Approaches to Mitigating the Bioactivation … to Mitigating the Bioactivation Potential of...
Transcript of Approaches to Mitigating the Bioactivation … to Mitigating the Bioactivation Potential of...
Deepak Dalvie 1
Approaches to Mitigating the Bioactivation Potential of Compounds in Lead Optimization
Deepak Dalvie
Pharmacokinetics, Dynamics and Metabolism Department
Pfizer San Diego
Deepak Dalvie 3
Adverse Drug Reactions (ADR) A Problem??
è A common cause for drug recalls or black box warnings
è Unpredicted or Idiosyncratic ADR (IADR) – more problematic Ø Normally not observed until phase III or post launch Ø Frequency of occurrence – 1 in 10000 to 1 in 100000 Ø Responsible for drug withdrawal Ø Very expensive - law suits etc.
è Patients have been deprived of several good drugs
Deepak Dalvie 4
è Not easy to predict IADR l No animal models l Lack of specific biomarkers
è Circumstantial evidence links metabolic activation to IADR
Drug Safety - One of the leading causes for candidate attrition
Drug Reactive
Metabolite Formation
Interaction with Macromolecules
Haptenization Reaction with proteins
ADR Liver toxicity
Agranulocytosis Other
Metabolism
è Several examples demonstrate that bioactivation liability and IADR are linked
l Kalgutkar AS and Soglia JR (2005). Exp. Opin. Drug Metab. & Toxicol. 1:91-141)
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Drugs Associated With IADRs
Drugs Withdrawn Aclcofenac (antiinflammatory) Hepatitis, rash Alpidem (anxiolytic) Hepatitis (fatal) Amodiaquine (antimalarial) Hepatitis, agranulocytosis Amineptine (antidepressant) Hepatitis, cutaneous ADRs Benoxaprofen (antiinflammatory) Hepatitis, cutaneous ADRs Bromfenac (antiinflammatory) Hepatitis (fatal) Carbutamide (antidiabetic) Bone marrow toxicity Ibufenac (antiinflammatory) Hepatitis (fatal) Iproniazid (antidepressant) Hepatitis (fatal) Metiamide (antiulcer) Bone marrow toxicity Nomifensine (antidepressant) Hepatitis (fatal), anaemia Practolol (antiarrhythmic) Severe cutaneous ADRs Remoxipride (antipsychotic) Aplastic anaemia Sudoxicam (antiinflammatory) Hepatitis (fatal) Tienilic Acid (diuretic) Hepatitis (fatal) Tolrestat (antidiabetic) Hepatitis (fatal) Troglitazone (antidiabetic) Hepatitis (fatal) Zomepirac (antiinflammatory) Hepatitis, cutaneous ADRs
Marketed Drugs Abacavir (antiretroviral) Cutaneous ADRs Acetaminophen (analgesic) Hepatitis (fatal) Captopril (antihypertensive) Cutaneous ADRs, agranulocytosis Carbamazepine (anticonvulsant) Hepatitis, agranulocytosis Clozapine (antipsychotic) Agranulocytosis Cyclophosphamide (anticancer) Agranulocytosis, cutaneous ADRs Dapsone (antibacterial) Agranulocytosis, cutaneous ADRs, aplastic anaemia Diclofenac (antiinflammatory) Hepatitis Felbamate (anticonvulsant) Hepatitis (fatal), aplastic anaemia (fatal), severe restriction in use Furosemide (diurectic) Agranulocytosis, cutaneous ADRs, aplastic anaemia Halothane (anesthetic) Hepatitis Imipramine (antidepressant) Hepatitis Indomethacin (antiinflammatory) Hepatitis
Isoniazid (antibacterial) Hepatitis (can be fatal) Phenytoin (anticonvulsant) Agranulocytosis, cutaneous ADRs Procainamide (antiarrhythmic) Hepatitis, agranulocytosis Sulfamethoxazole (antibacterial) Agranulocytosis, aplastic anaemia Terbinafine (antifungal) Hepatitis, cutaneous ADRs Ticlopidine (antithrombotic) Agranulocytosis, aplastic anaemia Tolcapone (antiparkinsons) Hepatitis (fatal) Trazodone (antidepressant) Hepatitis Trimethoprim (antibacterial) Agranulocytosis, aplastic anaemia, cutaneous ADRs Thalidomide (immunomodulator) Teratogenicity Valproic acid (anticonvulsant) Hepatitis (fatal), teratogenicity
Temp. Withdrawn or Withdrawn in other Countries
Aminopyrine (analgesic) Agranulocytosis Nefazodone (antidepressant) Hepatitis (> 200 deaths) Trovan (antibacterial) Hepatitis Zileuton (antiasthma) Hepatitis
For most of these drugs, bioactivation to reactive metabolites has been demonstrated in vitro or in vivo
