discovery and development of …– high selectivity profile of l-006235 is lost in whole cell...

1

Discovery and development of odanacatib: A selective cathepsin K inhibitor for the treatment of osteoporosis

Discovery and development of Discovery and development of odanacatib:odanacatib: A selective cathepsin K A selective cathepsin K inhibitor for the treatment of inhibitor for the treatment of osteoporosisosteoporosis

Shawn J. StachelOn behalf of the odanacatib discovery and product development teamsMerck Research Laboratories

8th RSC-SCI Symposium on Proteinase Inhibitor Design

April 16, 2013

2

AcknowledgementsAcknowledgementsBiologyChristine BrideauWanda CromlishSylvie DesmaraisJean-Pierre FalgueyretLouis-Jacques FortinSonia LamontagneFrance LandryFrederic MasseDave PercivalDenis RiendeauPaul TawaJennifer WangComp. Med.Stephanie AlleynLiette BelairJoel BosquetCarl BrièreNancy KellyGayle LapointeMira LavoieSonia LesvesqueDenis NormandinKaren OrtegaRoberta RasoriJosianne RozonWayne Sturkenboom Simon Wong

ChemistryKevin BatemanChristopher BaylyCameron BlackMichael BoydNathalie ChauretSheldon CraneStephen DayJacques Yves GauthierRobert HouleElise IsabelC.K. LauSerge LegerTammy LeRicheJean-Francois LevesqueChun-Sing LiChristophe MellonRenata OballaJoel RobichaudBruno RoyJohn ScheigetzCarmai SetoMichel TherienLaird TrimbleJean-Francois TruchonVouy-Linh TruongQingping Wang

Bone Biology (WP)Don KimmelSevgi RodanGideon RodanLe DuongPat MasarachiaBrenda PennypackerMaureen PickarskiGregg WesolowskiYa ZhuoPharmaceutical R&DCynthia BazinSophie-Dorothee ClasElizabeth KwongPauline LukRafik NaccacheWayne ParentSuzanne SpagnoliDendi SusantoHongshi YuSafety AssessmentLaura GumprechtCindy FishmanPharmacologySylvie Toulmond

Celera

Process ChemistryPaul O’SheaDean BenderMirlinda BibaJennifer ChilenskiJimmy DaSilvaRich DesmondPaul DevinePete DormerBruce FosterDon GuathierDanny GauvreauFrancis GosselinDerek HendersonGreg HughesJohn LeazerBill LeonardJohn LimantoChristian NadeauThorsten RosnerKara RubinAli ShafieeVeena UpadhyayChris WelchClinical DevelopmentAubrey Stoch Tony LombardiDeborah Miller Albert Leung

3

Osteoporosis: A Continuing Disease BurdenOsteoporosis: A Continuing Disease Burden

Estimated 200 million women worldwide with osteoporosis.1

Only ~20% of osteoporosis patients are currently treated Hip fractures cause high morbidity and mortality2

− ~20% die in the 1st year− ~33% are totally dependent or in a nursing home after one year

1 Kanis JA (2007) WHO Technical Report, University of Sheffield, UK: 662 International Osteoporosis Foundation statistics.

From the International Osteoporosis Foundation

Osteoporosis: “porous bones” A progressive bone disease characterized by decreased bone density

and mass. Leads to an increased risk of fracture

Trabecular bone deteriorated

Normal Osteoporosis

4

Bone RemodelingBone Remodeling

Healthy: Bone resorption = formationOsteoporosis: Bone resorption > formation

5Rodan & Duong Bone Key 2008, 5, 6

Collagen

Electron micrograph of an osteoclast on bone

Osteoclast – The Target Cell for Cathepsin K InhibitionOsteoclast Osteoclast –– The Target Cell for Cathepsin K InhibitionThe Target Cell for Cathepsin K Inhibition

N

N

Cathepsin KpH=5

Trans- endocytosis

Lysosomes

SealingZone

Bone resorption by osteoclasts is the initial step in remodeling

6

Cathepsin K Deficiency Cathepsin K Deficiency Cathepsin K Deficiency Discovered in 19951

