Whole-body distribution of tenofovir, emtricitabine and dolutegravir in non-human primates.

BACKGROUND: One major barrier to HIV cure is the persistence of virus, possibly linked to an insufficient antiretroviral drug (ARV) distribution into tissues. OBJECTIVES: To draw the whole-body distribution of three antiretroviral drugs-tenofovir disoproxil fumarate, emtricitabine and dolutegravir-in non-human primates (NHPs). METHODS: Eight uninfected NHPs received a single injection of a solution containing the three ARVs. Forty-five different tissues were sampled 24 h after injection. RESULTS: Median tissue penetration factors (TPFs) were 45.4, 5.8 and 0.5 for tenofovir, emtricitabine and dolutegravir, respectively, and were statistically different between the three ARVs. Tissues were grouped by system, because TPFs were consistent according to these groups, and ranked in order of decreasing TPFs. The digestive system was the system with the highest tissue concentrations. Next came the two main sites of elimination, the liver and the kidney, as well as the tissues of the cardiopulmonary and urinary systems. Then, it was the whole lymphatic system. The next group included the reproductive system, the adipose tissue and the skin. The last two systems were the muscle and the CNS. The intra-tissue variability was rather low with a median coefficient of variation of the concentrations around 15% and no value greater than 80%. CONCLUSIONS: Overall, this study determines the first whole-body distribution in a validated NHP model. These data have important implications for future preclinical and clinical studies for the development of novel HIV therapies towards an HIV cure.

Recruitment of FBXO22 for targeted degradation of NSD2.

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD.

Ube3a unsilencer for the potential treatment of Angelman syndrome.

Deletion of the maternal UBE3A allele causes Angelman syndrome (AS); because paternal UBE3A is epigenetically silenced by a long non-coding antisense (UBE3A-ATS) in neurons, this nearly eliminates UBE3A protein in the brain. Reactivating paternal UBE3A holds promise for treating AS. We previously showed topoisomerase inhibitors can reactivate paternal UBE3A, but their therapeutic challenges prompted our search for small molecule unsilencers with a different mechanism of action. Here, we found that (S)-PHA533533 acts through a novel mechanism to significantly increase paternal Ube3a mRNA and UBE3A protein levels while downregulating Ube3a-ATS in primary neurons derived from AS model mice. Furthermore, peripheral delivery of (S)-PHA533533 in AS model mice induces widespread neuronal UBE3A expression. Finally, we show that (S)-PHA533533 unsilences paternal UBE3A in AS patient-derived neurons, highlighting its translational potential. Our findings provide a lead for developing a small molecule treatment for AS that could be safe, non-invasively delivered, and capable of brain-wide unsilencing of paternal UBE3A.

Population Dose-Response-Time Analysis of Itch Reduction and Patient-Reported Tolerability Supports Phase III Dose Selection for Linerixibat.

Increase in serum bile acids (BAs) in patients with primary biliary cholangitis (PBC) may play a causal role in cholestatic pruritus (itch). Linerixibat is a selective small molecule inhibitor of the ileal bile acid transporter, which blocks re-absorption of BAs in the gastrointestinal tract thereby lowering BAs in the systemic circulation and reducing itch. One consequence is excess BAs in the colon, leading to diarrhea and abdominal pain. GLIMMER (NCT02966834) was a placebo-controlled phase IIb dose-ranging trial of linerixibat once (q.d.) or twice daily (b.i.d.) in adults with moderate to severe pruritus and PBC. To determine the optimal dose for maximum itch reduction while minimizing diarrhea, a kinetic-pharmacodynamic (k-PD) model was developed using data from GLIMMER. The PD end point modeled was worst daily itch, derived from itch score reported by patients b.i.d. A proportional odds model was developed post hoc to indicate the probability of diarrhea occurrence, a patient-reported outcome (GI-5) recorded weekly. The final k-PD model successfully described the effects of linerixibat and placebo on itch. Model simulations were consistent with the observed dose-dependent increase in the average number of itch responders (patients with a ≥ 2-point improvement in itch). This was paralleled by a dose-dependent increase in the probability of higher diarrhea frequency scores. The b.i.d. dosing regimens led to a modest increase in the number of itch responders as compared with q.d. dosing. This quantitative framework highlights the trade-off between benefit and tolerability and supported the selection of 40 mg b.i.d. in the phase III GLISTEN trial (NCT04950127).

