Heparan sulfate selectively inhibits the collagenase activity of cathepsin K.

Cathepsin K (CtsK) is a cysteine protease with potent collagenase activity. CtsK is highly expressed by bone-resorbing osteoclasts and plays an essential role in resorption of bone matrix. Although CtsK is known to bind heparan sulfate (HS), the structural details of the interaction, and how HS regulates the biological functions of CtsK, remains largely unknown. In this report, we discovered that HS is a multifaceted regulator of the structure and function of CtsK. Structurally, HS forms a highly stable complex with CtsK and induces its dimerization. Co-crystal structures of CtsK with bound HS oligosaccharides reveal the location of the HS binding site and suggest how HS may support dimerization. Functionally, HS plays a dual role in regulating the enzymatic activity of CtsK. While it preserves the peptidase activity of CtsK by stabilizing its active conformation, it inhibits the collagenase activity of CtsK in a sulfation level-dependent manner. These opposing effects can be explained by our finding that the HS binding site is remote from the active site, which allows HS to specifically inhibit the collagenase activity without affecting the peptidase activity. At last, we show that structurally defined HS oligosaccharides effectively block osteoclast resorption of bone in vitro without inhibiting osteoclast differentiation, which suggests that HS-based oligosaccharide might be explored as a new class of selective CtsK inhibitor for many diseases involving exaggerated bone resorption.

Highly potent inhibitors of cathepsin K with a differently positioned cyanohydrazide warhead: structural analysis of binding mode to mature and zymogen-like enzymes.

Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-ß-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.

Pharmacoinformatics and hypothetical studies on allicin, curcumin, and gingerol as potential candidates against COVID-19-associated proteases.

Medicinal plants have been known to provide the essential raw material for the majority of antiviral drugs. This study demonstrated the putative inhibitory potential of curcumin, allicin, and gingerol towards cathepsin K, COVID-19 main protease, and SARS-CoV 3 C-like protease. The pharmacokinetic properties were predicted through the SwissADME server while the corresponding binding affinity of the selected phytocompounds towards the proteins was computed using PyRx-Python Prescription 0.8 and the binding free energy were computed based on conventional molecular dynamics using LARMD server. The ADMET properties revealed all the drugs possess drug-like properties. Curcumin has the highest binding affinities with all the selected proteases while allicin has the lowest binding affinities towards the proteases. Moreover, it was observed that curcumin exhibited the highest binding free energy of -17.90 ± 0.23,  -18.21 ± 0.25, and -9.67 ± 0.08 kcal/mol for Cathepsin K, COVID-19 main protease, and SARS-CoV 3 C-like protease, respectively. Based on the activities of the phytocompounds against coronavirus target proteases involved in the viral entry as evident from the results, the study, therefore, suggests that these phytocompounds could be valuable for the development of drugs useful for the prevention of coronavirus entry and replication.Communicated by Ramaswamy H. Sarma.

Cathepsin K Regulates Intraocular Pressure by Modulating Extracellular Matrix Remodeling and Actin-Bundling in the Trabecular Meshwork Outflow Pathway.

The homeostasis of extracellular matrix (ECM) and actin dynamics in the trabecular meshwork (TM) outflow pathway plays a critical role in intraocular pressure (IOP) regulation. We studied the role of cathepsin K (CTSK), a lysosomal cysteine protease and a potent collagenase, on ECM modulation and actin cytoskeleton rearrangements in the TM outflow pathway and the regulation of IOP. Initially, we found that CTSK was negatively regulated by pathological stressors known to elevate IOP. Further, inactivating CTSK using balicatib, a pharmacological cell-permeable inhibitor of CTSK, resulted in IOP elevation due to increased levels and excessive deposition of ECM-like collagen-1A in the TM outflow pathway. The loss of CTSK activity resulted in actin-bundling via fascin and vinculin reorganization and by inhibiting actin depolymerization via phospho-cofilin. Contrarily, constitutive expression of CTSK decreased ECM and increased actin depolymerization by decreasing phospho-cofilin, negatively regulated the availability of active TGFß2, and reduced the levels of alpha-smooth muscle actin (αSMA), indicating an antifibrotic action of CTSK. In conclusion, these observations, for the first time, demonstrate the significance of CTSK in IOP regulation by maintaining the ECM homeostasis and actin cytoskeleton-mediated contractile properties of the TM outflow pathway.

