Kartogenin Induces Chondrogenesis in Cartilage Progenitor Cells and Attenuates Cell Hypertrophy in Marrow-Derived Stromal Cells.

INTRODUCTION: Kartogenin (KGN) is a synthetic small molecule that stimulates chondrogenic cellular differentiation by activating smad-4/5 pathways. KGN has been proposed as a feasible alternative to expensive biologic growth factors, such as transforming growth factor ß, which remain under strict regulatory scrutiny when it comes to use in patients. METHOD: This study reports the previously unexplored effects of KGN stimulation on cartilage- derived mesenchymal progenitor cells (CPCs), which have been shown to be effective in applications of cell-based musculoskeletal tissue regeneration. Our findings demonstrate that KGN treatment significantly increased markers of chondrogenesis, SOX9 and COL2 following 3-10 days of treatment in human CPCs. RESULT: KGN treatment also resulted in a significant dose-dependent increase in GAG production in CPCs. The same efficacy was not observed in human marrow-derived stromal cells (BM-MSCs); however, KGN significantly reduced mRNA expression of cell hypertrophy markers, COL10 and MMP13, in BM-MSCs. Parallel to these mRNA expression results, KGN led to a significant decrease in protein levels of MMP-13 both at 0-5 days and 5-10 days following KGN treatment. CONCLUSION: In conclusion, this study demonstrates that KGN can boost the chondrogenicity of CPCs and inhibit hypertrophic terminal differentiation of BM-MSCs.

Suppressing Chondrocyte Hypertrophy to Build Better Cartilage.

Current clinical strategies for restoring cartilage defects do not adequately consider taking the necessary steps to prevent the formation of hypertrophic tissue at injury sites. Chondrocyte hypertrophy inevitably causes both macroscopic and microscopic level changes in cartilage, resulting in adverse long-term outcomes following attempted restoration. Repairing/restoring articular cartilage while minimizing the risk of hypertrophic neo tissue formation represents an unmet clinical challenge. Previous investigations have extensively identified and characterized the biological mechanisms that regulate cartilage hypertrophy with preclinical studies now beginning to leverage this knowledge to help build better cartilage. In this comprehensive article, we will provide a summary of these biological mechanisms and systematically review the most cutting-edge strategies for circumventing this pathological hallmark of osteoarthritis.

Stable human cartilage progenitor cell line stimulates healing of meniscal tears and attenuates post-traumatic osteoarthritis.

Meniscal tearing in the knee increases the risk of post-traumatic osteoarthritis (OA) in patients. The therapeutic application of tissue-specific mesenchymal progenitor cells is currently being investigated as an emerging biologic strategy to help improve healing of musculoskeletal tissues like meniscal fibrocartilage and articular hyaline cartilage. However, many of these approaches involve isolating cells from healthy tissues, and the low yield of rare progenitor populations (< 1% of total cells residing in tissues) can make finding a readily available cell source for therapeutic use a significant logistical challenge. In the present study, we investigated the therapeutic efficacy of using expanded cartilage-derived and bone marrow-derived progenitor cell lines, which were stabilized using retroviral SV40, for repair of meniscus injury in a rodent model. Our findings indicate that these cell lines express the same cell surface marker phenotype of primary cells (CD54+, CD90+, CD105+, CD166+), and that they exhibit improved proliferative capacity that is suitable for extensive expansion. Skeletally mature male athymic rats treated with 3.2 million cartilage-derived progenitor cell line exhibited approximately 79% greater meniscal tear reintegration/healing, compared to injured animals that left untreated, and 76% greater compared to animals treated with the same number of marrow-derived stromal cells. Histological analysis of articular surfaces also showed that cartilage-derived progenitor cell line treated animals exhibited reduced post-traumatic OA associated articular cartilage degeneration. Stable cell line treatment did not cause tumor formation or off-target engraftment in animals. Taken together, we present a proof-of-concept study demonstrating, for the first time, that intra-articular injection of a stable human cartilage-derived progenitor cell line stimulates meniscus tear healing and provide chondroprotection in an animal model. These outcomes suggest that the use of stable cell lines may help overcome cell source limitations for cell-based medicine.