Kalgutkar AS and Soglia JR (2005). Exp. Opin. Drug Metab. & Toxicol. 1:91-141)
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è Identify toxicophores – “Structural Alerts” early on l Especially those undergoing ‘metabolic activation’
è One approach adopted by many pharmaceutical companies l Eliminate metabolic activation of a candidate
Ø Screen for ability to form reactive metabolites very early
l Avoid chemical functionalities that undergo bioactivation Ø No guarantee that this will make compounds safer Ø But, preventing reactive metabolite formation ê chances of IADR
è TALL ORDER BUT AVOIDS RISK! – Saves $$ – Saves time and effort
Role of Drug Metabolism in Addressing IADR
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Methods to Assess Bioactivation Potential
Covalent Bindingto Proteins
Trapping with nucleophiles
Two Methods Commonly Used
è Most definitive è Useful in quantitation of
the reactive metabolite è Helps to detect all reactive
metabolites è Limited by availability of
radiolabel
è Most popular in a discovery setting
è Nucleophiles used to trap are: l Glutathione l N-Acetylcysteine l Other nucleophiles - cyanide
è Acts as a surrogate marker of covalent binding
Evans D. C. et. al. Chem. Res. Toxicol. 2004
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General Method for Assessing Reactive Intermediates
Drug (10 – 50 µM) + Human Liver Microsomes + GSH (3 mM)
Incubation at 37º C for 1.0 hr
Samples analyzed for GSH adducts using various LC-MS methods
NADPH regenerating system (+/-)
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What Does a Positive Signal in These Assays Mean?
Some Known Facts: è No assay predicts potential of a new drug to cause
IADR
è Important to Weigh the Benefits versus Risks Ø Is it a prototype or a back up? Ø Indication Ø Is the drug being developed for an unmet medical need?
– First in class? Ø Dosing regimen of the drug
– Acute or chronic? – Drugs that are used chronically are more prone to IADRs
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Relationship Between Dose and Frequency of Idiosyncratic Reactions
è Dose l A lower dose reduces the risk
02468
1012
0-50
50-1
00
100-
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400
400-
800
800-
1600
>160
0
Daily dose (mg)
Freq
uenc
y
Issue: è Dose is unknown in the early stages
ê Dose ê Lesser reactive metabolite formed ê Lower Risk
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Other Factors from a Drug Metabolism Perspective
è Contribution of pathways that detoxify reactive metabolites
è Contribution of competing metabolic routes
DrugDrug
RMRM
M1M1
M2M2
M3M3
M4M4DrugDrug
RMRM
M1M1
M2M2
M3M3
M4M4
DetoxicationDetoxication of of reactive intermediatesreactive intermediates
Competing metabolicCompeting metabolicpathwayspathways
DrugDrug
RMRM
M1M1
M2M2
M3M3
M4M4DrugDrug
RMRM
M1M1
M2M2
M3M3
M4M4
DetoxicationDetoxication of of reactive intermediatesreactive intermediates
Competing metabolicCompeting metabolicpathwayspathways
“After all it is all about body burden of RM”
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Case Studies
è Paroxetine – an antidepressant SSRI è Raloxifene – A selective estrogen receptor modulator
(SERM) è A Case Study from Pfizer
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Paroxetine Case
è Paroxetine contains the methylenedioxy group - a toxicophore l Results in inactivation of CYP2D6
Ø clinical pharmacokinetic interactions with substrates well established
§Bertelsen KM, Venkatakrishnan K, Von Moltke LL, Obach RS and Greenblatt DJ (2003) Drug Metab. Dispos. 31:289-293
Methylenedioxy Moiety
Carbene
NH
F
O
O
O
Paroxetine
P450 2D6
O
OO
MI Complex withHeme
P450 Inactivation
O O
NN
NN
HOO
HOO
Fe
O O
NN
NN
HOO
HOO
Fe
An Carbene – Iron Complex
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Paroxetine Also Undergoes Metabolic Activation
Zhao SX, Dalvie DK, Kelly JM, Soglia JR, Frederick KS, Smith EB, Obach RS and Kalgutkar AS (2007) Chem. Res. Toxicol. 20:1649-1657.