An Activity-Based Probe for Cathepsin K Imaging with Excellent Potency and Selectivity.

The cysteine protease cathepsin K is a target for the treatment of diseases associated with high bone turnover. Cathepsin K is mainly expressed in osteoclasts and responsible for the destruction of the proteinaceous components of the bone matrix. We designed various fluorescent activity-based probes (ABPs) and their precursors that bind to and inactivate cathepsin K. ABP 25 exhibited extraordinary potency (kinac/Ki = 35,300 M-1s-1) and selectivity for human cathepsin K. Crystal structures of cathepsin K in complex with ABP 25 and its nonfluorescent precursor 21 were determined to characterize the binding mode of this new type of acrylamide-based Michael acceptor with the particular orientation of the dibenzylamine moiety to the primed subsite region. The cyanine-5 containing probe 25 allowed for sensitive detection of cathepsin K, selective visualization in complex proteomes, and live cell imaging of a human osteosarcoma cell line, underlining its applicability in a pathophysiological environment.

Synthesis and kinetic characterization of hyperbolic inhibitors of human cathepsins K and S based on a succinimide scaffold.

Cathepsins K and S are closely related papain-like cysteine peptidases and potential therapeutic targets for metabolic and inflammatory diseases such as osteoporosis and arthritis. Here we describe the reduction of a previously characterized succinimide (2,5-dioxopyrrolidine)-containing hyperbolic inhibitor of cathepsin K (methyl (RS)-N-[1-(4-methoxyphenyl)-2,5-dioxopyrrolidin-3-yl]glycinate), to obtain a better and more selective compound (compound 4a - methyl (2,5-dioxopyrrolidin-3-yl)glycinate), which acted as a hyperbolic mixed inhibitor/activator similar to already known allosteric effectors of cathepsin K. We then investigated the potential of the succinimide scaffold as inhibitors of cathepsins K and/or S and synthesized a library of such compounds by 1,4-addition of α-amino acid esters and related compounds to N-substituted maleimides. From the generated library, we identified the first small molecule hyperbolic inhibitors of cathepsin S (methyl ((R)-2,5-dioxopyrrolidin-3-yl)-l-threoninate (compound R-4c) and 3-{[(1S,2R,3'S)-2-hydroxycyclohexyl]amino}pyrrolidine-2,5-dione (compound (1S,2R,3'S-10)). The former acted via a similar mechanism to compound 4a, while the latter was a hyperbolic specific inhibitor of cathepsin S. Given the versatility of the scaffold, the identified compounds will be used as the basis for the development of high-affinity hyperbolic inhibitors of the individual peptidases and to explore the potential of hyperbolic inhibitors for the inhibition of cysteine cathepsins in in vitro models.

From Pathogenesis to Therapy in Knee Osteoarthritis: Bench-to-Bedside.

Osteoarthritis (OA) is currently the most widespread musculoskeletal condition and primarily affects weight-bearing joints such as the knees and hips. Importantly, knee OA remains a multifactorial whole-joint disease, the appearance and progression of which involves the alteration of articular cartilage as well as the synovium, subchondral bone, ligaments, and muscles through intricate pathomechanisms. Whereas it was initially depicted as a predominantly aging-related and mechanically driven condition given its clear association with old age, high body mass index (BMI), and joint malalignment, more recent research identified and described a plethora of further factors contributing to knee OA pathogenesis. However, the pathogenic intricacies between the molecular pathways involved in OA prompted the study of certain drugs for more than one therapeutic target (amelioration of cartilage and bone changes, and synovial inflammation). Most clinical studies regarding knee OA focus mainly on improvement in pain and joint function and thus do not provide sufficient evidence on the possible disease-modifying properties of the tested drugs. Currently, there is an unmet need for further research regarding OA pathogenesis as well as the introduction and exhaustive testing of potential disease-modifying pharmacotherapies in order to structure an effective treatment plan for these patients.

Cathepsin K inhibitors based on 2-amino-1,3,4-oxadiazole derivatives.