Recruitment of FBXO22 for Targeted Degradation of NSD2.

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here, we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain of function mutation p.E1099K, resulting in growth suppression, apoptosis, and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 in a covalent and reversible manner to recruit the SCF FBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCF FBXO22 . Overall, we present a highly potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a novel FBXO22-dependent TPD strategy.

Longitudinal efficacy and safety modeling and simulation framework to aid dose selection of belantamab mafodotin for patients with multiple myeloma.

Belantamab mafodotin, a monomethyl auristatin F (MMAF)-containing monoclonal antibody-drug conjugate (ADC), demonstrated deep and durable responses in the DRiving Excellence in Approaches to Multiple Myeloma (DREAMM)-1 and pivotal DREAMM-2 studies in patients with relapsed/refractory multiple myeloma. As with other MMAF-containing ADCs, ocular adverse events were observed. To predict the effects of belantamab mafodotin dosing regimens and dose-modification strategies on efficacy and ocular safety end points, DREAMM-1 and DREAMM-2 data across a range of doses were used to develop an integrated simulation framework incorporating two separate longitudinal models and the published population pharmacokinetic model. A concentration-driven tumor growth inhibition model described the time course of serum M-protein concentration, a measure of treatment response, whereas a discrete time Markov model described the time course of ocular events graded with the GSK Keratopathy and Visual Acuity scale. Significant covariates included baseline ß2 -microglobulin on growth rate, baseline M-protein on kill rate, extramedullary disease on the effect compartment rate constant, and baseline soluble B cell maturation antigen on maximal effect. Efficacy and safety end points were simulated for various doses with dosing intervals of 1, 3, 6, and 9 weeks and various event-driven dose-modification strategies. Simulations predicted that lower doses and longer dosing intervals were associated with lower probability and lower overall time with Grade 3+ and Grade 2+ ocular events compared with the reference regimen (2.5 mg/kg every 3 weeks), with a less-than-proportional reduction in efficacy. The predicted improved benefit-risk profiles of certain dosing schedules and dose modifications from this integrated framework has informed trial designs for belantamab mafodotin, supporting dose-optimization strategies.

Exposure-Response Analyses for Therapeutic Dose Selection of Belantamab Mafodotin in Patients With Relapsed/Refractory Multiple Myeloma.

Belantamab mafodotin is an antibody-drug conjugate comprising a humanized anti-B-cell maturation antigen (BCMA) monoclonal antibody conjugated to monomethyl auristatin F (MMAF) via a protease-resistant maleimidocaproyl linker. Single-agent belantamab mafodotin showed clinically meaningful activity and manageable safety in patients with heavily pretreated relapsed/refractory multiple myeloma (RRMM) in the phase I DREAMM-1 and phase II DREAMM-2 studies and is approved by the US Food and Drug Administration and European Medicines Agency for RRMM treatment. To support monotherapy dose selection, the relationship between Cycle 1 exposure (derived using a population pharmacokinetic model) and clinical response (for multiple efficacy and safety end points) was explored. In DREAMM-2, efficacy end points (probability of response (PoR) and progression-free survival (PFS)) were associated with exposure in univariate evaluation; however, once disease burden factors were included in the model (e.g., baseline soluble BCMA, ß2 -microglobulin), exposure was no longer significant. Patients with higher disease burden had lower exposure. In DREAMM-1, belantamab mafodotin exposure was the only variable to correlate with PoR and PFS. Probability of corneal events (keratopathy), but not dry eye or blurred vision, was strongly associated with belantamab mafodotin exposure (DREAMM-2). Higher cys-mcMMAF maximum plasma drug concentration (Cmax ) and lower baseline platelet count were associated with increased probability of thrombocytopenia (DREAMM-1 and DREAMM -2). In general, safety end points were more strongly associated with belantamab mafodotin exposure than efficacy end points, particularly after disease factors and patient characteristics were taken into account. Overall, these findings supported the monotherapy dose recommendation of belantamab mafodotin as 2.5 mg/kg every 3 weeks in patients with RRMM who have received four or more prior therapies.