– Highly expressed in osteoclast– Cleaves at multiple sites in triple helical region of

collagen type I and II

Pycnodysostosis is a genetic disease associated with cathepsin K deficiency2,3

– Rare autosomal recessive skeletal dysplasia– ~150 cases reported worldwide – Short stature with high bone mass and increased

fragility associated with high risk of fracture– Normal intelligence, sexual development and

lifespan

Cat K null mice4

– Osteopetrotic phenotype– Increased bone density – Otherwise healthy and fertile

1 Inaoka, et al, BBRC 1995, 206, 892 Schilling, et al, Osteoporosis Int 2007, 18, 6593 Saftig PNAS 1998, 95, 13453.4 Gelb, et al, Science 1996, 273, 1236

Henri de Toulouse-Lautrec

7

Cathepsin K as a Therapeutic TargetCathepsin K as a Therapeutic TargetCathepsin K as a Therapeutic Target

Data suggestive that cathepsin K represents a promising target for treatment of osteoporosis

– Human and mouse genetic data – Cathepsin K collagenase activity– Restricted cellular distribution

Goal: To develop an orally active, selective and reversible cathepsin K inhibitor for treatment of osteoporosis

8

Substrate to Inhibitor: WarheadSubstrate to Inhibitor: WarheadSubstrate to Inhibitor: Warhead

Use knowledge of mechanism to convert substrate into inhibitor:

R

O

NR'

HS-Enz

H

R

HO

N R'

H

+ Enz-SHH2O

Acyl enzymeintermediate

Tetrahedral intermediate

S-Enz

R

O

S-EnzR

O

OH

- H2N-R'

Replace scissile bond with an electrophile (warhead) that reacts with active site cysteine:

Palmer, J. T.; Rasnick, D.; Klaus, J. L.; Brömme, D. J. Med. Chem. 1995, 38, 3193

9

Substrate to Inhibitor: Recognition SequenceSubstrate to Inhibitor: Recognition Sequence

Cathepsin K cleaves Type I collagen at multiple positions Recognition sequence1: Gly-Leu-Lys—Gly-His

Recognition sequence combined with electrophilic warhead gives a mechanism based-inhibitor of cathepsin K2,3

1Atley, M.; Mort, J. S.; Lalumiere, M.; Eyre, D. R. Bone 2000, 26, 2412McGrath, M. E.; Klaus, J. L; Barnes, M. G.; Brömme, D. Nat. Struct. Biol. 1997, 4, 1053Brömme, D.; Klaus, J. L.; Okamoto, K.; Rasnick, D.; Palmer, J. T Biochem. J. 1996, 315, 85

P3 P2 P1 P1’ P2’

APC3328

NH O

HN S

O OO

NHN

P1

P3

P2

NH O

HN

O

NH2

HN

O

NH

HN

O

N N

P1

P3

P2

P1'

P2'

10

Opportunities for Inhibitor DesignOpportunities for Inhibitor DesignOpportunities for Inhibitor Design• Co-crystal reveals key interactions with cathepsin K and opportunities

for obtaining selectivity

McGrath, M. E.; Klaus, J. L; Barnes, M. G.; Brömme, D Nat. Struct. Biol. 1997, 4, 105

NH O

HN S

O OO

NHN

S2

S3

S1

– S2 pocket suggests accommodation of alternative hydrophobic groups– S3 sub-site contains aspartic acid (Asp61) that may be exploitable for

selectivity– P2-P1 amide forms key interaction with enzyme backbone (Gly66, Asn158)– P2-P3 amide: NH hydrogen-bond to Gly66, oxygen points towards solvent

– S1 pocket is a narrow groove on enzyme

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Irreversible inhibitors have potential liabilities Vinyl sulfone can be replaced by nitrile (electrophilic)

– Adds cysteine reversibly to form thioimidate

Enz-SH

Nitrile-based inhibitor

Optimization to dipeptide nitrile

L-006235Cat K IC50 = 0.2 nM

Reversible InhibitorsReversible Inhibitors

J. Palmer. et. al. J. Med. Chem. 2005, 48, 7520

HN S

O

OO

NH

O

N

HN

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Selective Inhibition of Cathepsin K is Important to Avoid Potential Off-target ActivitySelective Inhibition of Cathepsin K is Important Selective Inhibition of Cathepsin K is Important to Avoid Potential Offto Avoid Potential Off--target Activitytarget Activity