Two new series of hitherto unknown dipeptides, containing an electrophilic nitrile or a non-electrophilic 2-amino-1,3,4-oxadiazole moiety were synthesized and evaluated in vitro as Cathepsin K (Cat K) inhibitors. From 14 compounds obtained, the oxadiazole derivatives 10a, 10b, 10e, and 10g acted as enzymatic competitive inhibitors with Ki values between 2.13 and 7.33 µM. Molecular docking calculations were carried out and demonstrated that all inhibitors performed hydrogen bonds with residues from the enzyme active site, such as Asn18. The best inhibitors (10a, 10b, 10g) could also perform these bonds with Cys25, and 10a showed the most stabilizing interaction energy (-134.36 kcal mol-1) with the active cavity. For the first time, derivatives based in 2-amino-1,3,4-oxadiazole scaffolds were evaluated, and the results suggested that this core displays a remarkable potential as a building block for Cat K inhibitors.

1,2,4-Thiadiazole acyclic nucleoside phosphonates as inhibitors of cysteine dependent enzymes cathepsin K and GSK-3ß.

In analogy to antiviral acyclic nucleoside phosphonates, a series of 5-amino-3-oxo-1,2,4-thiadiazol-3(2H)-ones bearing a 2-phosphonomethoxyethyl (PME) or 3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) group at the position 2 of the heterocyclic moiety has been synthesized. Diisopropyl esters of PME- and HPMP-amines have been converted to the N-substituted ureas and then reacted with benzoyl, ethoxycarbonyl, and Fmoc isothiocyanates to give the corresponding thiobiurets, which were oxidatively cyclized to diisopropyl esters of 5-amino-3-oxo-2-PME- or 2-HPMP- 1,2,4-thiadiazol-3(2H)-ones. The phosphonate ester groups were cleaved with bromotrimethylsilane, yielding N5-protected phosphonic acids. The subsequent attempts to remove the protecting group from N5 under alkaline conditions resulted in the cleavage of the 1,2,4-thiadiazole ring. Similarly, compounds with a previously unprotected 5-amino-1,2,4-thiadiazolone base moiety were stable only in the form of phosphonate esters. The series of twenty-one newly prepared 1,2,4-thiadiazol-3(2H)-ones were explored as potential inhibitors of cysteine-dependent enzymes - human cathepsin K (CatK) and glycogen synthase kinase 3ß (GSK-3ß). Several compounds exhibited an inhibitory activity toward both enzymes in the low micromolar range. The inhibitory potency of some of them toward GSK-3ß was similar to that of the thiadiazole GSK-3ß inhibitor tideglusib, whereas others exhibited more favorable toxicity profile while retaining good inhibitory activity.

Inhibitory effect of cathepsin K inhibitor (ODN-MK-0822) on invasion, migration and adhesion of human breast cancer cells in vitro.

Approximately 90% of patients with advanced breast cancer develop bone metastases; an event that results in severe decrease of quality of life and a drastic deterioration in prognosis. Therefore, to increase the survival of breast cancer patients, the development of new therapeutic strategies to impair metastatic process and skeletal complications is critical. Previous studies on the role of cathepsin K (CTSK) in metastatic spreading led to several strategies for inhibition of this molecule such as MIV-711 (Medivir), balicatib and odanacatib (ODN) which were on trial in the past. The present study intended to assess the anti-metastatic efficacy of ODN in breast cancer cells. Human breast cancer cell lines MDA-MB-231 were treated with different concentrations of ODN and performed invasion, adhesion and migration assays and, RT-PCR and western blot to evaluate the effect of ODN on the metastatic potential of breast cancer cells. ODN markedly decreased wound healing cell migration, invasion and adhesion at a dose dependent manner. ODN inhibits cell invasion by decreasing the matrix metalloproteinase (MMP-9) with the upregulation of TIMP-1 expression. ODN effectively inhibited the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal Kinase (JNK), and blocked the expression of ß-integrins and FAK proteins. ODN also significantly inhibited PI3K downstream targets Rac1, Cdc42, paxillin and Src which are critical for cell adhesion, migration and cytoskeletal reorganization. ODN exerts anti-metastatic action through inhibition of signaling pathway for MMP-9, PI3K and MAPK. This indicates potential therapeutic effects of ODN in the treatment of metastatic breast cancer.

Preclinical evaluation of [11 C]L-235 as a radioligand for Positron Emission Tomography cathepsin K imaging in bone.

