Inhibitor In Clinical Development Research Articles

Long-acting drugs and formulations for the treatment and prevention of HIV infection

Long-acting and extended-release formulations represent one of the most important approaches to improving the treatment and prevention of chronic HIV infection. Long-acting small molecules and monoclonal antibodies have demonstrated potent anti-HIV activity in early- and late-stage clinical trials. Strategies to manage toxicity and falling drug concentrations after missed doses, as well as primary and secondary resistance to current drugs and monoclonal antibodies are important considerations. Long-acting injectable nanoformulations of the integrase inhibitor cabotegravir and the non-nucleoside reverse transcriptase inhibitor rilpivirine were safe, well tolerated and efficacious in large randomised phase 3 studies. Regulatory approval for this two-drug combination for HIV maintenance therapy was granted in Canada in 2020 and is expected in the USA during 2021. 4'-Ethynyl-2-fluoro-2'-deoxyadenosine (islatravir) is a novel nucleoside reverse transcriptase inhibitor in clinical development as a long-acting oral drug and as a long-acting subcutaneous polymer implant. GS-6207 is a novel HIV capsid inhibitor that is injected subcutaneously every 3 months. Broadly-neutralising monoclonal antibodies have potent antiviral activity in early human trials, however there is substantial baseline resistance and rapid development of resistance to these antibodies if used as monotherapy. Limitations of these antiretroviral approaches include management of toxicities and prevention of drug resistance when these drugs are discontinued and drug concentrations are slowly reduced over time. These approaches appear to be especially attractive for patients complaining of pill fatigue and for those experiencing HIV-associated stigma. As these formulations are shown to be safe, well tolerated and economical, they are likely to gain broader appeal.

Development of Pharmacodynamic Assays to Assess Ex Vivo Masp-2 Inhibition and Their Use to Characterize the Pharmacodynamics of Narsoplimab (OMS721) in Humans and Monkeys

Introduction Narsoplimab (OMS721) is a fully human monoclonal antibody that binds to and inhibits mannan-binding lectin-associated serine protease 2 (MASP-2). MASP-2 is the key enzyme responsible for activation of the lectin pathway of the complement system. The lectin pathway is activated when plasma is exposed to molecular patterns present on microbes and injured host cell surfaces, initiating a proteolytic cascade that results in the activation of complement components C4, C2, C3 and C5 to yield multiple biologically active products. Inhibiting MASP-2 activity prevents lectin-mediated generation of complement activation products, thereby reducing inflammation and tissue injury. Narsoplimab is the first lectin pathway-specific complement inhibitor in clinical development. In published clinical studies and case reports, improvement has been observed in narsoplimab-treated patients with hematopoietic stem cell transplant-associated thrombotic microangiopathy, immunoglobulin A (IgA) nephropathy and atypical hemolytic uremic syndrome. Here we describe the development of pharmacodynamic (PD) assays to quantify MASP-2 inhibition ex vivo and their use to study duration of action and to establish the pharmacokinetic (PK)/PD relationship of narsoplimab in monkeys and humans. Methods PD assays to measure lectin pathway activity, a measure of MASP-2 activity, in minimally diluted (90%) serum from human and monkey were developed using immobilized mannan as a lectin pathway-specific complement activator with C4b as the activation endpoint. ELISA and flow cytometry assay platforms were employed to assess the PD response of monkeys or normal healthy human volunteers administered narsoplimab, respectively. The PD response was used to characterize the time-course and dose-response of lectin pathway inhibition by narsoplimab. Drug concentration data obtained from matched serum samples were used to estimate the ex vivo EC50 of lectin pathway inhibition in monkeys and humans. Lectin pathway-specific complement activation assays applicable to minimally diluted serum samples were developed by evaluating the time-course of lectin-induced complement activation response under varying reaction temperatures and selecting optimized test conditions within the dynamic range of the assay platform. The test methods were used to characterize monkey and human lectin pathway inhibition profiles of narsoplimab in vitro, and the flow cytometric method was validated for analysis of human serum samples. Results The PD assays were used to characterize the PD response of monkeys administered a single intravenous (IV) bolus injection of narsoplimab at 0.05, 0.15, 0.5, 1.5 or 5 mg/kg, and of normal human healthy volunteers administered a single IV infusion of the drug at 0.25, 0.625 or 2 mg/kg, or 6 weekly IV infusions at 2 or 4 mg/kg. A clear, dose-dependent initial PD response was observed in monkeys in the dose range of 0.5 mg/kg to 5 mg/kg, which subsequently declined over time. In humans, a minimal PD response was observed at 0.025 mg/kg. A near maximal initial PD response was seen at 0.675 mg/kg or higher, followed by a decline over time. In the 6-week repeat-dose study, once-weekly dosing of narsoplimab at 2 mg/kg resulted in a mean trough PD response of ~55% lectin pathway inhibition, which increased to an ~80% mean trough response with once-weekly dosing of 4 mg/kg. PK sample analysis revealed a clear PK/PD relationship in monkeys and humans. Modeling of the PK/PD relationship in monkeys indicated an EC50 value of ~7 µg/mL (~ 46 nM), which is comparable to the IC50 value of lectin pathway inhibition in vitro (33 nM). In humans, modeling of the PK/PD relationship data indicated an EC50 value of ~500 ng/mL (3.3 nM), again very similar to the IC50 value of lectin pathway inhibition in vitro (3.4 nM). Conclusion We successfully developed PD assays to monitor blockade of lectin pathway activation in monkeys and humans administered narsoplimab. Using a validated PD assay, we demonstrate that a high level of lectin pathway blockade can be maintained in normal human healthy volunteers by once-weekly administration of narsoplimab at 4 mg/kg IV. We also demonstrate a consistent PK/PD relationship across the study population, indicating that narsoplimab concentration ranges can be used to target the desired level of lectin pathway inhibition. Figure 1 Disclosures Freeman: Omeros Corporation: Current Employment. Cummings:Omeros Corporation: Current Employment. Chuidian:Omeros Corporation: Current Employment. Dudler:Omeros Corporation: Current Employment.