Population pharmacokinetics of belantamab mafodotin, a BCMA-targeting agent in patients with relapsed/refractory multiple myeloma.

Belantamab mafodotin (belamaf) is an antibody-drug conjugate (ADC) targeting B-cell maturation antigen (BCMA). Nonlinear mixed-effects models were developed to characterize the population pharmacokinetics (PopPK) of ADC, total monoclonal antibody (mAb), and cysteine-maleimidocaproyl-MMAF (cys-mcMMAF) after 0.03-4.6 mg/kg dosing every 3 weeks in heavily pretreated patients with relapsed/refractory multiple myeloma (RRMM; DREAMM-1, n = 73; DREAMM-2, n = 218). Sequential modeling methodology was used. Individual post hoc parameter estimates from the final ADC model were used to develop total mAb and cys-mcMMAF models. Formal covariate selection used a modified stepwise forward inclusion method with backward elimination. A linear, two-compartment PopPK model with a time-varying clearance (CL) described ADC PK. Initial ADC typical value for CL for a DREAMM-2 patient was 0.936 L/day with a half-life of 11.5 days, over time CL was reduced by 28% resulting in a half-life of 14.3 days. Time to 50% maximal CL change was ~ 50 days. Baseline soluble BCMA (sBCMA), immunoglobulin (IgG), albumin, and bodyweight impacted ADC CL. Cys-mcMMAF concentrations were described with a linear two-compartment model linked to ADC; input rate was governed by deconjugation/intracellular proteolytic degradation of ADC represented by an exponentially decreasing MMAF:mAb (drug antibody ratio [DAR]) after each dose. Time to 50% DAR reduction was 10.3 days. Baseline sBCMA and IgG impacted cys-mcMMAF central volume of distribution. In conclusion, ADC, total mAb, and cys-mcMMAF concentration-time profiles in RRMM were well-described by PopPK models, and exposure was most strongly impacted by disease-related characteristics.

Population Pharmacokinetic-Pharmacodynamic Modeling of Inotersen, an Antisense Oligonucleotide for Treatment of Patients with Hereditary Transthyretin Amyloidosis.

A population pharmacokinetic (PK) and pharmacodynamic (PD) model was developed for inotersen to evaluate exposure-response relationships and to optimize therapeutic dosing regimen in patients with hereditary transthyretin (TTR) amyloidosis polyneuropathy (hATTR-PN). Inotersen PK and TTR level (PD) data were composed of one Phase 1 study in healthy subjects, one Phase 2/3 study in hATTR patients, and its one open-label extension study. Effects of intrinsic and extrinsic factors (covariates) on PK and PK/PD of inotersen were evaluated using a full model approach. Inotersen PK was characterized by a two-compartment model with elimination from the central compartment. The population PK analysis identified disease status and lean body mass (LBM) as significant covariates for inotersen PK. Nonetheless, the contribution of disease status and LBM on PK was small, as the difference in clearance (CL/F) was 11.1% between healthy subjects and patients with hATTR-PN and 38% between the lowest and highest LBM quartiles of the patient population. Age, race, sex, baseline renal function estimated glomerular filtration rate, and hepatic function markers (baseline albumin, bilirubin, and alanine aminotransferase values) were not statistically significant covariates affecting inotersen PK. An inhibitory effect indirect-response model (inhibition of TTR production) was used to describe the drug effect on TTR-time profiles, with baseline TTR included as a covariate. The overall population Imax and IC50, together with 95% confidence interval, was estimated to be 0.913 (0.899-0.925) and 9.07 (8.08-10.1) ng/mL, respectively. V30M mutation showed no effect on the estimated IC50 value for hATTR patients. The final population PK and PK/PD model was used to simulate four different treatment regimens. The population PK/PD model developed well described the PK and PD of inotersen in patients with hATTR-PN and has been used for label recommendation and trial simulations.