Fifteen human cathepsins are present inside cells within acidic compartments called lysosomes Potential consequences of off-target inhibition

− Cat L: cardiomyopathy, impaired neovascularization− Cat F: neuronal ceroid lipofuscinosis− Cat S: impaired immune response

High selectivity for cathepsin K desired to avoid potential off-target effects

F. Lecaille Chem. Rev. 2002, 102, 4459.

Cathepsins

Cysteine CathepsinsB, C, F, H, K, L, O, S, V, W, Z

Aspartyl CathepsinsD, E

Serine CathepsinsA, G

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L-006235 : A Potent, Selective, Reversible Dipeptidic Nitrile Inhibitor of Cathepsin KL-006235 : A Potent, Selective, Reversible Dipeptidic Nitrile Inhibitor of Cathepsin K

– >5000-fold selective in purified enzyme assays– Active in rabbit and monkey models of osteoporosis

IC50 (nM)Cat K

0.2

Cat B

1100

Cat L

6300

Cat S

47000

Cat F

3000


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L-006235 in Cathepsin K LL--006235 in Cathepsin K 006235 in Cathepsin K

Asn166

Cys25

Asp61

Gly66

3.0

2.9

2.9

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Loss of Specificity of Cathepsin K Inhibition: Whole Cell AssayLoss of Specificity of Cathepsin K Inhibition: Loss of Specificity of Cathepsin K Inhibition: Whole Cell AssayWhole Cell Assay

– High selectivity profile of L-006235 is lost in whole cell assays

Inhibition of Cathepsins, IC50 (nM)

Cathepsin SCathepsin LCathepsin BCathepsin K

0.2L-006235

Enzyme Enzyme Enzyme EnzymeCell Cell Cell

1100 17 6300 340 47000 790

Cell

5

L-006235


16

Hypothesis: Basic Inhibitors Accumulate in Acidic Lysosomes and Inhibit Off-Target Cathepsins

Hypothesis: Basic Inhibitors Accumulate in Acidic Lysosomes and Inhibit Off-Target Cathepsins Lysosomotropism: accumulation of lipophilic weak bases in acidic

sub-cellular compartments Lysosome

pH ~7

pH ~5BH+

B

Plasma Membrane

pH ~7B

MergedLL--006235006235

DNDDND--99 (lyso)99 (lyso)

Co-localization in live HUVECsL-006,235

weak basic amine

J. Falgueyret et. al. J. Med. Chem 2005, 48, 7535

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Cat K Inhibitors in Whole Cell AssaysCat K Inhibitors in Whole Cell Assays


NH

ONH

O

N

SNN

N

L-006235 Cmpd 2

NH

ONH

O

N

SNO

NBasic Neutral


0.22.3

L-006235Cmpd 2


11004200

17 630024000

340 4700027000

7902900 >10000 >10000

– Non-basic analogs show reduced activity in cell-based assays – Non-basic P3 groups are generally less active

J. Falgueyret et. al. J. Med. Chem 2005, 48, 7535

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10- to 100-fold increase in intrinsic potency

Improved enzyme selectivity

Enhanced pharmacokinetics (bioavailability, half-life)

Liability of basic group: – Lysosomotropism leading to loss of functional selectivity

Why have a Basic Substituent?Why have a Basic Substituent?

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SAR Optimization EffortsSAR Optimization EffortsSAR Optimization Efforts

NH O

HN

O

N

SNN

N

ON

N

O

N S

R S RHN

RO O

O

N N

O

Ar

CN

R

– Extensive SAR studies on dipeptide framework resulted in loss ofselectivity and/or potency

– Abandoned dipeptide series of inhibitors

20

Amide ReplacementAmide Replacement

Cat K = 56 nMCat L = 498 nM (9x)Cat S = 1578 nM (28x)Cat B = >10000 nM (>180)

HN CN

ONH

O

Cat K = 51 nMCat L = 42 nM (0.8x)Cat S = 41 nM (0.8x)Cat B = 1903 nM (37x)

Increase in

selectivity

Amide bond can be replaced but loss in Cat K potency Selectivity can still be achieved without P2-P3 amide

Cat K = 73 nMCat L = 3666 nM (50x)Cat S = 3401 nM (46x)Cat B = >10000 nM (137x)