Discovery and Evaluation of Entry Inhibitors for SARS-CoV-2 and Its Emerging Variants.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 19 (COVID-19) pandemic. Despite unprecedented research and developmental efforts, SARS-CoV-2-specific antivirals are still unavailable for the treatment of COVID-19. In most instances, SARS-CoV-2 infection initiates with the binding of Spike glycoprotein to the host cell ACE2 receptor. Utilizing the crystal structure of the ACE2/Spike receptor-binding domain (S-RBD) complex (PDB file 6M0J) in a computer-aided drug design approach, we identified and validated five potential inhibitors of S-RBD and ACE-2 interaction. Two of the five compounds, MU-UNMC-1 and MU-UNMC-2, blocked the entry of pseudovirus particles expressing SARS-CoV-2 Spike glycoprotein. In live SARS-CoV-2 infection assays, both compounds showed antiviral activity with IC50 values in the micromolar range (MU-UNMC-1: IC50 = 0.67 µM and MU-UNMC-2: IC50 = 1.72 µM) in human bronchial epithelial cells. Furthermore, MU-UNMC-1 and MU-UNMC-2 effectively blocked the replication of rapidly transmitting variants of concern: South African variant B.1.351 (IC50 = 9.27 and 3.00 µM) and Scotland variant B.1.222 (IC50 = 2.64 and 1.39 µM), respectively. Following these assays, we conducted "induced-fit (flexible) docking" to understand the binding mode of MU-UNMC-1/MU-UNMC-2 at the S-RBD/ACE2 interface. Our data showed that mutation N501Y (present in B.1.351 variant) alters the binding mode of MU-UNMC-2 such that it is partially exposed to the solvent and has reduced polar contacts. Finally, MU-UNMC-2 displayed high synergy with remdesivir, the only approved drug for treating hospitalized COVID-19 patients. IMPORTANCE The ongoing coronavirus infectious disease 2019 (COVID-19) pandemic is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). More than 207 million people have been infected globally, and 4.3 million have died due to this viral outbreak. While a few vaccines have been deployed, a SARS-CoV-2-specific antiviral for the treatment of COVID-19 is yet to be approved. As the interaction of SARS-CoV-2 Spike protein with ACE2 is critical for cellular entry, using a combination of a computer-aided drug design (CADD) approach and cell-based in vitro assays, we report the identification of five potential SARS-CoV-2 entry inhibitors. Out of the five, two compounds (MU-UNMC-1 and MU-UNMC-2) have antiviral activity against ancestral SARS-CoV-2 and emerging variants from South Africa and Scotland. Furthermore, MU-UNMC-2 acts synergistically with remdesivir (RDV), suggesting that RDV and MU-UNMC-2 can be developed as a combination therapy to treat COVID-19 patients.

Diversity in heat shock protein families: functional implications in virus infection with a comprehensive insight of their role in the HIV-1 life cycle.

Heat shock proteins (HSPs) are a group of cellular proteins that are induced during stress conditions such as heat stress, cold shock, UV irradiation and even pathogenic insult. They are classified into families based on molecular size like HSP27, 40, 70 and 90 etc, and many of them act as cellular chaperones that regulate protein folding and determine the fate of mis-folded or unfolded proteins. Studies have also shown multiple other functions of these proteins such as in cell signalling, transcription and immune response. Deregulation of these proteins leads to devastating consequences, such as cancer, Alzheimer's disease and other life threatening diseases suggesting their potential importance in life processes. HSPs exist in multiple isoforms, and their biochemical and functional characterization still remains a subject of active investigation. In case of viral infections, several HSP isoforms have been documented to play important roles with few showing pro-viral activity whereas others seem to have an anti-viral role. Earlier studies have demonstrated that HSP40 plays a pro-viral role whereas HSP70 inhibits HIV-1 replication; however, clear isoform-specific functional roles remain to be established. A detailed functional characterization of all the HSP isoforms will uncover their role in cellular homeostasis and also may highlight some of them as potential targets for therapeutic strategies against various viral infections. In this review, we have tried to comprehend the details about cellular HSPs and their isoforms, their role in cellular physiology and their isoform-specific functions in case of virus infection with a specific focus on HIV-1 biology.

Discovery and in-vitro evaluation of potent SARS-CoV-2 entry inhibitors.