DMD – ANIMAL MODELS & PRECLINICAL TREATMENT: P.221 Edasalonexent maintains bone density and bone strength in the mdx mouse model of Duchenne muscular dystrophy

Persistent NF-kB activation is a hallmark in DMD, which drives inflammation and fibrosis, leading to the loss of muscle function and disease progression. Reduced skeletal muscle force and function also reduces bone strength and mineralization via decreased muscle-bone strain. Steroids, which activate the glucocorticoid receptor (GR), further impact bone health, leading to short stature and an increased risk of vertebral and long bone fractures. Edasalonexent is a novel oral NF-kB inhibitor in clinical development that has the potential to slow disease progression in DMD without adversely impacting bone health.

Abstract LB-108: A potent and selective small molecule degrader of STAT5 for the treatment of hematological malignancies

Abstract STAT5 is a member of the signal transducer and activator of transcription family of proteins and is widely recognized as an oncogenic master regulator of hematological malignancies. To date, therapeutic approaches to attenuate aberrant activity have focused on upstream kinase targets such as JAK2, Bcr-Abl, Flt3, and Flt3-ITD. To date, there is no STAT5-selective inhibitor in clinical development. More recently, PROTAC approaches have been deployed successfully against the STAT3 isoform and demonstrated efficacy and safety in preclinical models. We herein report the identification of a potent and selective, non-PROTAC, small molecule degrader of STAT5 protein, JPX-0750. Upon binding, JPX-0750 covalently and selectively binds to a cysteine residue on STAT5 via an electrophilic warhead, which induces profound destabilization of STAT5. The unfolding/destabilization effect can be observed via Western blot, isothermal denaturation of purified protein and HDX exchange. In a range of AML and TPLL cell lines exposure to JPX-750 at nM concentrations leads to a rapid and dose-dependant degradation of both phospho-STAT5 and total STAT5 with resultant IC50's between 80-200 nM. Washout experiments determined that exposure to JPX-0750 at 1 µM for 2-4 h is sufficient to degrade STAT5, inhibit downstream targets, and induce apoptosis. Moreover, in the same washout experiments, 50% STAT5 recovery post-washout required >72 h suggesting a long pharmacodynamic effect. Against STAT3, degrading effects are observed later, 24 h post washout (6 h treatment), indicating that anti-STAT5 activity is more rapid in AML cells and in line with the inherent stability of each protein. No effect was observed on either STAT1 or pSTAT1 upon exposure to JPX-750. Importantly, compared to standard AML cell lines JPX-750 demonstrated similar low nM potency in 25/30 primary AML blasts and TPLL patient samples, including those with poor prognostic markers. JPX-750 exhibits a large therapeutic window for AML versus pooled human fibroblasts and hematopoietic stem cells (ca 100 fold). In a preclinical MV4;11 luciferase model, structurally related JPX-700, at 5 mg/kg (IP, qd) significantly reduced leukemic burden, suppressed tumour dissemination to both the lung and liver, and had no effect on body weight, organ histology or blood parameters. In summary, JPX-750 represents a new class of potent and selective small molecule degraders of STAT5 protein. Citation Format: Ji Sung Park, Gary Tin, Elvin D. de Araujo, Anna Orlova, Helena Sorger, Florian Grebien, Elizabeth Heyes, Mulu Geletu, Ruth Villalonga, Angel Sampedro, Abootaleb Sedighi, Marco Herling, Satu Mustjoki, Mohammad S. Eram, Siawash Ahmar, Richard Moriggl, Jeff A. O'Meara, John Proudfoot, Kay Noel, Dziyana Kraskouskaya, Roman Fleck, Patrick T. Gunning. A potent and selective small molecule degrader of STAT5 for the treatment of hematological malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-108.