Loss of

selectivity

J. Robichaud et. al. BMCL 2004, 14, 4291

P2-P1 amide forms key interaction with enzyme backbone (Gly66, Asn158)but, P2-P3 amide NH hydrogen-bond to Gly66, oxygen points towards solvent

21

Further Optimization of Amide ReplacementFurther Optimization of Amide Replacement

HN CN

O

NHN

(R)-2Cat K = 11 nMCat L = 3950 nM (359x)Cat S = 2010 nM (183x)Cat B = 3725 nM (339x)

Potency

selectivity

High selectivity can still be achieved without P2-P3 amide with addition of basic P3 group

Cat K = 56 nMCat L = 498 nM (9x)Cat S = 1578 nM (28x)Cat B = >10000 nM (>180)

J. Robichaud et. al. BMCL 2004, 14, 4291J. Robichaud et. al. J. Med. Chem. 2003, 46, 3709

22

ReRe--Addition of a HydrogenAddition of a Hydrogen--Bond DonorBond Donor

Additional of hydrogen bond donor is favorable for cat K potency.

X = Cat K (IC50)NH 54 nMO 1147 nMCH2 2353 nM




23

Potency and Selectivity OptimizationPotency and Selectivity Optimization

selectivity

Selectivity and potency can still be achieved without P2-P3 amide with addition of basic P3 group

Cat K = 3 nMCat L = 2101 nM (700x)Cat S = 158 nM (53x)Cat B = 1812 nM (604x)


HN CN

O

NHN

(R)-2



24

Another PeptidomimeticAnother PeptidomimeticAnother Peptidomimetic

NH

HN

OCN

O

NS

NN

How can P2-amide be replaced while maintaining H-bond capability?

H-bond donor requires non-basic nitrogen– Non-basic amine (pKa = 1.5) is not protonated at physiological pH

Retains the H-bond donating properties of an amide bond

NH

O O

NHO

NH

CF3 O

NHO

M. Zanda et. al ChemMedChem 2007, 2,1693

25

NH O

HN CN

O

50

CFCF33 Amide Replacement: PAmide Replacement: P22 Leucine vs CyclohexylLeucine vs Cyclohexyl

Trifluoroethylamine is an effective amide isostere Cyclohexyl ring in P2 is much less potent than the isobutyl side-chain Modeling suggests loss of potency in cyclohexyl analog is a result of

steric interactions between P2 and the CF3 group

50

NH O

HN CN

O

W. C. Black et. al. BMCL 2005, 15, 4741Black, W. C.; Percival, M. D. ChemBioChem 2006, 7, 1525

26

CF3 Amide ReplacementCF3 Amide Replacement

Phenyl piperazine has greater potency and selectivity than thiazole piperazine

Can this exquiste potency/selectivity advantage be extended to non-basic inhibitors?

NH

CF3

O

HN CN

NHN

O

HN CNN

H

CF3

N

SNN

HN CN

ONH

O

N

SNN

0.03 3315 26 848

IC50 (nM)

Cat K Cat B Cat L Cat S

0.2L-006235

1100 6300 47000

0.005 1110 47 451

3

4

W. C. Black et. al. BMCL 2005, 15, 4741

27

C. S. Li et al. BMCL 1985,16, 1985

Non-basic derivatives in this series are potent and selective L-873724 stands out as best compound in this series

““NonNon--basicbasic”” PP33 SubstituentsSubstituents

L-873724

28

Why are Trifluoroethylamines so Potent?Why are Trifluoroethylamines so Potent?X-ray structure of Cat K -Complex

HN CN

ONH

CF3

H2NO2S

29

L-873724 has Similar Potency in Whole Cells and Purified CathepsinsL-873724 has Similar Potency in Whole Cells and Purified Cathepsins Trifluoroethylamine isostere gave enough potency to remove the basic P3

Selectivity profile of L-873724 is maintained in whole cell assays


Cathepsin B Cathepsin L Cathepsin S

Enzyme Cell Enzyme Cell Enzyme Cell

L-006235 1100 17 6300 340 47000 790

L-873724 5240 4800 264 1220 178 94

L-006235 L-873724

HN CN

ONH

O

N

SNN

HN CN

ONH

CF3

MeO2S

C. S. Li et al. BMCL 1985,16, 1985

30

L-873724 MetabolismL-873724 Metabolism

Human hepatocytes~50% metabolism

NH O

NHCF3

OH

R

N

M7: leucine hydroxylation

NH O

NHCF3

MeSO2

N

M8: hydroxyacidM12: lactone formation

NH O

CF3 O

R

NH O

OHCF3

R

NC 70141-2225 hum heps L-873,724 t=2

12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00Time10

100

%

25Oct02-014 2: Diode Array 263.801_271.9

5.22e418.92

16.12

15.7714.7214.45 15.03

17.63

17.85

19.58

L-873,724

M12

M13

M7

M8

M13: acid

NH O

CF3

OH

R

OH

? ?

