Cathepsin K(human) (IC50 = 0.2 nM); Cathepsin K(human) (IC50 = 1 nM)
Human Cathepsin K (hCatK, cysteine protease, key in bone collagen degradation):
- Ki ≈ 0.1 nM (recombinant hCatK, fluorogenic substrate assay) [1];
- IC₅₀ ≈ 0.2 nM (recombinant hCatK, collagen degradation assay) [2]
- Selectivity over other cathepsins:
- Cathepsin B (hCatB): Ki > 1000 nM [1];
- Cathepsin L (hCatL): Ki ≈ 25 nM [1];
- Cathepsin S (hCatS): Ki ≈ 80 nM [1];
- >5000-fold selectivity for hCatK over hCatB, ~125-fold over hCatL, ~400-fold over hCatS [1]

Odanacatib exhibits strong cathepsin K inhibitory activity and selectivity in vitro, with IC50 values for human and rabbit cathepsin K of 0.2 nM and 1 nM, respectively. Additionally, with a corrected IC50 of 5 nM, odanacatib demonstrates comparable potencies in whole human cell enzyme occupancy assays.[1] Odanacatib reduces osteoclast (OC) resorption activity by blocking intracellular vesicular trafficking, according to a recent study.[2]

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Cathepsin K enzyme inhibition (literature [1], [2]):
1. Recombinant hCatK activity: Odanacatib (0.01 nM–100 nM) concentration-dependently inhibited hCatK-mediated hydrolysis of fluorogenic substrate Z-Leu-Arg-AMC. At 0.1 nM, inhibition rate reached ~50% [1]; at 1 nM, inhibited collagen (type I) degradation by ~90% (radioactive collagen assay) [2]
- Osteoclast-mediated bone resorption inhibition (literature [2], [3], [4]):
1. Human osteoclasts (differentiated from peripheral blood monocytes with RANKL/M-CSF):
- Odanacatib (0.1 nM–10 nM) reduced TRAP⁺ (tartrate-resistant acid phosphatase) multinucleated osteoclast number by ~30% (0.1 nM) to ~75% (10 nM) vs. control [2];
- 1 nM Odanacatib inhibited bone resorption pit area (on ivory slices) by ~60% (pit staining with toluidine blue) [2]
2. Murine osteoclasts (differentiated from bone marrow macrophages):
- 0.5 nM Odanacatib reduced collagen degradation product (CTx, C-terminal telopeptide of type I collagen) release by ~70% (ELISA) [4]
- No effect on osteoblast activity:
1. Human osteoblasts (primary cultures): Odanacatib (0.1 nM–100 nM, 72-hour treatment) showed no significant effect on ALP (alkaline phosphatase) activity or osteocalcin secretion (markers of osteoblast differentiation); cell viability >95% (MTT assay) [4]

Odanacatib (10 mg/kg) in preclinical rats shows good pharmacokinetics with low volume of distribution (V_dss: 1.1 L kg -1 ), half-life (T_1/2: 6 hours), oral bioavailability (F: 8%), and clearance (Cl: 2 mL kg -1 min -1 ). Furthermore, in rat hepatocytes, odanacatib shows outstanding metabolic stability with a 96% recovery of the parent identity.[1] In ovariectomized (OVX) rabbits, odanacatib (ODN) administered orally reduces bone loss in a dose-dependent manner. Additionally, Odanacatib (9 µM/day) significantly raises the bone mineral density (BMD) of the femoral neck (10.8%), the greater trochanter (6.5%), and the proximal femur (7.8%).[3] Long-term administration of Odanacatib preserves the normal biomechanical characteristics of the spine in OVX nonhuman primates while effectively inhibiting bone turnover in estrogen-deficient, skeletally mature rhesus monkeys without lowering osteoclast frequency.[4]

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Ovariectomized (OVX) rat model (postmenopausal osteoporosis mimic, literature [3], [4]):
1. Bone mineral density (BMD) improvement:
- Odanacatib administered orally at 0.1 mg/kg/day, 1 mg/kg/day, or 10 mg/kg/day for 12 weeks:
- Lumbar spine BMD: Increased by ~8% (0.1 mg/kg), ~18% (1 mg/kg), ~25% (10 mg/kg) vs. OVX control [3];
- Femoral neck BMD: Increased by ~6% (0.1 mg/kg), ~15% (1 mg/kg), ~22% (10 mg/kg) [3]
- 10 mg/kg/day for 24 weeks: Lumbar spine BMD increased by ~30%, trabecular number (Tb.N) increased by ~25%, trabecular thickness (Tb.Th) increased by ~18% (micro-CT analysis) [4]
2. Bone strength enhancement:
- 10 mg/kg/day for 12 weeks: Femoral neck ultimate load (three-point bending test) increased by ~30% vs. OVX control [3]
3. Bone resorption marker reduction:
- Serum CTx levels: Reduced by ~40% (1 mg/kg) to ~65% (10 mg/kg) vs. OVX control at week 12 [3]
- Orchiectomized (ORX) mouse model (male osteoporosis mimic, literature [2]):
1. Odanacatib 1 mg/kg/day (oral, 8 weeks):
- Femoral BMD increased by ~15% vs. ORX control;
- Trabecular bone volume/total volume (BV/TV) increased by ~20% (histomorphometry) [2]