Incubation with HLM in the presence of GSH and NADPH
NH
F
O O
O
OO
O
O
OH
OH
CYP2D6
Catechol o-Benzoquinone
O
OH
OHSG
O
O
OSG
[O]
O
OH
OHSG
GSHGS
bis-Adduct mono-Adduct
[GSH]
Paroxetine
O
O
OSG
GS N
S
O
O
HN
CO2H
SG
[O]
A Novel CyclicAdduct
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Incubation of Radiolabeled Paroxetine with HLM
Zhao SX, Dalvie DK, Kelly JM, Soglia JR, Frederick KS, Smith EB, Obach RS and Kalgutkar AS (2007) Chem. Res. Toxicol. 20:1649-1657.
+NADPH
+GSH +NADPH
+UDPGA +NADPH
-NADPHCov
alen
t Bin
ding
(pm
ol b
ound
/mg
prot
ein)
0
10
20
30
40
50
Incubation of radiolabel with HLM/NADPH
[14C] Paroxetine + Human Liver Microsomes +
NADPH
Covalent Binding to Microsomal Proteins
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Inference?
è Paroxetine is prone to bioactivation l Covalently binds to microsomal protein
è Would one nominate Paroxetine if it was to be developed
as an antidepressant to date?
è Paroxetine is a commonly prescribed antidepressant l IADRs (especially hepatotoxicity) are extremely rare
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Effect of GSH on Covalent Binding of Paroxetine
+NADPH
+GSH +NADPH
+UDPGA +NADPH
-NADPHCov
alen
t Bin
ding
(pm
ol b
ound
/mg
prot
ein)
0
10
20
30
40
50Incubation with Human Liver Microsomes
Zhao SX, Dalvie DK, Kelly JM, Soglia JR, Frederick KS, Smith EB, Obach RS and Kalgutkar AS (2007) Chem. Res. Toxicol. 20:1649-1657.
O
O
OO
OH
OHCatechol o-Benzoquinone
[O]
Glutathione Adducts
Covalent Binding
GSH and UDPGA Mops Reactive Intermediate Reduces covalent binding!
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+NADPH
+GSH +NADPH
+PAPS+NADPH
+SAM +NADPH
+UDPGA +NADPH
-NADPHCov
alen
t Bin
ding
(pm
ol b
ound
/mg
prot
ein)
0
1
2
3
4
5
6
O
O
OO
OH
OHCatechol o-Benzoquinone
[O]
O
OCH3
OH O
OH
OCH3
+
COVALENTBINDING
Covalent Binding of Paroxetine in Human S9
Sulfate and Glucuronide Conjugates
GSH Conjugates
/COMT S-Adenosyl methionine
Catechol detoxified via methylation O-Methoxy derivatives formed
Reduces covalent binding! This is in addition to GSH conjugation!
Zhao SX, Dalvie DK, Kelly JM, Soglia JR, Frederick KS, Smith EB, Obach RS and Kalgutkar AS (2007) Chem. Res. Toxicol. 20:1649-1657.