Abstract 6218: Discovery and preclinical evaluation of AMG 397, a potent, selective and orally bioavailable MCL1 inhibitor

Abstract MCL1 inhibitors are an emerging class of therapeutics targeting key protein-protein interactions between MCL1 and BH3 domain containing pro-apoptotic BCL-2 family members. Several MCL1 inhibitors have entered clinical trials including AMG 176, currently in Phase I clinical development for hematologic malignancies. However, all existing clinical stage MCL1 inhibitors are being administered intravenously. Here we describe the discovery and preclinical evaluation of AMG 397, the first orally administered MCL1 inhibitor in clinical development. The pursuit of an orally bioavailable MCL1 inhibitor with an acceptable human clinical dose required improvements to AMG 176 in both potency and pharmacokinetic properties. The chemistry effort to identify AMG 397 began with the observation that there were two conformations in AMG 176 bound to MCL1 with a difference of 2.8 kcal/mol in energy. A structure-guided approach in combination with ligand-based design to attempt to favor the more active conformation provided the advanced lead AM-3106 (Ki=100 pM) with significantly improved cell based potency (OPM2 viability IC50 = 19 nM) compared to AMG 176. Further refinements in potency and PK properties of AM-3106 culminated in the discovery of AMG 397. AMG 397 exhibited picomolar affinities for MCL1 (Ki=15 pM), selectively competing for binding to the BH3-binding groove of MCL1 with pro-apoptotic BCL-2 family members (e.g. BIM). In cells, AMG 397 potently disrupted the interaction between MCL1 and BIM. Treatment of OPM2 cells, an MCL1 dependent multiple myeloma cell line, induced clear increases in Caspase-3/7 activity within one hour. Subsequent effects on viability were observed across a treatment time course with maximal effects observed following 24 hours of continuous exposure (IC50=50 nM). Time course washout viability studies demonstrated that AMG 397 induced a committed step towards apoptosis and suggested that discontinuous dosing schedules would be sufficient to achieve antitumor effects in vivo. Cell lines from hematologic malignancies including AML, multiple myeloma and DLBCL exhibited greatest sensitivity to AMG 397 in a large tumor cell line profiling screen (n=300). In vivo, oral administration of AMG 397 demonstrated rapid and sustained dose dependent increases in activated BAK, cleaved Caspase-3 and cleaved PARP in established OPM2 xenografts. Discontinuous dosing schedules of once or twice weekly at 25 or 50 mg/kg of AMG 397 exhibited significant tumor regressions in mice bearing OPM2 xenografts, with 9 of 10 mice tumor free at the end of the study in both 50 mg/kg groups. AMG 397 was also tested in the MOLM-13 orthotopic model of AML where twice weekly administration at 10, 30 and 60 mg/kg achieved 47% tumor growth inhibition (TGI), 99% TGI and 75% regression respectively. Combination of AMG 397 at 10 mg/kg twice weekly and 50 mg/kg of venetoclax daily achieved 45% regression in the same model. Citation Format: Sean Caenepeel, Rex Karen, Brian Belmontes, Alla Verlinsky, Hong Tan, Yajing Yang, Xiaoyue Chen, Kexue Li, Jennifer Allen, Jan Wahlstrom, Jude Canon, Angela Coxon, Paul Hughes. Discovery and preclinical evaluation of AMG 397, a potent, selective and orally bioavailable MCL1 inhibitor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6218.

Abstract 3036: Intracellular phospho-flow reveals novel insights to pathway modulation by tirabrutinib in combination with entospletinib and obinutuzumab

Abstract Tirabrutinib is a second-generation, potent, selective irreversible Bruton tyrosine kinase (BTK) inhibitor in clinical development for relapsed/refractory chronic lymphocytic leukemia (CLL) in combination with entospletinib and obinutuzumab. To study the modulation or inhibition of different signaling pathways in response to drug treatment, we developed a phospho-flow cytometric assay that can measure up to 110 intracellular signaling proteins. This method was validated in an in vitro setting to develop the pre-analytical sample handling, assay setup and analysis parameters. The optimized procedure was implemented in a phase II clinical study (NCT02983617). For the in vitro experiments, whole blood was collected from healthy volunteers and CLL patients. Following a stimulation with anti-IgD/anti-IgM, the blood was processed and stained with a panel of 110 phospho-specific antibodies and evaluated by flow cytometry. A panel of 43 phosphoproteins was selected based on their relevance to the inhibited pathways and/or their responsiveness to B cell receptor cross-linking. In the clinical study, whole blood was collected at baseline, week 2 and 25 post-treatment from 23 patients treated with tirabrutinib in combination with entospletinib and obinutuzumab. The samples were stimulated with anti-IgM/anti-IgD and stained with surface markers to identify the cancer cells and their signaling fingerprint was evaluated with the selected panel of phosphoprotein markers. The data from the in vitro experiments showed, at baseline, both CLL and healthy B cells had no or minimal constitutive activation of downstream BCR signaling proteins. In normal B cells, of the phospho-proteins analyzed, most showed great increase after activation of BCR. However, BCR-induced phosphorylation of most signaling proteins was significantly lower in the CLL cells from the patients. In addition, the fraction of cells responding to the in vitro stimulation was highly variable among the CLL patient samples compared to normal donors. Down-regulation of phosphoproteins, including pBTK (Y223)/pITK (Y180), pSyk (Y348), pSyk (Y525/Y526) was observed in most of the patients both in stimulated or un-stimulated conditions at week 2 and 25. The level of the phosphorylation of pBTK (Y223)/pITK (Y180), pSyk (Y348), pSyk (Y525/Y526) from stimulated blood was highly correlated with the blast counts in the periphery at week 1 and 2. These findings support the utility of this multi-parameter flow assay to detect and monitor immune signaling inside different immune cells. Evaluating the effects of targeted therapeutics following clinical administration can inform on disease and drug mechanisms, combination strategies, and identify potential biomarkers for pharmacodynamic or patient stratification that are otherwise inaccessible using less sensitive or non-functional analyses. Citation Format: Xiaoyun Yang, Tom Wehrman, Justin Laboriante, Biao Li, Nadine Kutsch, Christian Pallasch, Barbara Eichhorst, Juliane M. Jürgensmeier. Intracellular phospho-flow reveals novel insights to pathway modulation by tirabrutinib in combination with entospletinib and obinutuzumab [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3036.

