Role of mechanistic transport studies in lead optimization'

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Role of mechanistic transport studies in lead
optimization. AAPS workshop on optimization of
drug-like properties Jerome H. Hochman,
Ph.D. Department of Drug Metabolism, Merck and
Co., Inc.
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Properties of drug transporting P-glycoprotein

• Pgp recognizes a wide variety of structurally
  diverse substrates
• Substrate interactions occur within the inner
  leaflet of the bilayer
• Membrane partitioning is essential for Pgp
  interaction
• Drug transport entails ATP hydrolysis and large
  conformational changes.
• Assays evaluating ATP hydrolysis by Pgp or
  inhibition of Pgp efflux of marker substrates
  does not detect Pgp substrates effectively.
• Interactions between Pgp and substrates entails
  polar interactions, in particular hydrogen bond
  acceptors.

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Tissue expression of P-glycoprotein

• Intestine
• Expressed on luminal brush border
• Barrier to drug absorption
• Elimination from blood
• synergistic interaction with intestinal CYP3A
• Liver
• Expressed on the canalicular membrane
• Elimination of drugs and metabolites into bile
• Kidney
• Expressed on the brush border of proximal tubule
  cells
• elimination of drug into urine.
• Brain
• Luminal side of Brain microvascular endothelial
  cells
• Integral component of the blood brain barrier
• Impact of Pgp is the greatest at the BBB.
• Other tissues Placenta, Lymphocytes, Testis,
  uterus, adrenal cortex

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CF-1 and -- mice studies showing impact of
P-gp on brain and plasma drug concentrations

• In vitro transport ratio in L-mdr1a 11

Brain drug concentrations
Plasma drug concentrations

• Plasma drug concentrations are not different
  between mdr1a deficient and competent mice.
• Brain concentrations are 13 fold higher in the
  mdr1a deficient mice.

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Impact of Pgp on oral absorption and CNS
penetration in Pgp deficient mice
1Data summarized from Kim et. al. J Clin. Invest.
(1998) 101 289-294 Schinkel et. al. J Clin.
Invest. (1996) 97 2517-2524 Mol Pharmacol
(2001) 59 806-813 Lankas et. al. Toxicol appl.
pharmacol. (1997) 143 357-365 Prueksaritanont
et. al. (2002) Xenobiotica 32207-20.

• The impact of Pgp on oral absorption is generally
  small compared to the effect on CNS penetration

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Impact of Pgp on oral absorption of discovery
compounds

• Several Pgp substrates show good oral absorption
  but restricted CNS penetration

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Transport across the blood brain barrier

• Passive permeability
• Continuous endothelium (no fenestrae)
• Low paracellular permeability
• Tight junction formation
• TER 1000-2000 Ocm2
• Sucrose permeability Papp 1X 10-7 cm/sec
• Attenuated transcytosis
• Efflux transporters
• Pump from the brain to the blood
• P-glycoprotein
• Anion transporter (MRP-2?, BSAT1 )
• BCRP (ABCG2)?

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P-glycoprotein at the blood brain barrier

• P-glycoprotein at the BBB can reduce CNS
  exposure to drugs10-50 fold

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Tools to evaluate drug transport by P-glycoprotein

• In vitro models
• Transport across cell monolayers
• Caco-2 cells- colon carcinoma
• LLC PK-1 cell- control cell line (porcine kidney)
• L-MDR1- LLC PK-1 cells expressing human
  P-glycoprotein
• L-mdr1a- LLC PK-1 cells expressing mouse
  P-glycoprotein
• In vivo models
• CF-1 -/- and / mice
• Spontaneous mutation in mdr-1a gene.

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P-glycoprotein transport studies
Apical solution
Basolateral solution
Direction of P-gp transport
P-glycoprotein (P-gp)
In vitro MDR1(human) and mdr1a (mouse)
transfected LLC-PK1 cells In vivo mdr1a
deficient and competent CF-1 mice.
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CF-1 and -- mice studies showing impact of
P-gp on brain and plasma drug concentrations

• In vitro transport ratio in L-mdr1a 11

Brain drug concentrations
Plasma drug concentrations

• Plasma drug concentrations are not different
  between mdr1a deficient and competent mice.
• Brain concentrations are 13 fold higher in the
  mdr1a deficient mice.

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In vivo impact of Pgp on brain distribution of
drugs versus in vitro transport in L-mdr1a and
L-MDR1
Discovery compounds
Model compound set
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1 to 1 mdr1a vs CF-1 1hr brain ratio Y5
15
10
indinavir
mdr1a(-/-)(/) CF-1 mice
(mouse, in vivo)
Kp,brain ratio of
423
ritonavir
5
L-365,260
diazepam
vinblastine
0
L-790070
0
5
10
15
20
J-120961
J-122360
B-to-A / A-to-B at in L-mdr1a
(mouse, in vitro)
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Species differences in P-glycoprotein transport
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Prediction of P-glycoprotein influence in man
Human
Mouse
L-MDR1
In vitro
L-mdr1a
?
In vivo
CF-1 /-- mice
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P-glycoprotein at the blood brain barrier

• P-glycoprotein at the BBB can reduce CNS
  exposure to drugs10-50 fold

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Influence of Passive permeability on brain and
spinal cord penetration in mdr1a deficient mice
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Properties of CNS drugs
J. Polli, et. al. JPET (2002) vol 303 1029-1037.
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Designing drugs with low Pgp efflux Developing
SAR for Pgp efflux

• Influence of specific functional groups on Pgp
  transport.
• Influence of physical chemical properties on Pgp
  transport.
• Molecular modeling.
• Calculated physical chemical properties
• knn (similarity of atom pair descriptors)
• Look for consensus trends.