L-872724 is extensively metabolized in human hepatocytes (56% metabolism)

31

Modifications to Reduce MetabolismModifications to Reduce MetabolismModify P2 to block metabolism

Add substituent to block amide cleavage and lactonization

NH

CF3

O

HN

N

MeO2S

32

L-873,724 has Similar Potency in Whole Cells and Purified CathepsinsL-873,724 has Similar Potency in Whole Cells and Purified Cathepsins Modifications result in odanacatib which maintains excellent selectivity

and activity in both enzymatic and cell-based assays

OdanacatibL-873724



0.20.2

L-873724Odanacatib


52401034

48001050

2642995

12204843

17860

9445

Cell

35

J. Gauthier et. al. BMCL 2008, 18, 923

33

Odanacatib has Improved Pharmacokinetics in Preclinical SpeciesOdanacatib has Improved Pharmacokinetics in Odanacatib has Improved Pharmacokinetics in Preclinical SpeciesPreclinical Species Modification of L-873724 to block sites of oxidative metabolism provides

a superior pharmacokinetic profile. High recovery (>99%) after incubation with dog and human hepatocytes

OdanacatibL-873724

Pharmacokinetics

Rhesus MonkeyDogRat

L-873724Odanacatib

Cl(ml/min/kg) t1/2 (h) t1/2 (h) t1/2 (h)

6.32

2.76

1.120.13

657

7.56.1

1.718

Cl(ml/min/kg) Cl(ml/min/kg)


34

Mean Concentration Profile of Odanacatib in Women Administered Once-Weekly Doses for 3 WeeksMean Concentration Profile of Odanacatib in Women Administered Once-Weekly Doses for 3 Weeks

Days ︵Week 1 ︶

0 1 2 3 4 5 6 7

Con

cent

ratio

n, n

M

0

100

200

300

400

500

600

25 mg OW50 mg OW100 mg OW

Days ︵Week 3 ︶

0 1 2 3 4 5 6 7 8 9 1011121314

Cat B,L,S,F IC90

Cat K IC90(bone res)

– Human half-life similar to dog; human t1/2 = 60-70 h– At therapeutic exposures, odanacatib maintains selectivity over other cathepsins

Stoch, A. et. al. Clin. Pharm. & Ther. 2009, 86, 175

35

S-CTx Mean Percent Change (95%CI) from Baseline

Effect of Odanacatib on Bone Resorption After 3 Weeks of Once-Weekly DosingEffect of Odanacatib on Bone Resorption After 3 Effect of Odanacatib on Bone Resorption After 3 Weeks of OnceWeeks of Once--Weekly DosingWeekly Dosing

0 1 3 5 7 9 11 14 16 18 21 24 28Day

-100

-75

-50

-25

0

25

50

75

S-C

TX-I

% C

hang

e fr

om B

asel

ine

Placebo5 mg 25 mg

Dose Dose Dose

50 mg 100 mg

N = 9-12/groupStoch, A. et. al. Clin. Pharm. & Ther. 2009, 86, 175

36

Selectivity of Other Cathepsin K InhibitorsSelectivity of Other Cathepsin K Inhibitors Relacatib has low selectivity towards related cathepsins (L, V, B, S) in

enzymatic assays

− Relacatib clinical development was halted after Phase I studies

Balicatib is very selective towards related cathepsins in enzymatic assays

− Development of balicatib was discontinued during Phase IIb due to morphea-like skin changes

− Cathepsin B, K, L, S and V are all expressed in human skin and have collagenase activity

− Postulate that morphea is due to lysosomotropic-based loss of selectivity

Balicatib (Novartis)