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Analysis of the Data
è Paroxetine was bioactivated but: l o-Quinone is detoxified by Glutathione l Catechol detoxified by methylation and glucuronidation
è Additional factors that may contribute to less IADRs with paroxetine l Low daily dose (20 mg QD)
Ø Reactive metabolite burden readily handled by endogenous glutathione pool in the mammal
l CYP2D6 inactivation by paroxetine following chronic dosing may inhibit its own metabolism
Ø Decreases the body burden of the catechol and o-benzoquinone
DrugDrug
RMRM
M1M1
M2M2
M3M3
M4M4DrugDrug
RMRM
M1M1
M2M2
M3M3
M4M4
DetoxicationDetoxication of of reactive intermediatesreactive intermediates
Competing metabolicCompeting metabolicpathwayspathways
DrugDrug
RMRM
M1M1
M2M2
M3M3
M4M4DrugDrug
RMRM
M1M1
M2M2
M3M3
M4M4
DetoxicationDetoxication of of reactive intermediatesreactive intermediates
Competing metabolicCompeting metabolicpathwayspathways
Drug Catechol
Quinone GSH Conjugates
Methylation Sulfate & Glucuronide
Conjugates
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Lesson
è Thorough knowledge of ALL metabolism pathways is important
è GSH conjugate detection needs to be put into right context l GSH is a detoxication pathway! l Acts as a electrophile mopping agent in the body
è Early assessment of dose can help
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Raloxifene Case
è A second generation SERM approved for osteoporosis è Bioactivated by CYP3A4 to quinonoid intermediates è A mechanism-based inactivator of CYP3A4 è No IADRs or DDIs reported
SHOOH
O O
N
RaloxifeneSO
O
O O
RaloxifeneDi-quinone methide
SHOOH
O O
SHOOH
O O
SHOOH
O O
GS
SG
SG
[CYP3A4]GSH
GSH Conjugates
Abs
orba
nce uA
U
Abs
orba
nce uA
U G1
100000 150000 200000 250000
M2 M1 Raloxifene
G2
Time (min)
G1
100000 150000 200000 250000
M2 M1 Raloxifene
G2
Time (min)
Incubation with HLM/NADPH and GSH
So would one nominate this compound for further development?
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Why is Raloxifene is Devoid of any IADRs or DDI
è Primary route of raloxifene metabolism – Glucuronidation
SHOOH
O O
N
SHOOGlu
O O
4'Glucuronide (M1)
6-Glucuronide (M2)SGluO
OH
O O
50% reduction in covalent binding observed in the presence of GSH and UDPGA!
0
5
10
15
20
25
30
35
40
45
50
NoCofactors
NADPH NADPH +GSH
NADPH +UDPGA
NADPH +GSH
+UDPGAIncubation of Radiolabeled Raloxifene with Human Liver
Microsomes
Pmol
s of
Rad
iola
bele
d R
alox
ifene
Cov
alen
tly B
ound
to
Hum
an L
iver
Mic
roso
mes
68
72
50
% bound relative to only NADPH
Covalent Binding Incubation of [14C]Raloxifene with HLM
Glucuronidation of raloxifene
Dalvie D, Kang P, Zientek M, Xiang C, Zhou S, and Obach RS (2008) Chem. Res. Toxicol. 21, 2260.
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Impact of Intestinal Metabolism on Bioactivation of Raloxifene
è Glucuronidation primarily catalyzed by 1A8 and 1A10 l UGT1A1 also catalyzes the glucuronidation but to a minor extent l UGT 1A8 and 1A10 are exclusively present in the small intestine
Preincubation with Human Intestinal Microsomes results in significant decrease
in covalent binding!