KRAS(G12C)\u2013AMG\xa0510 interaction dynamics revealed by all-atom molecular dynamics simulations

The first KRAS(G12C) targeting inhibitor in clinical development, AMG 510, has shown promising antitumor activity in clinical trials. On the molecular level, however, the interaction dynamics of this covalently bound drug–protein complex has been undetermined. Here, we disclose the interaction dynamics of the KRAS(G12C)–AMG 510 complex by long timescale all-atom molecular dynamics (MD) simulations (total of 75 μs). Moreover, we investigated the influence of the recently reported post-translational modification (PTM) of KRAS’ N-terminus, removal of initiator methionine (iMet1) with acetylation of Thr2, to this complex. Our results demonstrate that AMG 510 does not entrap KRAS into a single conformation, as one would expect based on the crystal structure, but rather into an ensemble of conformations. AMG 510 binding is extremely stable regardless of highly dynamic interface of KRAS’ switches. Overall, KRAS(G12C)–AMG 510 complex partially mimic the native dynamics of GDP bound KRAS; however, AMG 510 stabilizes the α3-helix region. N-terminally modified KRAS displays similar interaction dynamics with AMG 510 as when Met1 is present, but this PTM appears to stabilize β2–β3-loop. These results provide novel conformational insights on the molecular level to KRAS(G12C)–AMG 510 interactions and dynamics, providing new perspectives to RAS related drug discovery.

Pamiparib is a potent and selective PARP inhibitor with unique potential for the treatment of brain tumor

Pamiparib, an investigational Poly (ADP-ribose) polymerase (PARP) inhibitor in clinical development, demonstrates excellent selectivity for both PARP1 and PARP2, and superb anti-proliferation activities in tumor cell lines with BRCA1/2 mutations or HR pathway deficiency (HRD). Pamiparib has good bioavailability and is 16-fold more potent than olaparib in an efficacy study using BRCA1 mutated MDA-MB-436 breast cancer xenograft model. Pamiparib also shows strong anti-tumor synergy with temozolomide (TMZ), a DNA alkylating agent used to treat brain tumors. Compared to other PARP inhibitors, pamiparib demonstrated improved penetration across the blood brain barrier (BBB) in mice. Oral administration of pamiparib at a dose as low as 3 mg/kg is sufficient to abrogate PARylation in brain tumor tissues. In SCLC-derived, TMZ-resistant H209 intracranial xenograft model, combination of pamiparib with TMZ overcomes its resistance and shows significant tumor inhibitory effects and prolonged life span. Our data suggests that combination of pamiparib with TMZ has unique potential for treatment of brain tumors. Currently, the combination therapy of pamiparib with TMZ is evaluated in clinical trial [NCT03150862].

Development and validation of an LC-MS/MS method for monitoring larotrectinib, a tropomyosin-related kinase inhibitor, in mouse and human plasma and application to pharmacokinetic studies

Larotrectinib is an orally administered drug and the first and only selective pan-tropomyosin receptor kinase (TRK) inhibitor in clinical development to treat cancer patients harboring a neurotrophic receptor tyrosine kinase gene fusion. In this study, an analytical method to quantify the TRK inhibitor in mouse and human plasma was developed and validated using LC-MS/MS following protein precipitation with acetonitrile. Larotrectinib and an internal standard (carbamazepine) were separated from endogenous substances using an Xterra C18 column with acetonitrile containing 0.1% formic acid as the mobile phase. The ions m/z 429.8 → 342.8 for larotrectinib and m/z 237.0 → 194.0 for carbamazepine detected in multiple reaction monitoring mode were used for the quantitation. The detector response of larotrectinib was linear within the concentration range 5–10,000 ng/mL with a correlation coefficient (r2) of not less than 0.999. The intra- and inter-day precision and accuracy were less than 10.48% and within − 8.99%, respectively, in mouse and human plasma. Larotrectinib was stable under various storage and handling conditions, and no significant matrix effect was observed in both mouse and human plasma. Finally, the assay was successfully applied to the pharmacokinetic study of larotrectinib after its intravenous and oral administration to mice.