SARS-CoV-2 infection initiates with the attachment of spike protein to the ACE2 receptor. While vaccines have been developed, no SARS-CoV-2 specific small molecule inhibitors have been approved. Herein, utilizing the crystal structure of the ACE2/Spike receptor binding domain (S-RBD) complex in computer-aided drug design (CADD) approach, we docked ∼8 million compounds within the pockets residing at S-RBD/ACE2 interface. Five best hits depending on the docking score, were selected and tested for their in vitro efficacy to block SARS-CoV-2 replication. Of these, two compounds (MU-UNMC-1 and MU-UNMC-2) blocked SARS-CoV-2 replication at sub-micromolar IC 50 in human bronchial epithelial cells (UNCN1T) and Vero cells. Furthermore, MU-UNMC-2 was highly potent in blocking the virus entry by using pseudoviral particles expressing SARS-CoV-2 spike. Finally, we found that MU-UNMC-2 is highly synergistic with remdesivir (RDV), suggesting that minimal amounts are needed when used in combination with RDV, and has the potential to develop as a potential entry inhibitor for COVID-19.

Post-Traumatic Osteoarthritis Assessment in Emerging and Advanced Pre-Clinical Meniscus Repair Strategies: A Review.

Surgical repair of meniscus injury is intended to help alleviate pain, prevent further exacerbation of the injury, restore normal knee function, and inhibit the accelerated development of post-traumatic osteoarthritis (PTOA). Meniscus injuries that are treated poorly or left untreated are reported to significantly increase the risk of PTOA in patients. Current surgical approaches for the treatment of meniscus injuries do not eliminate the risk of accelerated PTOA development. Through recent efforts by scientists to develop innovative and more effective meniscus repair strategies, the use of biologics, allografts, and scaffolds have come into the forefront in pre-clinical investigations. However, gauging the extent to which these (and other) approaches inhibit the development of PTOA in the knee joint is often overlooked, yet an important consideration for determining the overall efficacy of potential treatments. In this review, we catalog recent advancements in pre-clinical therapies for meniscus injuries and discuss the assessment methodologies that are used for gauging the success of these treatments based on their effect on PTOA severity. Methodologies include histopathological evaluation of cartilage, radiographic evaluation of the knee, analysis of knee function, and quantification of OA predictive biomarkers. Lastly, we analyze the prevalence of these methodologies using a systemic PubMed® search for original scientific journal articles published in the last 3-years. We indexed 37 meniscus repair/replacement studies conducted in live animal models. Overall, our findings show that approximately 75% of these studies have performed at least one assessment for PTOA following meniscus injury repair. Out of this, 84% studies have reported an improvement in PTOA resulting from treatment.

Drug Repurposing Approaches to Combating Viral Infections.

Development of novel antiviral molecules from the beginning costs an average of $350 million to $2 billion per drug, and the journey from the laboratory to the clinic takes about 10-15 years. Utilization of drug repurposing approaches has generated substantial interest in order to overcome these drawbacks. A drastic reduction in the failure rate, which otherwise is ~92%, is achieved with the drug repurposing approach. The recent exploration of the drug repurposing approach to combat the COVID-19 pandemic has further validated the fact that it is more beneficial to reinvestigate the in-practice drugs for a new application instead of designing novel drugs. The first successful example of drug repurposing is zidovudine (AZT), which was developed as an anti-cancer agent in the 1960s and was later approved by the US FDA as an anti-HIV therapeutic drug in the late 1980s after fast track clinical trials. Since that time, the drug repurposing approach has been successfully utilized to develop effective therapeutic strategies against a plethora of diseases. Hence, an extensive application of the drug repurposing approach will not only help to fight the current pandemics more efficiently but also predict and prepare for newly emerging viral infections. In this review, we discuss in detail the drug repurposing approach and its advancements related to viral infections such as Human Immunodeficiency Virus (HIV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

The barley lectin, horcolin, binds high-mannose glycans in a multivalent fashion, enabling high-affinity, specific inhibition of cellular HIV infection.

N-Linked glycans are critical to the infection cycle of HIV, and most neutralizing antibodies target the high-mannose glycans found on the surface envelope glycoprotein-120 (gp120). Carbohydrate-binding proteins, particularly mannose-binding lectins, have also been shown to bind these glycans. Despite their therapeutic potency, their ability to cause lymphocyte proliferation limits their application. In this study, we report one such lectin named horcolin (Hordeum vulgare lectin), seen to lack mitogenicity owing to the divergence in the residues at its carbohydrate-binding sites, which makes it a promising candidate for exploration as an anti-HIV agent. Extensive isothermal titration calorimetry experiments reveal that the lectin was sensitive to the length and branching of mannooligosaccharides and thereby the total valency. Modeling and simulation studies demonstrate two distinct modes of binding, a monovalent binding to shorter saccharides and a bivalent mode for higher glycans, involving simultaneous interactions of multiple glycan arms with the primary carbohydrate-binding sites. This multivalent mode of binding was further strengthened by interactions of core mannosyl residues with a secondary conserved site on the protein, leading to an exponential increase in affinity. Finally, we confirmed the interaction of horcolin with recombinant gp120 and gp140 with high affinity and inhibition of HIV infection at nanomolar concentrations without mitogenicity.