Population Pharmacokinetic/Pharmacodynamic Analysis of Intravenous Zanamivir in Healthy Adults and Hospitalized Adult and Pediatric Subjects With Influenza.

Zanamivir is a potent and highly selective inhibitor of influenza neuraminidase in which the inhibition of this enzyme prevents the virus from infecting other cells and specifically prevents release of the new virion from the host cell membrane. It is available as an oral powder for inhalation and intravenous formulations. The current population pharmacokinetic model based on data from eight studies of subjects treated with the intravenous formulation (125 healthy adults and 533 hospitalized adult and pediatric subjects with suspected or confirmed influenza) suggested a decreased zanamivir clearance in pediatric and renal impairment adult subjects. It also indicates that b.i.d. dosing is necessary to keep the exposure in influenza infected subjects above the 90% inhibitory concentration values of recently circulating viruses over the dosing interval. In the exposure-response analysis (phases II and III studies), no apparent relationship was found between zanamivir exposure and clinically relevant pharmacodynamic end points.

Discovery and Development of TMPRSS6 Inhibitors Modulating Hepcidin Levels in Human Hepatocytes.

Iron overload disorders are characterized by the body's inability to regulate iron absorption and its storage which can lead to organ failures. Accumulated evidence has revealed that hepcidin, the master regulator of iron homeostasis, is negatively modulated by TMPRSS6 (matriptase-2), a liver-specific type II transmembrane serine protease (TTSP). Here, we report that treatment with a peptidomimetic inhibitor affecting TMPRSS6 activity increases hepcidin production in hepatic cells. Moreover, similar effects were observed when using non-peptidic inhibitors obtained through optimization of hits from high-throughput screening. Using HepG2 cells and human primary hepatocytes, we show that TMPRSS6 inhibitors block TMPRSS6-dependent hemojuvelin cleavage and increase HAMP expression and levels of secreted hepcidin.

Comparison of tenofovir plasma and tissue exposure using a population pharmacokinetic model and bootstrap: a simulation study from observed data.

Sparse tissue sampling with intensive plasma sampling creates a unique data analysis problem in determining drug exposure in clinically relevant tissues. Tissue exposure may govern drug efficacy, as many drugs exert their actions in tissues. We compared tissue area-under-the-curve (AUC) generated from bootstrapped noncompartmental analysis (NCA) methods and compartmental nonlinear mixed effect (NLME) modeling. A model of observed data after single-dose tenofovir disoproxil fumarate was used to simulate plasma and tissue concentrations for two destructive tissue sampling schemes. Two groups of 100 data sets with densely-sampled plasma and one tissue sample per individual were created. The bootstrapped NCA (SAS 9.3) used a trapezoidal method to calculate geometric mean tissue AUC per dataset. For NLME, individual post hoc estimates of tissue AUC were determined, and the geometric mean from each dataset calculated. Median normalized prediction error (NPE) and absolute normalized prediction error (ANPE) were calculated for each method from the true values of the modeled concentrations. Both methods produced similar tissue AUC estimates close to true values. Although the NLME-generated AUC estimates had larger NPEs, it had smaller ANPEs. Overall, NLME NPEs showed AUC under-prediction but improved precision and fewer outliers. The bootstrapped NCA method produced more accurate estimates but with some NPEs > 100%. In general, NLME is preferred, as it accommodates less intensive tissue sampling with reasonable results, and provides simulation capabilities for optimizing tissue distribution. However, if the main goal is an accurate AUC for the studied scenario, and relatively intense tissue sampling is feasible, the NCA bootstrap method is a reasonable, and potentially less time-intensive solution.