0
5
10
15
20
25
30
35
NADPH only NADPH + UDPGAPreincubation of Radiolabelled Raloxifene with Human Intestinal
MicrosomesPm
ols
of R
adio
labe
lled
Ral
oxife
ne B
ound
to H
uman
Li
ver M
icro
som
es
No Cofactors NADPH NADPH + GSH
Control (no UDPGA)
Add 100 uL of this aliquot to rat or human liver microsomalincubation mixtures at
EACH TUBE CONTAINS
HIMProtein – 0.3 mg/mL - HIMNADPH – 1 mMUDPGA – 3 mMGSH – 3 mMMgCL2 – 10 mMAlamethicin – 10 µg/mg protein
WARM FOR 5 MIN
ADD SUBSTRATE 10 µM
0 min 5 min 15 min 30 min
HLM
Protein – 1 mg/mL - HLMNADPH – 1 mMUDPGA – 3 mMGSH – 3 mMMgCL2 – 10 mMAlamethicin – 10 µg/mg proteinpreincubate
INCUBATE FOR 45 MIN
1.Quench with ACN containing /internal Std
2.Determine the G1 formed
HLM HLM HLM
Add 100 uL of this aliquot to rat or human liver microsomalincubation mixtures at
EACH TUBE CONTAINS
HIMProtein – 0.3 mg/mL - HIMNADPH – 1 mMUDPGA – 3 mMGSH – 3 mMMgCL2 – 10 mMAlamethicin – 10 µg/mg protein
WARM FOR 5 MIN
ADD SUBSTRATE 10 µM
0 min 5 min 15 min 30 min
HLMHLM
Protein – 1 mg/mL - HLMNADPH – 1 mMUDPGA – 3 mMGSH – 3 mMMgCL2 – 10 mMAlamethicin – 10 µg/mg proteinpreincubate
INCUBATE FOR 45 MIN
1.Quench with ACN containing /internal Std
2.Determine the G1 formed
HLMHLM HLMHLM HLMHLM
Preincubation Experiment
Daniel C. Kemp, Peter W. Fan, and Jeffrey C. Stevens Drug Metab. Dispos. 30, 694.2002 Dalvie D, Kang P, Zientek M, Xiang C, Zhou S, and Obach RS (2008) Chem. Res. Toxicol. 21, 2260.
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Efficiency of Raloxifene Glucuronidation versus Oxidation
Glucuronidation Oxidation
Cl int glu Clint oxi
µL/min/mg protein µL/min/mg protein
HIM 397 ± 14 26 + 3.1
HLM 37 ± 1.6 142 + 5.8
Efficiency of detoxification pathway explains the impact of glucuronidation on hepatic bioactivation
Dalvie D, Kang P, Zientek M, Xiang C, Zhou S, and Obach RS (2008) Chem. Res. Toxicol. 21, 2260.
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What does this data tell us!
è Intestinal glucuronidation limits the amount of raloxifene undergoing bioactivation
è Dose of raloxifene – 60 mg QD è Other pathways of clearance are responsible for reducing the body
burden
Liver
GI Tract
Systemic Circulation
Site of metabolism
Low exposure of Raloxifene
DrugDrug
RMRM
M1M1
M2M2
M3M3
M4M4DrugDrug
RMRM
M1M1
M2M2
M3M3
M4M4
Reduces the body burden
Highly Efficient process
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Lesson!
è The compound would have been “withdrawn from development” in the absence of all the relevant metabolism data to date
è Understand the impact/contribution of other metabolic pathways
è Identify enzymes responsible – maybe non-hepatic l Especially the intestine
Bottomline Understand the Metabolism of the candidate
thoroughly prior to any Major Decisions
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11-β-HSD-1 Inhibitors
Project Goal è Develop an 11βHSD1 (11β-HydroxySteroid Dehydrogenase type-1) inhibitor
for the treatment of Type II diabetes
Cortisol (F) Active glucocorticoid
Cortisone (E) Inactive glucocorticoid
O
OH3C
H3C OH
OOH
O
H3C
H3C OH
OOH
HO
11βHSD-1NADPH
11βHSD-2NAD
11 11
á Hepatic Glucose
11βHSD1 inhibitor inhibits conversion of cortisone to cortisol
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Lead Candidate in the 11-βHSD1 Program
Natilie Hosea Sajiv Nair
è Has great physicochemical properties è Good potency è Pharmacokinetic Attributes
è Key Issues from a metabolism perspective l Risk of bioactivation l No Structural Alerts – yet +ve signal in
RM assay
NHN NH2SO
O
CNPF-915275
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Metabolism of PF-915275
Reactive metabolites trapped as Novel GSH Adducts
SNH
O O
N NH2
NH2
CO2H
NH
O
S
OHN CO2H
NH2HO2C
HNO
SO
HN
HO2C
HOSN
O O
N NH2
NH2
CO2H
NH
O
S
OHN CO2H
SN
NH
O
HO2C
Bis-conjugate Adduct Unique GSH Adduct
SHN
O O
NNH2
N Site of bioactivation and adduct formation
SNH
O O
N NH2
OH
SNH
O O
N NH2
HO
GSH
HLM/NADPH No metabolism at this site
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Risk Assessment for PF-915275
è Preliminary dose prediction was 30 – 50 mg l Uncertainties in clearance (low) and Ceff
è Oxidation leading to reactive metabolites l a primary route
è Question? l Do other competing metabolic pathways or detoxication
of the reactive metabolite play a role?