N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) inhibits HIV-1 by suppressing the activity of viral protease.

Human Immunodeficiency Virus (HIV), the etiological agent for Acquired Immunodeficiency Syndrome (AIDS), continues to kill humans despite stupendous advances in antiviral research. With the presently available combination antiretroviral therapeutic arsenal, AIDS is now a manageable disease but with no cure available till date. The development of novel antivirals consumes an extensive amount of time and resources. Hence, repurposing of the established gold standard molecules for their anti-HIV application is enormously advantageous. In this study, we report that N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) inhibits HIV-1 replication in a highly-conserved manner. Further, TPCK inhibits HIV-1 replication at the late stages of its life cycle by impeding viral protease (PR) enzyme activity. Additionally, our results demonstrate that the combination of TPCK with established HIV-1 PR inhibitors shows significant synergistic inhibitory potential, suggesting the potential use of TPCK in cART regimen. Collectively, we report the anti-HIV activity of TPCK, which should be further characterized for its translational applications.

Recent Advances in the Development of Integrase Inhibitors for HIV Treatment.

PURPOSE OF THE REVIEW: The complex multistep life cycle of HIV allows it to proliferate within the host and integrate its genome in to the host chromosomal DNA. This provirus can remain dormant for an indefinite period. The process of integration, governed by integrase (IN), is highly conserved across the Retroviridae family. Hence, targeting integration is not only expected to block HIV replication but may also reveal new therapeutic strategies to treat HIV as well as other retrovirus infections. RECENT FINDINGS: HIV integrase (IN) has gained attention as the most promising therapeutic target as there are no equivalent homologues of IN that has been discovered in humans. Although current nano-formulated long-acting IN inhibitors have demonstrated the phenomenal ability to block HIV integration and replication with extraordinary half-life, they also have certain limitations. In this review, we have summarized the current literature on clinically established IN inhibitors, their mechanism of action, the advantages and disadvantages associated with their therapeutic application, and finally current HIV cure strategies using these inhibitors.

A novel isothiocyanate derivative inhibits HIV-1 gene expression and replication by modulating the nuclear matrix associated protein SMAR1.

The essential role of SMAR1 in HIV-1 transcription and LTR driven gene expression suggests SMAR1 as an HIV dependency factor (HDF) and a potential anti-HIV therapeutic target. Here, we report for the first time, anti-HIV activity of 8 novel isothiocyanate (ITC) derivatives that differentially stabilise SMAR1. Out of 8 novel ITC derivatives, SCS-OCL-381 was observed to inhibit HIV-1 replication most significantly at the noncytotoxic concentration in reporter T-cell line, CEM-GFP. Further, the highly conserved anti-HIV activity of SCS-OCL-381 is a cell type, virus isolate and viral load independent phenomena and is approximately 3 fold more effective than the representative ITC, Sulforaphane (SFN). Further, SCS-OCL-381 does not hamper the activity of viral enzymes reverse transcriptase, integrase and protease. Mechanistically, SCS-OCL-381 stabilises SMAR1 which, otherwise undergoes proteasomal degradation upon HIV-1 infection in T-cells. This stabilisation results in the recruitment of repressor complex on HIV-1 LTR resulting in repression of LTR mediated transcription and gene expression. These inhibitory consequences were further confirmed by reporter based LTR activity assays in different cell lines. Taken together, these findings highlight the anti-HIV potential of novel ITC derivatives by the stabilisation of SMAR1 and strongly support further in vivo characterisation and potential translational applications of SCS-OCL-381.

Discovery of 2-isoxazol-3-yl-acetamide analogues as heat shock protein 90 (HSP90) inhibitors with significant anti-HIV activity.