A Randomized, Placebo- and Positive-Controlled, Single-Dose, Crossover Thorough QT/QTc Study Assessing the Effect of Daprodustat on Cardiac Repolarization in Healthy Subjects.

Daprodustat (GSK1278863) is a prolyl hydroxylase inhibitor in phase 3 clinical studies for the treatment of anemia associated with chronic kidney disease. This study was conducted to evaluate the effect of daprodustat on cardiac repolarization and enrolled 55 healthy adult male (29) and female (26) subjects who received single-dose 75 and 500 mg daprodustat, 400 mg moxifloxacin, and placebo. Mean placebo-corrected change from baseline QT interval for daprodustat showed no statistically significant increase. However, statistically significant decreases in the ΔΔQTcF were observed for both doses of daprodustat, reaching a lowest value of -2.74 milliseconds for 75 mg and -5.93 milliseconds for 500 mg daprodustat; this minor shortening effect is not considered clinically significant. The moxifloxacin group showed a statistically significant increase in the ΔΔQTcF value, reaching a maximal increase of 11.47 milliseconds at 4 hours. Forty subjects (73%) reported at least 1 adverse event, with the highest incidence with 500 mg daprodustat. This group had a higher incidence of gastrointestinal adverse events compared to the other treatment groups. These results suggest that 500 mg daprodustat was not well tolerated; however, daprodustat at 75 mg was generally well tolerated. No new safety concerns were identified in subjects who received 500 mg daprodustat.

Exploration of phenylpropanoic acids as agonists of the free fatty acid receptor 4 (FFA4): Identification of an orally efficacious FFA4 agonist.

The long chain free fatty acid receptor 4 (FFA4/GPR120) has recently been recognized as lipid sensor playing important roles in nutrient sensing and inflammation and thus holds potential as a therapeutic target for type 2 diabetes and metabolic syndrome. To explore the effects of stimulating this receptor in animal models of metabolic disease, we initiated work to identify agonists with appropriate pharmacokinetic properties to support progression into in vivo studies. Extensive SAR studies of a series of phenylpropanoic acids led to the identification of compound 29, a FFA4 agonist which lowers plasma glucose in two preclinical models of type 2 diabetes.

Identification of diarylsulfonamides as agonists of the free fatty acid receptor 4 (FFA4/GPR120).

The exploration of a diarylsulfonamide series of free fatty acid receptor 4 (FFA4/GPR120) agonists is described. This work led to the identification of selective FFA4 agonist 8 (GSK137647A) and selective FFA4 antagonist 39. The in vitro profile of compounds 8 and 39 is presented herein.

Enzymatic- and iridium-catalyzed asymmetric synthesis of a benzothiazepinylphosphonate bile acid transporter inhibitor.

A synthesis of the benzothiazepine phosphonic acid 3, employing both enzymatic and transition metal catalysis, is described. The quaternary chiral center of 3 was obtained by resolution of ethyl (2-ethyl)norleucinate (4) with porcine liver esterase (PLE) immobilized on Sepabeads. The resulting (R)-amino acid (5) was converted in two steps to aminosulfate 7, which was used for construction of the benzothiazepine ring. Benzophenone 15, prepared in four steps from trimethylhydroquinone 11, enabled sequential incorporation of phosphorus (Arbuzov chemistry) and sulfur (Pd(0)-catalyzed thiol coupling) leading to mercaptan intermediate 18. S-Alkylation of 18 with aminosulfate 7 followed by cyclodehydration afforded dihydrobenzothiazepine 20. Iridium-catalyzed asymmetric hydrogenation of 20 with the complex of [Ir(COD)2BArF] (26) and Taniaphos ligand P afforded the (3R,5R)-tetrahydrobenzothiazepine 30 following flash chromatography. Oxidation of 30 to sulfone 31 and phosphonate hydrolysis completed the synthesis of 3 in 12 steps and 13% overall yield.