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Influence of substituents on Pgp transport
moderate
high
Low
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• decreasing the number of hydrogen bond acceptors
  lowers the potential that a compound will subject
  to P-gp efflux.

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Knn analysis

• Cut-off for experimental data 2
• subset of relevant atom pair descriptors is
  determined.
• susceptibility of test compound determined based
  on properties of compound with closest
  similarity of atom pair descriptors.
• Consensus from five models
• Accuracy for Prediction 84 (majority of
  incorrect assignments have transport ratios
  between 2 and 3)
• identification of sites conferring interactions
  with Pgp is consistent with SAR developed from
  empirical structural comparison.

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SAR for Pgp transport

• All three approaches indicated that reducing
  hydrogen bond acceptors reduced Pgp efflux.
• SAR was established to effectively overcome Pgp
  as an issue.
• Avoidance of functional groups with high
  potential hydrogen bond acceptors.
• How do we improve properties of compounds in
  which groups conferring P-gp efflux are essential
  for pharmacological activity?

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Influence of lipophilicity on Pgp transport
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Influence of lipophilicity on Pgp transport
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Properties of drug transporting P-glycoprotein

• Pgp recognizes a wide variety of structurally
  diverse substrates
• Substrate interactions occur within the inner
  leaflet of the bilayer
• Membrane partitioning is essential for Pgp
  interaction
• Drug transport entails ATP hydrolysis and large
  conformational changes.
• Assays evaluating ATP hydrolysis by Pgp or
  inhibition of Pgp efflux of marker substrates
  does not detect Pgp substrates effectively.
• Interactions between Pgp and substrates entails
  polar interactions, in particular hydrogen bond
  acceptors.

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Approaches to limit the influence of essential
groups conferring Pgp efflux

• Steric hindrance
• Decrease hydrogen bond acceptor potential
• decrease electron density of hydrogen bond
  acceptors.

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Pgp transport versus Passive membrane
permeabilityinfluence of amide bond
modifications
amide
Modified amide
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Effect of amide modifications on Pgp transport

• Removal of CF3 did not reduce Pgp efflux.

• Replacement or direct modifications to amide
  reduce Pgp efflux, but at the expense of potency.
• Steric hindrance to amide reduced Pgp but at the
  expense of potency.

• Electron withdrawing groups adjacent to the amide
  reduced Pgp efflux

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Effect of amide modification of directional
transport by mouse and human Pgp
50
40
30
mdr1a transport ratio
20
10
amide
Modified amide
0
0
10
20
30
40
50
MDR1 transport ratio

• Within a structural series subtle structural
  changes can have disproportionate influences on
  transport by human and mouse Pgp.

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• 96 amino acid identity between human and monkey
  Pgp
• Majority of changes occur in first extracellular
  loop

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Summary

• Low passive permeability and Pgp efflux can limit
  distribution of drugs into the CNS.
• A structure based approach can be used to
  minimize Pgp efflux.
• Decrease hydrogen bond acceptors.
• Introduce steric hindrance to hydrogen bond
  acceptors.
• Reduce electron density of hydrogen bond
  acceptors.
• Evaluation of SAR for Pgp transport should
  consider mechanistic aspects of Pgp transport.
• In vitro models can supplement in vivo studies to
  evaluate the impact of P-glycoprotein efflux on
  CNS exposure in man.
• Animal models for P-glycoprotein drug
  interactions should take into account species
  differences.

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Hepatic and renal uptake transporters

• Facilitate uptake of compounds from the blood
  into the liver and kidney.
• Multiple families of transporters including OATs,
  OCTs, OATPs , and nutrient uptake transport
  systems.
• Multiple transporters with related functions.
• Many compounds are subject transport by more than
  1 transporter.

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Tools for studying uptake transporters

• Few well characterized specific inhibitors.
• Few ( if any ) inhibitory monoclonal antibodies.
• Transporters expressed in mammalian cells.
• Used by several labs to evaluate mechanisms of
  drug transport
• Tells you if a drug is subject to transport by a
  specific transporter.
• Does not indicate the relative contribution of
  the transporter.
• RNAi silencing of transporters.
• Promising tools for specific reduction of
  transporter expression.
• Depletes the cell of specific mRNA.
• Specificity of siRNA is determined by homology to
  the mRNA.
• Binding of siRNA to mRNA signal docking of an
  endonuclease which cleaves mRNA.
• Multiple technical approaches to application of
  RNAi strategies.

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RNAi knockdown of OATP4 mRNA and activity in
transfected cells
mRNA
Uptake

• RNAi mediated supression of activity corresponds
  with reduction in OATP4 mRNA
• Oatp4 mediated uptake CCK-8 uptake is reduced
  70-90 48 hours after Stealth RNAi transfection.

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Conclusions

• Stealth RNAi is effective in knock-down of OATP4
  in stably transfected HEK cells.
• RNAi is a promising approach to study transport
  and metabolism issues
• Rapid and inexpensive design of specific probes
  if cDNA sequence is known.
• Multiple strategies for achieving siRNA knockdown
  including synthetic duplexes and stable
  expression vectors.
• Knock-down specific transporters in whole cells.
• Potential extension to in vivo knock-down in
  rodents.
• More rapid less expensive and shorter term
  response than knockout animals.
• Application of the approach to drug discovery and
  development issues requires stable expression of
  the transporters in cell models in a manner that
  reflects expression in vivo.

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Contributors

• Scott Fauty
• Todd Killino
• Anne Taylor
• Tamara Pittman
• Janice Brunner
• Thomayant Prueksaritanont
• Jiunn H Lin

• Mark Bock
• Doug Pettibone
• Sookhee Ha
• Qin Mei
• Cuyue Tang
• Sergey Krymgold
• Xiadong Shen
• I-Wu Chen
• Masayo Yamazaki
• Yan Xu
• Bing Li