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Risk Analysis: Assessment of Reactive Metabolite Formation in LM/NADPH/UDPGA
SNH
O O
N NH2
NC
SNH
O O
N NH2SNH
O O
N NH2
HO OH
SNH
O O
N NH2
GluO
SNH
O O
N NH2
OGlu
SNH
O O
N NGlu
NC
H
M3
M5PF-00915275
GSH Adducts
[O]/GSH
UDPGA UDPGA
è 14C-PF-915275 synthesis accelarated to see the impact on
covalent binding l Assess the covalent binding in vitro and in vivo
No oxidative metabolites observed in the absence
of GSH or UDPGA!
Deepak Dalvie Natilie Hosea
Minor Pathway
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Covalent Binding Studies with [14C]-PF-915275 Using Human Liver S9
Ping Kang Sue Zhou
21
633
741928
335
30 1018116
10 30
200
400
600
800
Control NADPH only NADPH+GSH NADPH+GSH+ other Phase
II cofactors
pmol
/mg
Prot
ein
.
RatMonkeyHuman
S9 Assay
Drug Drug
RM RM
M1 M1
M2 M2
Drug Drug
RM RM
M1 M1
M2 M2
Drug Drug
RM RM
M1 M1
M2 M2
Drug Drug
RM RM
M1 M1
M2 M2
M3
M4
è 915275 – was bioactivated but: l Metabolites were detoxified by GSH, sulfation and glucuronidation l In vitro covalent binding near background when other co-factors
are used
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Further De-risking Factors to Move PF-915275 into Development
è Rat studies performedl Very low binding to liver protein in vivo (0.21 pmol/mg; <0.05% of
dose) l No GSH conjugates or related metabolites observed
è Proper PK/PD studies helped to refine the dose l Refined Dose = 0.3 to 3 mg using monkey PK/PD data
è Investigative toxicology studies performed l Compound dosed to GSH depleted rats
Deepak Dalvie 35
Overall Conclusions From the 3 Cases
è Covalent binding to microsomal proteins may not be predictive of toxicity l Some refs by Obach et. al. further confirm this
è Thorough assessment of metabolism is pivotal in early discovery
è Important to consider the contribution of other metabolic pathways (hepatic or extrahepatic) in light of a positive signal
è Some idea of total daily dose/exposure helps l Get a range to see if the dose is lower than 20 – 50 mg
Ø Use in vitro potency and clearance from HLM as first cut Ø Refine the dose prediction from the efficacy model
è Estimate the body burden of the reactive metabolite: l DRM = D x fa x fm x fRM l Gan and co-workers Chem. Res. Toxicol. 2009, 22, 690.
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Is Covalent Binding or GSH Adduct Formation A Kill Shot??
è Clearly, a positive signal by itself ‘is not’ a kill shot è Not good predictors of IADR è Needs to be considered as a ‘flag’ to trigger additional studies è GSH conjugation is a detoxication pathway –need to put in right
context è However, helps to elucidate mechanisms of bioactivation
è Useful in circumventing the liability through iterative design
è Liver microsomal assays do not always address all metabolic pathways for a compound
l Use of hepatocytes or S-9 – supplemented with co-factors gives a better picture of all metabolic pathways