Discovery of a highly potent, nonabsorbable apical sodium-dependent bile acid transporter inhibitor (GSK2330672) for treatment of type 2 diabetes.

The apical sodium-dependent bile acid transporter (ASBT) transports bile salts from the lumen of the gastrointestinal (GI) tract to the liver via the portal vein. Multiple pharmaceutical companies have exploited the physiological link between ASBT and hepatic cholesterol metabolism, which led to the clinical investigation of ASBT inhibitors as lipid-lowering agents. While modest lipid effects were demonstrated, the potential utility of ASBT inhibitors for treatment of type 2 diabetes has been relatively unexplored. We initiated a lead optimization effort that focused on the identification of a potent, nonabsorbable ASBT inhibitor starting from the first-generation inhibitor 264W94 (1). Extensive SAR studies culminated in the discovery of GSK2330672 (56) as a highly potent, nonabsorbable ASBT inhibitor which lowers glucose in an animal model of type 2 diabetes and shows excellent developability properties for evaluating the potential therapeutic utility of a nonabsorbable ASBT inhibitor for treatment of patients with type 2 diabetes.

Inhibition of apical sodium-dependent bile acid transporter as a novel treatment for diabetes.

Bile acids are recognized as metabolic modulators. The present study was aimed at evaluating the effects of a potent Asbt inhibitor (264W94), which blocks intestinal absorption of bile acids, on glucose homeostasis in Zucker Diabetic Fatty (ZDF) rats. Oral administration of 264W94 for two wk increased fecal bile acid concentrations and elevated non-fasting plasma total Glp-1. Treatment of 264W94 significantly decreased HbA1c and glucose, and prevented the drop of insulin levels typical of ZDF rats in a dose-dependent manner. An oral glucose tolerance test revealed up to two-fold increase in plasma total Glp-1 and three-fold increase in insulin in 264W94 treated ZDF rats at doses sufficient to achieve glycemic control. Tissue mRNA analysis indicated a decrease in farnesoid X receptor (Fxr) activation in small intestines and the liver but co-administration of a Fxr agonist (GW4064) did not attenuate 264W94 induced glucose lowering effects. In summary, our results demonstrate that inhibition of Asbt increases bile acids in the distal intestine, promotes Glp-1 release and may offer a new therapeutic strategy for type 2 diabetes mellitus.

Liver X receptor protects against liver injury in sepsis caused by rodent cecal ligation and puncture.

BACKGROUND: Liver X receptor (LXR) is a transcription factor of the nuclear receptor family, regulating genes involved in metabolism, inflammation, and apoptosis. In the present investigation, we examined the role of LXR in organ injury and systemic inflammation in rodent models of polymicrobial peritonitis caused by cecal ligation and puncture (CLP). METHODS: Rats were subjected to CLP sepsis or a sham operation. Some were treated with the synthetic LXR agonist GW3965 0.3 mg/kg 30 min prior to the CLP procedure, and organs and plasma were harvested at 3, 10, 18, or 24 h. Organs were analyzed for RNA expression by quantitative polymerase chain reaction or for morphologic differences by histologic review. Organ injury and inflammatory markers were measured in plasma. RESULTS: Expression of the LXRα gene was decreased in the livers of CLP rats compared with sham-operated rats. Administration of a synthetic agonist of LXR (GW3965) reduced biochemical indices of liver injury in the blood of CLP rats. We also demonstrated that liver injury associated with CLP is aggravated in LXRα- and LXRαß-deficient mice compared with wild-type and LXRß-deficient mice, indicating a role for LXRα in protecting the liver. The enhanced liver injury in LXR-deficient mice was associated with elevated plasma concentrations of high mobility group box 1, a late mediator of inflammation and a known factor in the pathology of this model. CONCLUSIONS: Collectively, these results argue in favor of a role for LXRα in protection against liver injury in experimental sepsis induced by CLP.