Evaluation of the effect of UGT1A1 polymorphisms on the pharmacokinetics of oral and long-acting injectable cabotegravir

BackgroundCabotegravir is an HIV integrase inhibitor in clinical development with both oral and long-acting (LA) injectable formulations. Cabotegravir is primarily metabolized by uridine 5′-diphospho-glucuronosyltransferase (UGT) 1A1, a known polymorphic enzyme with functional variants that can affect drug metabolism and exposure.ObjectivesTo investigate the pharmacogenetic effects of the reduced-function alleles UGT1A1*6, UGT1A1*28 and/or UGT1A1*37 on steady-state pharmacokinetics (PK) and safety of oral cabotegravir (30 mg/day) and intramuscular cabotegravir LA (400 mg every 4 weeks or 600 mg every 8 weeks).MethodsPlasma cabotegravir PK was assessed in 346 UGT-genotyped participants with and without UGT1A1 functional variants across six studies (four Phase I and two Phase II) of oral cabotegravir, including 215 HIV-infected participants who received oral cabotegravir followed by cabotegravir LA. Changes from baseline in total bilirubin and ALT were assessed in one study (LATTE; NCT01641809).ResultsStatistically significant (P < 0.05) associations were observed between UGT1A1 genotype and plasma cabotegravir PK parameters, with 28%–50% increases following oral cabotegravir [plasma cabotegravir concentration at the end of the dosing interval (Ctau), 1.50-fold; AUCtau, 1.41-fold; and Cmax, 1.28-fold] and 16%–24% increases following cabotegravir LA administration (48 week Ctau, 1.24-fold; AUCtau, 1.16-fold; and Cmax, 1.18-fold) among those with low-versus-normal genetically predicted UGT1A1 activity. A statistically significant (P < 10−5) association between predicted UGT1A1 activity and maximum change in total bilirubin was also observed (2.45-fold asymptomatic increase for low versus normal) without a corresponding change in ALT.ConclusionsThis modest increase in oral and parenteral cabotegravir exposure associated with a reduced function of UGT1A1 is not considered clinically relevant based on accumulated safety data; no dose adjustment is required.

Penetration of Ibrexafungerp (Formerly SCY-078) at the Site of Infection in an Intra-abdominal Candidiasis Mouse Model.

Ibrexafungerp (IBX), formerly SCY-078, is a novel, oral and intravenous, semisynthetic triterpenoid glucan synthase inhibitor in clinical development for treating multiple fungal infections, including invasive candidiasis. Intra-abdominal candidiasis (IAC) is one of the most common types of invasive candidiasis associated with high mortality largely due to poor drug exposure in infected lesions. To better understand the potential of IBX to treat such infections, we investigated its penetration at the site of infection. Using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) and laser capture microdissection (LCM)-directed high-pressure liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), we investigated tissue distribution and lesion-specific drug exposure of IBX in a clinically relevant IAC mouse model. After a single-dose treatment, IBX quickly distributed into tissues and efficiently accumulated within lesions. Drug concentrations of IBX within the liver abscesses were almost 100-fold higher than the serum concentration. In addition, drug penetration after repeated treatment of IBX was compared with micafungin. IBX exhibited robust and long-lasting lesion penetration after repeated treatment. These data indicate that IBX penetrates into intra-abdominal abscesses highly efficiently and holds promise as a potential therapeutic option for IAC patients.

P157 FBXO3-FBXL2 AXIS MODULATORS AS A NOVEL CLASS OF ORAL SMALL MOLECULE COMPOUNDS FOR THE TREATMENT OF CROHN’S DISEASE

Abstract Background Ubiquitination is a common post-translational modification, tagging proteins for degradation. The ubiquitin proteasome system is activated in Crohn’s Disease (CD), and modulation of its components might be a novel strategy for therapeutic intervention to control inflammation. The conjugation of ubiquitin to a target protein is orchestrated by a series of enzymatic reactions, the last step being catalyzed by a selective ubiquitin E3 ligase. Among ubiquitin E3 ligases, Fbxl2 serves as a sentinel gatekeeper to limit inflammation by targeting and enhancing the degradation of tumor necrosis factor receptor associated factors (TRAF) proteins, which link cell surface signals (through NFkB signaling) to cytokine secretion. In addition, Fbxl2 controls the ubiquitination of the inflammasome, NOD-like receptor protein 3 (NLRP3), which mediates the release of interleukin (IL)1β and IL18 (Fig.1). Fbxl2 protein itself is ubiquitinated and degraded by another protein, called Fbxo3. Inhibition of Fbxo3 results in increased Fbxl2 levels and decreased TRAF and NLRP3. Individuals with a natural occurring polymorphism within Fbxo3V221Ihave decreased lipopolysaccharide(LPS)-induced cytokine (TNFα, IL1β &amp; IL6) production, Fbxo3 and TRAF levels and increased Fbxl2, providing human genetic target validation for Fbxo3-Fbxl2 axis modulation in inflammatory conditions. BC1261 is a first-in-class, selective, orally available Fbxo3 inhibitor in clinical development for the treatment of CD. Methods and Results In vitro data demonstrated that BC1261 binds to Fbxo3, prevents Fbxo3/Fbxl2 association and produces a concentration-dependent inhibition of LPS-induced release of TNFα and IL1β from human peripheral blood mononuclear cells. In an acute DSS-mouse model, administration of BC-1261 via drinking water ad libitum (30 μg/ml) or by daily intraperitoneal (IP) injection (150 μg) for 5 days resulted in the attenuation of the shortening of colonic length, tissue damage (Fig2.A), and TNFα and IL6 tissue levels (Fig.2B). In a repeated DSS-mouse model, BC-1261 (10 mg/kg bid) given orally for 19 days was comparable to the positive control (anti-p40) in the DAI composite (Fig2.C), stool consistency (Fig2.D), and histopathology score in the proximal colon (Fig2.E). BC-1261 also lead to clinically relevant reductions on endoscopy score (Fig2.F). Conclusion BC1261, a Fbxo3 inhibitor, was efficacious in both acute and chronic preclinical models of colitis. Taken together, these results suggest the potential utility of selective Fbxo3-Fbxl2 modulation in the treatment of CD.