The recent burst of explorations on heat shock protein 90 (HSP90) in virus research supports its emergence as a promising target to overcome the drawbacks of current antiviral therapeutic regimen. In continuation of our efforts towards the discovery of novel anti-retroviral molecules, we designed, synthesized fifteen novels 2-isoxazol-3-yl-acetamide based compounds (2a-o) followed by analysis of their anti-HIV activity and cytotoxicity studies. 2a-b, 2e, 2j, and 2l-m were found to be active with inhibitory potentials >80% at their highest non-cytotoxic concentration (HNC). Further characterization of anti-HIV activity of these molecules suggests that 2l has ∼3.5 fold better therapeutic index than AUY922, the second generation HSP90 inhibitor. The anti-HIV activity of 2l is a cell type, virus isolate and viral load independent phenomena. Interestingly, 2l does not significantly modulate viral enzymes like Reverse Transcriptase (RT), Integrase (IN) and Protease (PR) as compared to their known inhibitors in a cell free in vitro assay system at its HNC. Further, 2l mediated inhibition of HSP90 attenuates HIV-1 LTR driven gene expression. Taken together, structural rationale, modeling studies and characterization of biological activities suggest that this novel scaffold can attenuate HIV-1 replication significantly within the host and thus opens a new horizon to develop novel anti-HIV therapeutic candidates.

Prediction of Acute-Phase Treatment Outcomes by Adding a Single-Item Measure of Activity Impairment to Symptom Measurement: Development and Validation of an Interactive Calculator from the STAR*D and CO-MED Trials.

BACKGROUND: Day-to-day functioning is impaired in major depressive disorder. Yet there are no guidelines to systematically assess these functional changes. This report evaluates prognostic utility of changes in activity impairment to inform clinical decision-making at an individual level. METHODS: Mixed model analyses tested changes in activity impairment (sixth item of Work and Activity Impairment scale, rated 0-10) at mid-point (week 6) and end of step 1 (weeks 12-14) in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) trial (n = 2697) after controlling for depression severity [Quick Inventory of Depressive Symptomatology Self-Report (QIDS-SR)]. Interactive calculators for end of step 1 remission (QIDS-SR ≤5) and no meaningful benefit (<30% QIDS-SR reduction from baseline) were developed for participants with complete data (n = 1476) and independently replicated in the Combining Medications to Enhance Depression Outcomes trial (n = 399). RESULTS: Activity impairment improved independently with acute-phase treatment in STAR*D (F = 7.27; df = 2,2625; P < .001). Baseline to mid-point activity impairment change significantly predicted remission (P < .001, model area under the curve = 0.823) and no meaningful benefit (P < .001, area under the curve = 0.821) in the STAR*D trial. Adding activity impairment variables to depression severity measures correctly reclassified 28.4% and 15.8% remitters and nonremitters (net reclassification improvement analysis, P < .001), and 11.4% and 16.8% of those with no meaningful benefit and meaningful benefit (net reclassification improvement analysis, P < .001). The STAR*D trial model estimates accurately predicted remission (area under the curve = 0.80) and no meaningful benefit (area under the curve = 0.82) in the Combining Medications to Enhance Depression Outcomes trial and was used to develop an interactive calculator. CONCLUSION: A single-item self-report measure of activity impairment changes independently with antidepressant treatment. Baseline to week 6 changes in activity impairment and depression severity can be combined to predict acute-phase remission and no meaningful benefit at an individual level.

Design, synthesis, docking studies and biological screening of 2-thiazolyl substituted -2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazines as potent HIV-1 reverse transcriptase inhibitors.

1,3-oxazine nucleus and thiazolyl group features prominently in many biologically important natural products as well as bioactive molecules. A series of novel 2-thiazolyl substituted-2,3-dihydro-1H-naphtho [1,2-e][1,3]oxazine derivatives were designed and synthesized based on their structure-activity relationships (SARs) from 2-naphthol, substituted thiazolyl amines and formalin through ring closure by one-pot three component reaction. These derivatives were first evaluated for their inhibitory effect on HIV-1 Reverse Transcriptase (RT) enzyme activity. Out of 14 compounds, 4 showed potent inhibition of HIV-1 RT activity at significantly low concentration. Docking studies of these molecules revealed their high affinity binding to several amino acids of HIV-1 RT which are less sensitive to point mutations. Furthermore, anti-HIV activity of these molecules was analysed in a CD4+ T cell-line, which indicates that Therapeutic Index (TI) of some of these compounds is better than Zidovudine and Efavirenz, known HIV-1 RT inhibitors. Taken together, our studies report for the first time some novel naphthoxazine derivatives with significant TI, which is through inhibition of HIV-1 RT activity.

Gene expression profiling reveals Nef induced deregulation of lipid metabolism in HIV-1 infected T cells.