The cathepsin K (CatK) enzyme is abundantly expressed in osteoclasts, and CatK inhibitors have been developed for the treatment of osteoporosis. In our effort to support discovery and clinical evaluations of a CatK inhibitor, we sought to discover a radioligand to determine target engagement of the enzyme by therapeutic candidates using positron emission tomography (PET). L-235, a potent and selective CatK inhibitor, was labeled with carbon-11. PET imaging studies recording baseline distribution of [11 C]L-235, and chase and blocking studies using the selective CatK inhibitor MK-0674 were performed in juvenile and adult nonhuman primates (NHP) and ovariectomized rabbits. Retention of the PET tracer in regions expected to be osteoclast-rich compared with osteoclast-poor regions was examined. Increased retention of the radioligand was observed in osteoclast-rich regions of juvenile rabbits and NHP but not in the adult monkey or adult ovariectomized rabbit. Target engagement of CatK was observed in blocking studies with MK-0674, and the radioligand retention was shown to be sensitive to the level of MK-0674 exposure. [11 C]L-235 can assess target engagement of CatK in bone only in juvenile animals. [11 C]L-235 may be a useful tool for guiding the discovery of CatK inhibitors.

Inhibition of Cathepsin K Alleviates Autophagy-Related Inflammation in Periodontitis-Aggravating Arthritis.

Rheumatoid arthritis (RA) and periodontitis share many epidemiological and pathological features, with emerging studies reporting a relationship between the two diseases. Recently, RA and periodontitis have been associated with autophagy. In the present study, we investigated the effects of cathepsin K (CtsK) inhibition on RA with periodontitis in a mouse model and its immunological function affecting autophagy. To topically inhibit CtsK periodontitis with arthritis in the animal model, adeno-associated virus (AAV) transfection was performed in periodontal and knee joint regions. Transfection of small interfering RNA (siRNA) was performed to inhibit CtsK in RAW264.7 cells. The effects of CtsK inhibition on the autophagy pathway were then evaluated in both in vivo and in vitro experiments. RA and periodontitis aggravated destruction and inflammation in their respective lesion areas. Inhibition of CtsK had multiple effects: (i) reduced destruction of alveolar bone and articular tissue, (ii) decreased macrophage numbers and inflammatory cytokine expression in the synovium, and (iii) alleviated expression of the autophagy-related transcription factor EB (TFEB) and microtubule-associated protein 1A/1B-light chain 3 (LC3) at the protein level in knee joints. Inhibition of CtsK in vitro reduced the expression of autophagy-related proteins and related inflammatory factors. Our data revealed that the inhibition of CtsK resisted the destruction of articular tissues and relieved inflammation from RA with periodontitis. Furthermore, CtsK was implicated as an imperative regulator of the autophagy pathway in RA and macrophages.

A phase 1 pooled PK/PD analysis of bone resorption biomarkers for odanacatib, a Cathepsin K inhibitor.

To develop a framework for evaluating the resorption effects of Cathepsin K (CatK) inhibitors and to inform dose regimen selection, a pharmacokinetic/pharmacodynamic (PK/PD) model for odanacatib (ODN) was developed based upon data from Phase 1 studies. Pooled PK/PD data from 11 studies (N = 249) were fit reasonably to a population inhibitory sigmoid Emax model. Body weight on E0 (baseline uNTx/Cr, urinary N-terminal telopeptide normalized by creatinine) and age on Emax (fractional inhibition of the biomarker response) were significant covariates for biomarker response. Simulations of typical osteoporosis patients (by age, sex and weight) indicated minimal differences between sexes in concentration-uNTx/Cr relationship. There was no evidence that regimen (daily vs. weekly dosing) influenced the PK/PD relationship of resorption inhibition for odanacatib. PK/PD models based on data from odanacatib (ODN) Phase 1 studies demonstrated that uNTx/Cr was an appropriate bone resorption biomarker for assessment of the effects of a CatK inhibitor. The models also identified the determinants of response in the PK/PD relationship for ODN (body weight on E0 and age on Emax).

Green asymmetric synthesis of epoxypeptidomimetics and evaluation as human cathepsin K inhibitors.