Biotransformation Pathways and Metabolite Profiles of Oral [14C]Alisertib (MLN8237), an Investigational Aurora A Kinase Inhibitor, in Patients with Advanced Solid Tumors.

Alisertib (MLN8237) is an investigational, orally available, selective aurora A kinase inhibitor in clinical development for the treatment of solid tumors and hematologic malignancies. This metabolic profiling analysis was conducted as part of a broader phase 1 study evaluating mass balance, pharmacokinetics, metabolism, and routes of excretion of alisertib following a single 35-mg dose of [14C]alisertib oral solution (∼80 μCi) in three patients with advanced malignancies. On average, 87.8% and 2.7% of the administered dose was recovered in feces and urine, respectively, for a total recovery of 90.5% by 14 days postdose. Unchanged [14C]alisertib was the predominant drug-related component in plasma, followed by O-desmethyl alisertib (M2), and alisertib acyl glucuronide (M1), which were present at 47.8%, 34.6%, and 12.0% of total plasma radioactivity. In urine, of the 2.7% of the dose excreted, unchanged [14C]alisertib was a negligible component (trace), with M1 (0.84% of dose) and glucuronide conjugate of hydroxy alisertib (M9; 0.66% of dose) representing the primary drug-related components in urine. Hydroxy alisertib (M3; 20.8% of the dose administered) and unchanged [14C]alisertib (26.3% of the dose administered) were the major drug-related components in feces. In vitro, oxidative metabolism of alisertib was primarily mediated by CYP3A. The acyl glucuronidation of alisertib was primarily mediated by uridine 5'-diphospho-glucuronosyltransferase 1A1, 1A3, and 1A8 and was stable in 0.1 M phosphate buffer and in plasma and urine. Further in vitro evaluation of alisertib and its metabolites M1 and M2 for cytochrome P450-based drug-drug interaction (DDI) showed minimal potential for perpetrating DDI with coadministered drugs. Overall, renal elimination played an insignificant role in the disposition of alisertib, and metabolites resulting from phase 1 oxidative pathways contributed to >58% of the alisertib dose recovered in urine and feces over 192 hours postdose. SIGNIFICANCE STATEMENT: This study describes the primary clearance pathways of alisertib and illustrates the value of timely conduct of human absorption, distribution, metabolism, and excretion studies in providing guidance to the clinical pharmacology development program for oncology drugs, for which a careful understanding of sources of exposure variability is crucial to inform risk management for drug-drug interactions given the generally limited therapeutic window for anticancer drugs and polypharmacy that is common in cancer patients.

Safety, pharmacokinetics, and antiretroviral activity of islatravir (ISL, MK-8591), a novel nucleoside reverse transcriptase translocation inhibitor, following single-dose administration to treatment-naive adults infected with HIV-1: an open-label, phase 1b, consecutive-panel trial

Islatravir (also known as ISL and MK-8591) is a unique nucleoside reverse transcriptase translocation inhibitor in clinical development for treatment of people with HIV-1 infection. In preclinical studies, intracellular islatravir-triphosphate exhibits a long half-life and prolonged virological effects. In this study, we aimed to assess islatravir safety, pharmacokinetics, and antiretroviral activity in treatment-naive adults with HIV-1 infection. This open-label, consecutive-panel, phase 1b trial was done at Charité Research Organisation (Berlin, Germany) and included men and women (aged 18-60 years, inclusive) with HIV-1 infection who were ART naive. Participants were required to have plasma HIV-1 RNA counts of at least 10 000 copies per mL within 30 days before the trial treatment phase, without evidence of resistance to nucleoside reverse transcriptase inhibitors. Participants were enrolled in one of five consecutive dosing panels, receiving a single oral dose of islatravir (0·5-30 mg). The primary outcomes were safety and tolerability of islatravir and change from baseline in HIV-1 plasma RNA; secondary outcomes were islatravir plasma and islatravir-triphosphate intracellular pharmacokinetics. We obtained descriptive safety and pharmacokinetics statistics, and estimated efficacy results from a longitudinal data analysis model. This study is registered with ClinicalTrials.gov, NCT02217904, and EudraCT, 2014-002192-28. Between Sept 17, 2015, and May 11, 2017, we enrolled 30 participants (six per panel). Islatravir was generally well tolerated. 27 (90%) participants had 60 adverse events after receipt of drug, of which 21 (35%) were deemed to be drug related. The most common (n>1) drug-related adverse events were headache (in nine [30%] participants) and diarrhoea (in two [7%]). No serious adverse events were reported, and no participants discontinued due to an adverse event. Plasma islatravir pharmacokinetics and intracellular islatravir-triphosphate pharmacokinetics were approximately dose proportional. The islatravir-triphosphate intracellular half-life was 78·5-128·0 h. Least-squares mean HIV-1 RNA at 7 days after dose decreased from 1·67 log10 copies per mL (95% CI 1·42-1·92) at 10 mg dose to 1·20 log10 copies per mL (0·95-1·46) at 0·5 mg dose. No genetic changes consistent with development of viral resistance were detected. Single doses of islatravir as low as 0·5 mg significantly suppressed HIV-1 RNA by more than 1·0 log at day 7 in treatment-naive adults with HIV-1 infection and were generally well tolerated, supporting the further development of islatravir as a flexible-dose treatment for individuals with HIV-1 infection. Merck Sharp & Dohme Corp, a subsidiary of Merck & Co Inc, Kenilworth, NJ, USA.

