N                       N
            H                       H                O
  O N               O


                                                    Dragovich, P. et al (2003). Journal of Medicinal Chemistry, 46(21), 4572-4585.
Remove or block the vulnerable site of metabolism (Benzylic oxidation)


                                                              N
                                   Br
                                                                         N

                                                                             O

                                            Ki = 66 nM, AUC 0-6h = 40 ng/ml hr




                O                                                                           O
                    N                                                                           N
                                                                                                                              _
                                                                                                                         O
                                                                                                        N                 N
                                                                                                                              +
                               N
   Br                                                                            Br
                                        N                                                                        N

                                             O                                                                       O


                                                                                      Ki = 2.1 nM, AUC 0-6h = 6500 ng/ml hr
        Ki = 2 nM, AUC 0-6h = 1400 ng/ml hr




                        Palani, A. et al (2001) Journal of Medicinal Chemistry, 44(21), 3339-3342.
Remove or block the vulnerable site of metabolism (Allylic oxidation)


                                                    N
                                                            NH2
                                                     N                   IC50 = 0.06 mg/ml, Cmax = 14-140 ng/ml
                                                O S O




                                                    N
                                                            NH2
                                                     N
                                                O S O                 IC50 = 0.02 mg/ml, Cmax = 70-300 ng/ml




           Victor F et al (1997). Journal of medicinal chemistry, 40(10), 1511-8.
                  Remove or block the vulnerable site of metabolism (Glucuronidation)

       Effect of linker
                                           O                                                                     OH
              O                                                                        O                         N        NH2
                                     N         NH2

 F                                   OH                                                                               O
                                                                          F
UDPGA rate (nmol/min/mg protein) = 0.19, t1/2 = 4.7 hr
                                                                       UDPGA rate (nmol/min/mg protein) = 0.05, t1/2 = 5.5 hr
      Effect of template
                                       OH                                                                            OH
              O                        N       NH2                                                S                  N        NH2

                                           O                                                                              O
  F
                                                                          F
  UDPGA rate (nmol/min/mg protein) = 0.05, t1/2 = 5.5 hr
                                                                         UDPGA rate (nmol/min/mg protein) = 0.012, t1/2 = 14.5 hr

      Effect of stereochemistry
                                                                                                                OH
                                   OH
                                                                                              O                 N         NH2
                   O               N        NH2
                                                                                       O
             O
                                                                                                                     O
                                       O
  F                                                                       F

UDPGA rate (nmol/min/mg protein) = 0.02, t1/2 = 7.7 hr
                                                                         UDPGA rate (nmol/min/mg protein) = 0.01, t1/2 = 8.7 hr


             Bouska J J. et al (1997) Drug metabolism and disposition: biological fate of chemicals, 25(9), 1032-8.
      Examples to improve PK properties through
       structural modification of drug candidates
Minimize First-pass effect/prodrug approach
•Oral dosage of propranolol (Hasegawa et al 1978) produces a low bioavailability and a
wide variation from patient to patient when compared to intravenous administration; this
difference is attributed to first-pass elimination of the drug.

•Hemisuccinate ester of propranolol was selected as a potential prodrug with the hypothesis
that propranolol hemisuccinate ester administration would avoid glucuronide formation
during absorption and subsequently be released in the blood by hydrolysis.
                                                                                     HO         COOH
                                                           Propranolol       +
                                                                                          O

                                                                Hydrolysis

         O          N
                    H
               OH                                                  O                 N
                                                                                     H
                                                                             O                COOH
                          Glucuronidation
                                                                                 O

 Propranolol   AUC 0-6 = 132 ng/ml.h        Hemisuccinate ester of propranolol   AUC 0-6 = 1075 ng/ml.h
        Examples to improve PK properties through
     structural modification of drug candidates (cont’d)
Half-life
•ABT-418, an analogue of (S)-nicotine in which the pyridine ring is replaced by the 3-
methyl-5-isoxazole moiety, has been shown to possess cognitive-enhancing and anxiolytic-
like activities in animal models with an improved safety profile compared to that of nicotine
(Lin et al. 1997).

•One shortcoming of ABT-418 was its very poor bioavailability (%F = 1.2), with a short
plasma half-life (t1/2 = 0.21 h). Research on structural modification led to the identification
of ABT-089, 2-Methyl-3-(2(S)-pyrrolidinylmethoxy)pyridine, with a vastly improved oral
bioavailability (%F = 61.5) with t1/2 = 1.6 h.



                       O                                              O
            N              N                              N
                                                          H
                                                                               N

       5 pyrollidine ABT-418   t1/2 = 0.2 h                 ABT-089       t1/2 = 1.6 h
                              Conclusions
 In-silico and in vitro techniques are available to screen compounds for key
ADME characteristics.

 Structural information on metabolites is a great help in enhancing as well as
streamlining the process of developing new drug candidates.

 By improving our ability to identify both helpful and harmful metabolites,
suggestions for structural modifications will optimize the likelihood that other
compounds in the series are more successful.

 Structural modifications to solve a metabolic stability problem may not
necessarily lead to a compound with an overall improvement in PK properties.

 Solving metabolic stability problems at one site could result in the increase in
the rate of metabolism at another site, a phenomenon known as metabolic
switching. Further, reduction in hepatic clearance may lead to increased renal or
biliary clearance of a parent drug or inhibition of one or more drug-metabolizing
enzymes. Therefore, it is advisable that in vitro metabolic stability data be
integrated with other ADME screening.
                             Conclusions (cont’d)
 An accurate measurement of the pharmacokinetic parameters and a good
understanding of the factors that affect the pharmacokinetics will guide drug
design.

 High metabolic liability usually leads to poor bioavailability and high
clearance, and formation of active or toxic metabolites will have an impact on
the pharmacological and toxicological outcomes.

 Drug candidates should have little or none of the following:
•gut/hepatic-first-pass metabolism
•inhibition/induction of drug-metabolizing enzymes
•biologically active metabolites
•metabolism by polymorphically expressed drug-metabolizing enzymes
•formation of reactive metabolites
•Also, it is important to have the most desirable plasma half-life and ratio of
metabolic to renal clearance.

				
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