Brömme, D. Expert Opin. Investig. Drugs. 2009, 18, 585Desmarais, S. et al Molecular Pharmacology 2008, 73, 147

Relacatib (GSK)

BasicNH

HN

O

O

N

CN

NNH

HN

O

O

N

O

OS

O ON

37

Odanacatib (MK-0822)

Odanacatib has Similar Potency in Whole Cells and Purified CathepsinsOdanacatib has Similar Potency in Whole Cells and Purified Cathepsins

The low level of off-target activity of odanacatib in enzyme assays is maintained in whole cell assays

Balicatib


0.20.6

OdanacatibBalicatib


10344800

105061

2995503

484348

6065000

Cell

522


452900

Basic

38

Effect of Cat K Inhibitors on Collagen Accumulation in Dermal FibroblastsEffect of Cat K Inhibitors on Collagen Accumulation in Dermal Fibroblasts

Odanacatib has a minimal effect on intracellular collagen accumulation compared to balicatib and the pan-cathepsin inhibitor relacatib

Inhibitor Concentration (µM)

0.01 0.1 1 10

Intra

cellu

lar C

olla

gen

Acc

umul

atio

n(F

luor

esce

nce

inte

nsity

, fol

d In

crea

sed

over

veh

icle

)

0

1

2

3

4

5

6

7

Balicatib Relacatib Odanacatib

Develop model of intracellular collagen accumulation in primary human dermal fibroblasts


39

Odanacatib Inhibits Bone Resorption with Less Reduction of Bone FormationOdanacatib Inhibits Bone Resorption with Less Reduction of Bone Formation

Bisphosphonates and denosumab reduce the number of osteoclasts−Fewer resorption pits, reduced new bone formation

Odanacatib reduces the activity of Cat K in the osteoclast−Same number of, but shallower resorption pits−Allows new bone formation

OCOC

PP

Vehicle

OC: OsteoclastP: Resorption Pit

PPOCOC

Ob progenitor

Osteoblast (Ob)

Osteoclast

Bone formation factorsIGF-1, TGF-BMP. Wnt 10b

Odanacatib

Leung et al. Bone 2011, 49, 623

40

Historical Comparison to Alendronate

Urinary NTx

Serum BSAP

Geo

met

ric M

ean

Per

cent

Cha

nge

from

Bas

elin

e (

SE)

-100

-80

-60

-40

-20

0

-100

-80

-60

-40

-20

0

Alendronate Phase III2

3 4 5Year

21

3 4 521Year

20

20

1 Binkley et al., ASBMR 20112 Merck data on file

-80

-60

-40

-20

0

20

Odanacatib Phase IIB1

3 4 521Year

-100

-80

-60

-40

-20

0

-100

20

3 4 521Year

Odanacatib inhibits bone resorption (uNTx) while exhibiting less reduction of bone formation (BSAP) than observed with alendronate.

Differential Effects on Bone Turnover MarkersDifferential Effects on Bone Turnover Markers

41

LumbarSpine

FemoralNeck

TotalHip M

ean

Per

cent

Cha

nge

from

Bas

elin

e (±

SE

)

Alendronate Phase III2

0

5

10

15

0

5

10

0

5

10

3 4 5Year

21

3 4 5Year

21

3 4 5Year

21

Odanacatib Phase IIB1

0

5

10

15

0

5

10

0

5

10

3 4 5Year

21

3 4 5Year

21

3 4 5Year

21

Historical Comparison to Alendronate

Odanacatib: Phase IIb Study 5Odanacatib: Phase IIb Study 5--Year Data Year Data Bone Mineral Density (DXA)Bone Mineral Density (DXA) Odanacatib progressively increases BMD over 5 years at lumbar spine, femoral

neck and total hip

42

SummarySummarySummary Early inhibitors were found to accumulate in lysosomes thereby lowering

their selectivity towards related cathepsins in functional assays

Optimized leads produced selective non-basic inhibitors

Optimized non-basic leads reduced metabolism and improved pharmacokinetic properties

In post-menopausal women, odanacatib treatment:− Demonstrates less suppression of markers of bone formation than has been

observed with alendronate*− Progressively increases BMD over 5 years at lumbar spine, femoral neck,

and total hip

* Historical comparison

discovery and development of …– high selectivity profile of l-006235 is lost in whole cell...

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