P190 - The effect of CYP2C9 and CYP2C19 genotype on the pharmacokinetics of PF 04965842, A JAK1 inhibitor in clinical development

P190 - The effect of CYP2C9 and CYP2C19 genotype on the pharmacokinetics of PF 04965842, A JAK1 inhibitor in clinical development

Novel deazaflavin tyrosyl-DNA phosphodiesterase 2 (TDP2) inhibitors

Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a DNA repair enzyme that removes 5′-phosphotyrosyl blockages resulting from topoisomerase II (TOP2)-DNA cleavage complexes trapped by TOP2 inhibitors. TDP2 is a logical target for the development of therapeutics to complement existing treatments based on inhibition of TOP2. There is, however, no TDP2 inhibitor in clinical development at present. Of the reported TDP2 inhibitors, the deazaflavins are the most promising chemical class centered around the lead compound SV-5-153. Recently we reported new subtypes derived within the deazaflavin family with improved membrane permeability properties. In this work we characterize two representative analogues from two new deazaflavin subtypes based on their biochemical TDP2 inhibitory potency and drug-likeness. We demonstrate that the ZW-1288 derivative represents a promising direction for the development of deazaflavins as therapeutic agents. ZW-1288 exhibits potent inhibitory activity at low nanomolar concentrations against recombinant and cellular human TDP2 with profile similar to that of the parent analog SV-5-153 based on high resistance against murine TDP2 and human TDP2 mutated at residue L313H. While expressing weak cytotoxicity on its own, ZW-1288 potentiates the clinical TOP2 inhibitors etoposide (ETP) and mitoxantrone in human prostate DU145 and CCRF-CEM leukemia and chicken lymphoma DT40 cells while not impacting the activity of the topoisomerase I (TOP1) inhibitor camptothecin or the PARP inhibitor olaparib. ZW-1288 increases the uptake of ETP to a lesser extent than SV-5-153 and remained active in TDP2 knockout cells indicating that the deazaflavin TDP2 inhibitors have additional cellular effects that will have to be taken into account for their further development as TDP2 inhibitors.

724. The In Vitro Activity of Gepotidacin and Comparator Agents Against Anaerobic Bacterial Isolates

BackgroundGepotidacin (GSK2140944) is a first in class novel triazaacenaphthylene bacterial type II topoisomerase inhibitor in clinical development for the treatment of gonorrhea and uncomplicated UTI (acute cystitis). Gepotidacin selectively inhibits bacterial DNA gyrase and topoisomerase IV by a unique mechanism not utilized by any currently approved therapeutic agent and demonstrates in vitro activity against most target pathogens resistant to established antibacterials, including fluoroquinolones. This study assessed the in vitro activity of gepotidacin and comparators against a collection of 649 Gram-positive and Gram-negative anaerobic bacterial clinical isolates.MethodsA total of 649 clinically significant Gram-negative (333) and Gram-positive (316) anaerobic clinical isolates previously collected and frozen at −70°C were tested. Isolates came from North America (315/48.5%) and Europe (334/51.5%) and were collected between 2000 and 2017. Minimum inhibitory concentrations (MICs) for gepotidacin and 6 comparators were determined by agar dilution (AD) for all isolates, and by both AD and broth microdilution (BMD) for Bacteroides spp. according to CLSI guidelines (CLSI M11-A8). Most Lactobacillus spp. tested in this study require anaerobic conditions for growth and were tested by anaerobic AD.ResultsThe in vitro activity results of gepotidacin and comparators are shown in the table below. The gepotidacin MIC90 for all Gram-negative anaerobic isolates tested in this study was 4 µg/mL and for the comparators tested was as follows: ceftriaxone 512 μg/mL, clindamycin >8 μg/mL, imipenem 0.5 μg/mL, metronidazole 2 μg/mL, moxifloxacin 8 μg/mL and piperacillin–tazobactam 16 μg/mL. Gepotidacin had the lowest MIC90 (2 μg/mL) for the Gram-positive anaerobic isolates compared with the other antibiotics tested, with the exception of metronidazole (MIC90 = 0.5 µg/mL).ConclusionGepotidacin showed in vitro activity against a collection of 649 anaerobic Gram-negative and Gram-positive clinical isolates, with an MIC90 value against all Gram-negative anaerobic isolates of 4 µg/mL, and against all Gram-positive anaerobic isolates of 2 µg/mL. Disclosures All authors: No reported disclosures.