Human Immunodeficiency Virus-1 (HIV-1) encodes a 27 kDa Negative Factor or Nef protein, which is increasingly proving to be a misnomer. Nef seems to be crucial for AIDS progression as individuals infected with nef-deleted strain of HIV were reported to become Long Term Non Progressors (LTNP). These findings necessitate tracing of Nef's footprint on landscape of cellular transcriptome favoring HIV-1 pathogenesis. We have tried to explore effect of Nef on cellular gene expression profile in conjunction with rest of HIV-1 proteins. Our results show that 237 genes are differentially regulated due to the presence of Nef during infection, which belong to several broad categories like "signaling", "apoptosis", "transcription" and "lipid metabolism" in gene ontology analysis. Furthermore, our results show that Nef causes disruption of lipid content in HIV-1 infected T cells. Molecular inhibitors of lipid metabolism like Atorvastatin and Ranolazine were found to have profound effect on wild type virus as compared to nef-deleted HIV-1. Thus our results suggest that interference in lipid metabolism is a potential mechanism through which Nef contributes in enhancing HIV-1 pathogenesis.

Discovery of potent, nonsystemic apical sodium-codependent bile acid transporter inhibitors (Part 1).

Elevated plasma levels of low-density lipoprotein (LDL) cholesterol are a major risk factor for atherosclerosis leading to coronary artery disease (CAD), which remains the main cause of mortality in Western society. We believe that by preventing the reabsorption of bile acids, a minimally absorbed apical sodium-codependent bile acid transporter (ASBT) inhibitor would lower the serum cholesterol without the potential systemic side effects of an absorbed drug. A series of novel benzothiepines (3R,3R'-2,3,4,5-tetrahydro-5-aryl-1-benzothiepin-4-ol 1,1-dioxides) were synthesized and tested for their ability to inhibit the apical sodium dependent bile acid transport (ASBT)-mediated uptake of [(14)C]taurocholate (TC) in H14 cells. A 3R,4R,5R/3S,4S,5S racemate was found to have greater potency than the other three possible racemates. Addition of electron-donating groups such as a dimethylamino substituent at the 7 position greatly enhanced potency, and incorporation of a long-chain quaternary ammonium substituent on the 5-phenyl ring was useful in minimizing systemic exposure of this locally active ASBT inhibitor while also increasing water solubility and maintaining potency. The reported results describe the synthesis and SAR development of this benzothiepine class of ASBT inhibitors resulting in an 6000-fold improvement in ASBT inhibition with desired minimal systemic exposure of this locally acting drug candidate.

Discovery of potent, nonsystemic apical sodium-codependent bile acid transporter inhibitors (Part 2).

In the preceding paper several compounds were reported as potent apical sodium-codependent bile acid transporter (ASBT) inhibitors. Since the primary site for active bile acid reabsorption is via ASBT, which is localized on the luminal surface of the distal ileum, we reasoned that a nonsystemic inhibitor would be desirable to minimize or eliminate potential systemic side effects of an absorbed drug. To ensure bioequivalency and product stability, it was also essential that we identify a nonhygroscopic inhibitor in its most stable crystalline form. A series of benzothiepines were prepared to refine the structure-activity relationship of the substituted phenyl ring at the 5-position of benzothiepine ring and to identify potent, crystalline, nonhygroscopic, and efficacious ASBT inhibitors with low systemic exposure.

Effect of physicochemical and formulation variables on the in vivo absorption of ABT-761.

ABT-761 is a 5-lipoxygenase inhibitor developed for the treatment of asthma. The present study was undertaken to evaluate different crystal forms of ABT-761 and their impact on in vitro and in vivo performance in capsule formulations. Two crystal forms of ABT-761, hemihydrate and non-solvate from different sources, were characterized by thermal analysis, x-ray powder diffraction, moisture sorption, and intrinsic dissolution studies. An in vitro test was performed to assess the effect of formulation and drug from different sources on drug release. Crossover design was also used to evaluate oral bioavailability of ABT-761 hemihydrate formulations in beagle dogs. Plasma concentrations of ABT-761 were analyzed using a reverse-phase high-performance liquid chromatography (HPLC) assay. It was found that in vivo oral absorption as well as in vitro dissolution of ABT-761 were influenced by different formulations. Capsule formulations of ABT-761 hemihydrate are bioequivalent to the solution formulation in terms of extent of absorption, butformulation and the method of granulation preparation can have a major impact on the absorption rate. In conclusion, a single crystal form of ABT-761, i.e., hemihydrate, is preferred for subsequent product development. However, exposure of the drug to conditions that may facilitate phase transformation should be avoided.