Cathepsin K (CatK) is a cysteine protease known for its potent collagenolytic activity, being recognized as an important target to the development of therapies for the treatment of bone disorders. Epoxypeptidomimetics have been reported as potent inhibitors of cathepsins, thus in this work we present a green synthesis of new peptidomimetics by using a one-pot asymmetric epoxidation/Ugi multicomponent reaction. The compounds were evaluated against CatK showing selectivity when compared with cathepsin L, with an inhibition profile in the low micromolar IC50 range. Investigation of the mechanism of action carried out for compounds LSPN428 and LSPN694 suggested a mixed inhibition mode and docking studies allowed a better understanding about interactions of inhibitors with the enzyme.

Population Pharmacokinetic Analysis of the Cathepsin K Inhibitor Odanacatib: Insights Into Intrinsic and Extrinsic Factor Effects on Exposure in Postmenopausal and Elderly Women.

This analysis developed a population pharmacokinetic (PK) model for odanacatib, characterized demographic and concomitant medication covariates effect, and provided odanacatib exposure estimates for subjects in phase 2/3 studies. Data from multiple phase 1 (P005, P025, and P014), phase 2b (P004 and P022), and phase 3 (Long-Term Odanacatib Fracture Trial; P018) studies were pooled to create a data set of 1280 postmenopausal women aged 45 to 91 years (102 from phase 1, 514 from phase 2b, and 664 from phase 3) who received weekly oral odanacatib doses ranging from 3 to 100 mg. A 1-compartment model with first-order absorption, dose-dependent relative bioavailability (F1), and first-order elimination best described odanacatib PK. F1 decreased from the 100% reference bioavailability for a 3-mg oral dose to 24.5% for a 100-mg dose. Eight statistically significant covariates were included in the final PK model: body weight, age, race, and concomitant cytochrome P450 (CYP)3A inhibitors on apparent clearance; body weight on apparent central volume of distribution; and concomitant hydrochlorothiazide, high-fat breakfast, and a study effect on F1. All fixed- and random-effects parameters were estimated with good precision (%standard error of the mean ≤29.5%). This population PK analysis provides insights into intrinsic- and extrinsic-factor effects on odanacatib exposure in postmenopausal and elderly women with osteoporosis. The magnitude of the intrinsic-factor effects was generally modest (odanacatib exposure geometric mean ratios, 0.80-1.21) even in subjects aged >80 years, or in subsets with multiple combinations of factors.

Cathepsin K inhibition-induced mitochondrial ROS enhances sensitivity of cancer cells to anti-cancer drugs through USP27x-mediated Bim protein stabilization.

Cathepsin K (Cat K) is expressed in cancer cells, but the effect of Cat K on apoptosis is still elusive. Here, we showed that inhibition of Cat K sensitized the human carcinoma cells to anti-cancer drug through up-regulation of Bim. Inhibition of Cat K increased USP27x expression, and knock down of USP27x markedly blocked Cat K-induced up-regulation of Bim expression. Furthermore, inhibition of Cat K induced proteasome-dependent degradation of regulatory associated protein of mammalian target of rapamycin (Raptor). Down-regulation of Raptor expression increased mitochondrial ROS production, and mitochondria specific superoxide scavengers prevented USP27x-mediated stabilization of Bim by inhibition of Cat K. Moreover, combined treatment with Cat K inhibitor (odanacatib) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) reduced tumor growth and induced cell death in a xenograft model. Our results demonstrate that Cat K inhibition enhances anti-cancer drug sensitivity through USP27x-mediated the up-regulation of Bim via the down-regulation of Raptor.

Inhibition of Ctsk modulates periodontitis with arthritis via downregulation of TLR9 and autophagy.

OBJECTIVES: The mechanisms underlying the effects of Toll-like receptor 9 (TLR9) and autophagy on rheumatoid arthritis (RA)-aggravated periodontitis are unclear. We aimed to explore a novel target, cathepsin K (Ctsk)-mediated TLR9-related autophagy, during the progress of periodontitis with RA. MATERIALS AND METHODS: DBA/J1 mouse model of periodontitis with RA was created by local colonization of Porphyromonas gingivalis (Pg) and injection of collagen. The expression of Ctsk was inhibited by adeno-associated virus (AAV). Micro-CT, immunohistochemistry (IHC), Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of TLR9-related autophagy in periodontitis with RA. Small interfering RNA (siRNA) and CpG oligodeoxynucleotides (CpG ODN) were applied in macrophages. Western blot, immunofluorescence (IF) and qRT-PCR were used to verify the in vivo results. RESULTS: RA can promote periodontitis bone destruction in the lesion area, while inhibiting Ctsk could effectively alleviate this effect. The infiltration of macrophages, TLR9, autophagy proteins (TFEB and LC3) and inflammatory cytokines increased in the periodontitis-with-RA group and was reduced by the inhibition of Ctsk in the periodontal region. Macrophage stimulation confirmed the in vivo results. With the activation of TLR9 by CpG ODN, inhibition of Ctsk could suppress both TLR9 downstream signalling proteins and autophagy-related proteins. CONCLUSIONS: This study advanced a novel role for Ctsk in TLR9 and autophagy to explain the interaction between periodontitis and RA.