701. Comparison of MIC Results for Gepotidacin by Agar Dilution and Broth Microdilution Methods

BackgroundGepotidacin (GSK2140944) is a novel triazaacenaphthylene bacterial type II topoisomerase inhibitor in clinical development for the treatment of gonorrhea and uncomplicated UTI (acute cystitis). Gepotidacin selectively inhibits bacterial DNA gyrase and topoisomerase IV by a unique mechanism not utilized by any currently approved therapeutic agent and demonstrates in vitro activity against most target pathogens resistant to established antibacterials, including fluoroquinolones. This study tested the equivalency of minimal inhibitory concentrations (MICs) obtained by 2 reference susceptibility testing methods, agar dilution (AD) and broth microdilution (BMD), for gepotidacin against Gram-positive and Gram-negative organisms.MethodsSusceptibility testing for both methods was performed on a total of 733 clinical isolates recovered largely in 2016 from over 120 medical centers worldwide. For N. gonorrhoeae, only the AD method is recommended by CLSI, therefore BMD was performed using Fastidious Broth for comparison purposes. Essential agreement (EA) based on evaluable results was calculated as the number of isolates with MICs within one 2-fold dilution of the reference method divided by the total number of results. Equivalency was defined using the 95% criteria from the FDA’s class II controls document.ResultsThe EA observed for gepotidacin with these 2 methods was 85.8% overall and 98.3% when H. influenzae and N. gonorrhoeae isolates were excluded. Slightly higher gepotidacin MICs were observed when tested by BMD for each of these species/groups; this trend was especially prominent for E. coli and S. pyogenes. Gepotidacin tested against H. influenzae (73.1%) or N. gonorrhoeae (28.6%) species had much lower EAs.ConclusionEquivalency (EA >95%) was established between AD and BMD methods for determining gepotidacin susceptibility results against Staphylococcus spp., Streptococcus spp. and E. coli. However, for N. gonorrhoeae and H. influenzae, equivalency between the 2 methods was not established; therefore, future antimicrobial susceptibility testing for gepotidacin against these organisms should adhere to the methods for which quality control ranges and breakpoints are approved. Disclosures All authors: No reported disclosures.

714. Analysis of the Effect of Urine on the In Vitro Activity of Gepotidacin and Levofloxacin Against Escherichia coli, Staphylococcus epidermidis, and Staphylococcus saprophyticus

BackgroundGepotidacin (GSK2140944) is a first in class novel triazaacenaphthylene bacterial type II topoisomerase inhibitor in clinical development for the treatment of gonorrhea and uncomplicated UTI (acute cystitis). Gepotidacin selectively inhibits bacterial DNA gyrase and topoisomerase IV by a unique mechanism not utilized by any currently approved therapeutic agent and demonstrates in vitro activity against most target pathogens resistant to established antibacterials, including fluoroquinolones. This study was undertaken to determine the effect of various urine parameters on the in vitro activity of gepotidacin and a comparative agent, levofloxacin, against a variety of bacteria.MethodsStudy strains were tested according to the reference CLSI broth microdilution method using cation-adjusted Mueller–Hinton broth (CAMHB) and the following method variations: CAMHB with 25%, 50%, and 100% urine (not pH adjusted) and 100% urine (pH adjusted to 7.2–7.4, and 8). Quality control strains were concurrently tested each day.ResultsMIC endpoints in the reference method and in 100% urine were easily determined (i.e., clear buttons of growth up to the first well of no growth). Gepotidacin MIC results are summarized in the table. For E. coli and S. saprophyticus, there was a trend for higher gepotidacin MIC results with the addition of increasing amounts of urine. However, the increase was minimal such that mean dilution differences were ≤ 1.54. Against S. epidermidis, gepotidacin MICs were not significantly impacted by the addition of urine as 100% of urine condition MICs were within ±1 doubling dilution of the reference method MIC. The gepotidacin results for E. coli indicate that the average 1–2 dilution MIC increase observed in the unadjusted 100% pooled urine MIC may be associated with lower pH. A similar increase in levofloxacin MIC results for E. coli were also associated with pH, but at the higher pH of 8.0. In contrast, the increase in MIC observed in both the gepotidacin and levofloxacin S. saprophyticus results in 100% pooled urine do not appear to be a function of pH.ConclusionOverall, the effect of urine on the gepotidacin and levofloxacin MICs was minimal and not inclusive of all strains tested. Disclosures All authors: No reported disclosures.

Salsalate is An Active Agent in Multiple Myeloma

Multiple Myeloma (MM) is an incurable plasma cell neoplasm for which novel therapeutic options are required. Constitutive activity of the nuclear factor kappa B (NF-κB) pathway is critical to the pathogenesis of MM. NF-κB activation is mediated by inducible phosphorylation and degradation of the IkB members by IkB kinase (IKK). Inhibiting NF-kB by interfering with IKK activity induces apoptosis in MM. However, there is as yet no IKK inhibitor in clinical development. Salsalate is an IKK inhibitor used historically as an anti-inflammatory drug.