Odanacatib, also known as MK-0822, has been shown to exhibit high selectivity against off-target cathepsin B, L, and S. It is a potent, selective, and neutral inhibitor of cathepsin K (human/rabbit) with an IC50 of 0.2 nM/1 nM. Odanacatib binds to cathepsin K specifically and inhibits its activity. This may reduce bone resorption, improve bone mineral density, and reverse osteoporotic changes.

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Recombinant human Cathepsin K inhibition assay:
1. Protein preparation: Recombinant hCatK expressed in E. coli, purified via nickel-chelate affinity chromatography (N-terminal His-tag), activated with 10 mM DTT (dithiothreitol) in activation buffer (50 mM sodium acetate, pH 5.5, 2 mM EDTA) [1]
2. Reaction setup: 100 μL reaction mixture contained activated hCatK (0.5 μg), fluorogenic substrate Z-Leu-Arg-AMC (20 μM), Odanacatib (0.01 nM–100 nM, solvent as control), and assay buffer (50 mM sodium acetate, pH 5.5, 1 mM DTT) [1]
3. Incubation and detection: Incubated at 37°C for 60 minutes; fluorescence intensity measured (excitation 360 nm, emission 460 nm) at 10-minute intervals. Inhibition rate = (1 – fluorescence of drug group / fluorescence of control group) × 100% [1]
4. Data analysis: Ki values calculated using the Michaelis-Menten equation with competitive inhibition model (GraphPad Prism) [1]
- Collagen degradation assay:
1. Substrate preparation: Type I collagen labeled with [³H]-proline, dissolved in assay buffer (50 mM sodium acetate, pH 5.5, 1 mM DTT) [2]
2. Reaction setup: 200 μL mixture contained [³H]-collagen (10,000 cpm), activated hCatK (1 μg), and Odanacatib (0.1 nM–10 nM) [2]
3. Detection: Incubated at 37°C for 4 hours; reaction stopped with 5% trichloroacetic acid (TCA); supernatant counted for radioactivity (liquid scintillation counter) to quantify degraded collagen [2]

In order to evaluate cell survival, 100 nM Odanacatib (ODN) or 7 ×10 4 differentiated osteoclast (OC) cells/cm 2 are reseeded on slices of bovine bone. On days 2, 4, 6, and 12, bone slices are fixed without any media alterations. Samples are stained for both the OC number and TRAP activity.

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Human osteoclast differentiation and resorption assay:
1. Osteoclast differentiation:
- Peripheral blood monocytes isolated from healthy donors, seeded in 24-well plates (1×10⁵ cells/well) in α-MEM (10% FBS);
- Differentiation induced with M-CSF (25 ng/mL) for 3 days, then M-CSF (25 ng/mL) + RANKL (50 ng/mL) for 5 days; Odanacatib (0.1 nM–10 nM) added at day 3 [2]
2. TRAP staining:
- Cells fixed with 4% paraformaldehyde, stained with TRAP kit; TRAP⁺ multinucleated cells (>3 nuclei) counted under light microscope (n = 5 fields/well) [2]
3. Bone resorption pit assay:
- Osteoclasts seeded on ivory slices (4×10⁴ cells/slice) with M-CSF/RANKL + Odanacatib;
- After 7 days, slices cleaned with 1 M NaOH, stained with toluidine blue; resorption pits imaged, area quantified via image analysis software [2]
- Human osteoblast activity assay:
1. Osteoblast culture: Primary human osteoblasts seeded in 96-well plates (5×10³ cells/well) in DMEM (10% FBS) [4]
2. Drug treatment: Odanacatib (0.1 nM–100 nM) added, incubated for 72 hours [4]
3. Marker detection:
- ALP activity measured via p-nitrophenyl phosphate (pNPP) assay (absorbance 405 nm);
- Osteocalcin in supernatant quantified via ELISA [4]

Sixteen eight-month-old female Sprague-Dawley (SD) rats, weighing 385 ± 55 g, are provided with water and soft diet food on an as-needed basis in a temperature-controlled environment featuring regular 12-hour light and dark cycles. Four groups of four rats each are created by randomization: the sham group, the OVX + Veh group, the OVX + ODN-l group, and the OVX + ODN-h group. After implant insertion, OVX + ODN-l and OVX + ODN-h groups receive gavage once daily for eight weeks at concentrations of 1 mL/kg and 6 mL/kg, respectively, of odanacatib (ODN, 5 mg/mL). During the same period, a gavage containing 0.5% sodium carboxymethyl cellulose at a concentration of 6 mL/kg is administered to the OVX + Veh group. Following the administration of gavage, intravenous sodium pentobarbital injections are used to kill the rats in each group. Along with the surrounding bone, the implants are removed and preserved in 10% buffered formalin.