Disease-Modifying Effects of a Novel Cathepsin K Inhibitor in Osteoarthritis: A Randomized Controlled Trial.

Background: MIV-711 is a novel selective cathepsin K inhibitor with beneficial effects on bone and cartilage in preclinical osteoarthritis models. Objective: To evaluate the efficacy, safety, and tolerability of MIV-711 in participants with symptomatic, radiographic knee osteoarthritis. Design: 26-week randomized, double-blind, placebo-controlled phase 2a study with a 26-week open-label safety extension substudy. (EudraCT: 2015-003230-26 and 2016-001096-73). Setting: Six European sites. Participants: 244 participants with primary knee osteoarthritis, Kellgren-Lawrence grade 2 or 3, and pain score of 4 to 10 on a numerical rating scale (NRS). Intervention: MIV-711, 100 (n = 82) or 200 (n = 81) mg daily, or matched placebo (n = 77). Participants (46 who initially received 200 mg/d and 4 who received placebo) received 200 mg of MIV-711 daily during the extension substudy. Measurements: The primary outcome was change in NRS pain score. The key secondary outcome was change in bone area on magnetic resonance imaging (MRI). Other secondary end points included cartilage thickness on quantitative MRI and type I and II collagen C-telopeptide biomarkers. Outcomes were assessed over 26 weeks. Results: Changes in NRS pain scores with MIV-711 were not statistically significant (placebo, -1.4; MIV-711, 100 mg/d, -1.7; MIV-711, 200 mg/d, -1.5). MIV-711 significantly reduced medial femoral bone area progression (P = 0.002 for 100 mg/d and 0.004 for 200 mg/d) and medial femoral cartilage thinning (P = 0.023 for 100 mg/d and 0.125 for 200 mg/d) versus placebo and substantially reduced bone and cartilage biomarker levels. Nine serious adverse events occurred in 6 participants (1 in the placebo group, 3 in the 100 mg group, and 2 in the 200 mg group); none were considered to be treatment-related. Limitation: The trial was relatively short. Conclusion: MIV-711 was not more effective than placebo for pain, but it significantly reduced bone and cartilage progression with a reassuring safety profile. This treatment may merit further evaluation as a disease-modifying osteoarthritis drug. Primary Funding Source: Medivir.

Formulation of self-microemulsifying drug delivery system (SMEDDS) by D-optimal mixture design to enhance the oral bioavailability of a new cathepsin K inhibitor (HL235).

HL235 is a new cathepsin K inhibitor designed and synthesized to treat osteoporosis. Since HL235 has poor aqueous solubility, a self-microemulsifying drug delivery system (SMEDDS) was formulated to enhance its oral bioavailability. A solubility study of HL235 was performed to select a suitable oil, surfactant and cosurfactant. Pseudoternary phase diagrams were plotted to identify the microemulsion region and to determine the range of components in the isotropic mixture. D-optimal mixture design and a desirability function were introduced to optimize the SMEDDS formulation for the desired physicochemical characteristics, i.e., high drug concentration at 15 min after dilution with simulated gastric fluid (SGF) and high solubilization capacity. The optimized HL235-loaded SMEDDS formulation consisted of 5.0% Capmul MCM EP (oil), 75.0% Tween 20 (surfactant) and 20.0% Carbitol (cosurfactant). The droplet size of the microemulsion formed by the optimized formulation was 10.7 ±â€¯1.6 nm, and the droplets were spherical in shape. Pharmacokinetic studies in rats showed that the relative oral bioavailability of the SMEDDS formulation increased up to 3.22-fold compared to its solution in DMSO:PEG400 (8:92, v/v). Thus, the formulation of SMEDDS optimized by D-optimal mixture design could be a promising approach to improve the oral bioavailability of HL235.