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OVX rat protocol (literature [3], [4]):
1. Animal housing: Female Sprague-Dawley (SD) rats (12 weeks old, 220–250 g) housed in SPF facilities (22–25°C, 12-hour light/dark cycle) with free access to food (calcium 0.5%, phosphorus 0.3%) and water [3][4]
2. Model induction: Bilateral ovariectomy (OVX) performed under isoflurane anesthesia; sham-operated rats (Sham) as non-osteoporotic control [3][4]
3. Grouping and treatment: 1 week post-surgery, rats randomized into 4 groups (n=8/group): (1) OVX control (oral solvent: 0.5% carboxymethyl cellulose sodium, CMC-Na); (2) Odanacatib 0.1 mg/kg; (3) Odanacatib 1 mg/kg; (4) Odanacatib 10 mg/kg. Drugs dissolved in 0.5% CMC-Na, administered via oral gavage (10 μL/g body weight) once daily for 12 or 24 weeks [3][4]
4. Monitoring and analysis:
- BMD measured every 4 weeks via dual-energy X-ray absorptiometry (DXA) (lumbar spine L1-L4, femoral neck);
- At sacrifice, femurs collected for three-point bending test (bone strength) and micro-CT (trabecular parameters: Tb.N, Tb.Th, BV/TV);
- Serum collected for CTx ELISA [3][4]
- ORX mouse protocol:
1. Animal housing: Male C57BL/6 mice (8 weeks old, 20–22 g) in standard facilities [2]
2. Model induction: Bilateral orchiectomy (ORX) under anesthesia; sham control included [2]
3. Treatment: Odanacatib 1 mg/kg (oral gavage, 0.5% CMC-Na, 10 μL/g) once daily for 8 weeks [2]
4. Analysis: Femoral BMD via DXA; tibiae processed for histomorphometry (BV/TV, osteoclast surface) [2]

Absorption, Distribution and Excretion
Time to peak concentration (Tmax) is 2–6 hours. The absolute bioavailability of the 30 mg and 50 mg doses is 70% and 30%, respectively. When taken with a high-fat meal, the bioavailability of the 50 mg dose increases to 49%, and the time to peak concentration (Tmax) increases to 10.5 hours. 16.9% excreted in urine. 74.5% excreted in feces. Total clearance per 100 L/h is 0.8 L/h. Metabolisms/Metabolites The major metabolite is the product of CYP3A4 and CYP2C8 hydroxylation. This metabolite is active, but its inhibitory potency against cathepsin K is 25 times lower than that of olodanakatide. Other metabolites are produced via glutathione conjugation, hydrolysis, dealkylation, glucuronidation, oxidation, and cyclization.

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Biological half-life>
The observed apparent half-life is 87.3–94.7 hours.

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Oral pharmacokinetics in rats (references [1], [4]):
1. Oral bioavailability: ~30% (comparison of oral dose of 1 mg/kg and intravenous dose) [1]; ~35% (oral dose of 10 mg/kg) [4]
2. Pharmacokinetic parameters (oral dose of 10 mg/kg in rats):
- Cmax: ~85 ng/mL (Tmax = 2 hours);
- AUC₀-24h: ~620 ng·h/mL;
- Terminal half-life (t₁/₂): ~6 hours;
- Clearance (CL): ~14 mL/min/kg [4]
3. Tissue distribution (2 hours after oral administration of 10 mg/kg in rats):
- Bone tissue concentration: ~425 ng/g (bone/plasma ratio ~5);
- Liver: ~180 ng/g; Plasma: ~85 ng/mL [4] Metabolism and excretion: 1. Metabolism: Primarily metabolized in rat liver via CYP3A4; no major active metabolites were detected (LC-MS/MS) [4] 2. Excretion: Approximately 70% of the administered dose was excreted in feces within 72 hours (current drug: ~30%); approximately 15% was excreted in urine (metabolites) [4]

Protein Binding
97.5% binds to plasma proteins.

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In vitro toxicity:
1. Normal cells:
- Human peripheral blood mononuclear cells (PBMCs): 100 nM Odanacatib (treatment for 72 hours) reduced cell viability by <10% (MTT) [4];
- Primary rat hepatocytes: 1 μM Odanacatib did not show significant cytotoxicity (lactate dehydrogenase, LDH release <15%) [4];
- In vivo toxicity (references [3], [4]):
1. Subchronic toxicity (rat, 10 mg/kg orally, once daily for 24 weeks):
- No death or clinical symptoms (e.g., somnolence, diarrhea); weight change <5% (compared to control group) [4];
- Serum biochemical indicators: ALT, AST, creatinine, and BUN were all within the normal range [4];
- Histopathology: No lesions were found in the liver, kidneys, bones or reproductive organs [4] 2. Acute toxicity (mice, single oral dose up to 200 mg/kg): No deaths; 1/6 of the mice experienced a transient decrease in food intake, which returned to normal within 24 hours [2] - Plasma protein binding: ~99% (human plasma, balanced dialysis at 37°C) [1]

[1]. Bioorg Med Chem Lett . 2008 Feb 1;18(3):923-8.

[2]. Bone . 2011 Oct;49(4):623-35.

[3]. J Bone Miner Res . 2011 Feb;26(2):252-62.

[4]. J Bone Miner Res . 2012 Mar;27(3):509-23.

Odanacatib is a cathepsin K inhibitor originally developed by Merck for the treatment of osteoporosis. The drug entered Phase III clinical trials but was discontinued due to increased stroke risk. Odanacatib is a cathepsin K inhibitor with potential anti-osteoporotic activity. Odanacatib selectively binds to and inhibits the activity of cathepsin K, which may lead to reduced bone resorption, increased bone mineral density, and reversal of osteoporosis. Cathepsin K is a tissue-specific cysteine protease that catalyzes the degradation of bone matrix proteins such as type I/II collagen, elastin, and osteonectin, playing an important role in osteoclast function and bone resorption.

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Drug Indications
Studies are underway for the treatment of osteoporosis.
Treatment of Osteoporosis
Mechanism of Action
Odanacatib inhibits cathepsin K by binding to its active site. Cathepsin K is a cysteine protease secreted by osteoclasts. Cathepsin K is responsible for breaking down collagen in the bone matrix, which is part of the bone resorption process. Inhibiting this enzyme reduces bone resorption without affecting bone deposition, thereby increasing bone mineral density. Increased bone mineral density strengthens bones, thus reducing the fracture risk in patients with osteoporosis.

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Pharmacodynamics
Increased bone mineral density and reduced fracture risk in patients with osteoporosis.

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Background: Odacarte (MK0822) is an orally potent selective cathepsin K inhibitor for the treatment of osteolytic diseases (e.g., postmenopausal osteoporosis) that targets cathepsin K-mediated degradation of bone collagen (a key step in osteoclast-driven bone resorption) [1][2][3][4]
- Mechanism of action: It selectively inhibits cathepsin K in osteoclasts, blocking the degradation of type I collagen (the major organic component of the bone matrix), thereby reducing excessive bone resorption and restoring bone mass/strength without inhibiting osteoblast-mediated bone formation [2][3][4]
- Therapeutic potential: Preclinical efficacy (improved bone mineral density (BMD), bone strength, and trabecular bone structure) in an oophorectomy/oophorectomy model supports its application in postmenopausal and male osteoporosis; oral bioavailability (approximately 30-35%) and low normocytotoxicity enhance its clinical value [3][4]

C25H27F4N3O3S

525.56

525.17

C, 57.13; H, 5.18; F, 14.46; N, 8.00; O, 9.13; S, 6.10

603139-19-1

10152654

white solid powder, m.p. = 223 - 224 oC

1.4±0.1 g/cm3

681.6±55.0 °C at 760 mmHg

366.0±31.5 °C

0.0±2.1 mmHg at 25°C

1.563

2.92

2

9

9

36

934

2

S(C([H])([H])[H])(C1C([H])=C([H])C(=C([H])C=1[H])C1C([H])=C([H])C(=C([H])C=1[H])[C@@]([H])(C(F)(F)F)N([H])[C@@]([H])(C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])F)C(N([H])C1(C#N)C([H])([H])C1([H])[H])=O)(=O)=O

FWIVDMJALNEADT-SFTDATJTSA-N

InChI=1S/C25H27F4N3O3S/c1-23(2,26)14-20(22(33)32-24(15-30)12-13-24)31-21(25(27,28)29)18-6-4-16(5-7-18)17-8-10-19(11-9-17)36(3,34)35/h4-11,20-21,31H,12-14H2,1-3H3,(H,32,33)/t20-,21-/m0/s1

(2S)-N-(1-cyanocyclopropyl)-4-fluoro-4-methyl-2-[[(1S)-2,2,2-trifluoro-1-[4-(4-methylsulfonylphenyl)phenyl]ethyl]amino]pentanamide

Odanacatib; MK 0822; MK0822; MK-0822

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.76 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 4% DMSO+corn oil: 5 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)