A 28-Day Toxicity Study of Tenofovir Alafenamide Hemifumarate by Subcutaneous Infusion in Rats and Dogs.

The toxicity of tenofovir alafenamide (TAF) hemifumarate (HF) was evaluated when administered by continuous subcutaneous (s.c.) infusion via an external infusion pump for 28 days to rats and dogs. The toxicokinetics of TAF and two metabolites, tenofovir (TFV) and tenofovir diphosphate (TFV-DP) were also evaluated. After administration of TAF HF in rats and dogs, primary systemic findings supported an inflammatory response that was considered minimal to mild. Gross pathology and histopathologic evaluation of tissue surrounding the s.c. infusion site revealed signs of inflammation, including edema, mass formation, fibrosis, and mononuclear cell inflammation in groups receiving ≥300 µg/kg/day in rats and ≥25 µg/day in dogs. Although these changes were observed in animals receiving vehicle, the severity was greater in animals receiving TAF HF. Changes in the local tissue were considered a TAF HF-mediated exacerbation of an inflammatory response to the presence of the catheter. In rats, systemic and local findings were considered not adverse due to their low severity and reversibility; therefore, the "no observed adverse effect level" (NOAEL) was set at 1,000 µg/kg/day. Because none of the systemic findings were related to systemic exposure to TAF, the systemic NOAEL was set at 250 µg/kg/day in dogs. Due to the severity of the observations noted, a NOAEL for local toxicity could not be established. Although these results might allow for exploration of tolerability and pharmacokinetics of s.c. administered TAF HF in humans, data suggest a local reaction may develop in humans at doses below a clinically relevant dose. IMPORTANCE Human immunodeficiency virus (HIV) infection continues to be a serious global human health issue, with ∼38 million people living with HIV worldwide at the end of 2019. HIV preexposure prophylaxis (PrEP) has introduced the use of antiretroviral therapies as another helpful tool for slowing the spread of HIV worldwide. One possible solution to the problem of inconsistent access and poor adherence to HIV PrEP therapies is the development of subcutaneous (s.c.) depots or s.c. implantable devices that continuously administer protective levels of an HIV PrEP therapy for weeks, months, or even years at a time. We evaluate here the toxicity of tenofovir alafenamide, a potent inhibitor or HIV replication, after continuous s.c. infusion in rats and dogs for HIV PrEP.

Tenofovir Alafenamide for HIV Prevention: Review of the Proceedings from the Gates Foundation Long-Acting TAF Product Development Meeting.

The ability to successfully develop a safe and effective vaccine for the prevention of HIV infection has proven challenging. Consequently, alternative approaches to HIV infection prevention have been pursued, and there have been a number of successes with differing levels of efficacy. At present, only two oral preexposure prophylaxis (PrEP) products are available, Truvada and Descovy. Descovy is a newer product not yet indicated in individuals at risk of HIV-1 infection from receptive vaginal sex, because it still needs to be evaluated in this population. A topical dapivirine vaginal ring is currently under regulatory review, and a long-acting (LA) injectable cabotegravir product shows strong promise. Although demonstrably effective, daily oral PrEP presents adherence challenges for many users, particularly adolescent girls and young women, key target populations. This limitation has triggered development efforts in LA HIV prevention options. This article reviews efforts supported by the Bill & Melinda Gates Foundation, as well as similar work by other groups, to identify and develop optimal LA HIV prevention products. Specifically, this article is a summary review of a meeting convened by the foundation in early 2020 that focused on the development of LA products designed for extended delivery of tenofovir alafenamide (TAF) for HIV prevention. The review broadly serves as technical guidance for preclinical development of LA HIV prevention products. The meeting examined the technical feasibility of multiple delivery technologies, in vivo pharmacokinetics, and safety of subcutaneous (SC) delivery of TAF in animal models. Ultimately, the foundation concluded that there are technologies available for long-term delivery of TAF. However, because of potentially limited efficacy and possible toxicity issues with SC delivery, the foundation will not continue investing in the development of LA, SC delivery of TAF products for HIV prevention.

Development and Regulatory Challenges for Peptide Therapeutics.

There has been an increased interest in and activity for the use of peptide therapeutics to treat a variety of human diseases. The number of peptide drugs entering clinical development and the market has increased significantly over the past decade despite inherent challenges of peptide therapeutic discovery, development, and patient-friendly delivery. Disparities in interpretation and application of existing regulatory guidances to innovative synthetic and conjugated peptide assets have resulted in challenges for both regulators and sponsors. The Symposium on Development and Regulatory Challenges for Peptide Therapeutics at the 40th Annual Meeting of the American College of Toxicology held in November of 2019 focused on the following specific topics: (1) peptide therapeutic progress and future directions, and approaches to discover, optimize, assess, and deliver combination peptide therapeutics for treatment of diseases; (2) toxicological considerations to advance peptide drug-device combination products for efficient development and optimal patient benefit and adherence; (3) industry and regulatory perspectives on the regulation of synthetic and conjugated peptide products, including exploration of regulatory classifications, interpretations, and application of the existing guidances International Council for Harmonisation (ICH) M3(R2) and ICH S6(R1) in determining nonclinical study recommendations; and (4) presentation of the 2016 Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee working group assessment of genotoxicity testing requirements. Perspectives were shared from industry and regulatory scientists working in the peptide therapeutics field followed by an open forum panel discussion to discuss questions drafted for the peptide therapeutics scientific community, which will be discussed in more detail.

Insights into the Chemical Discovery of Remifentanil.

Design, Synthesis, and Pharmacological Evaluation of Ultrashort- to Long-acting Opioid Analgetics. By Feldman PL, James MK, Brackeen MF, Bilotta JM, Schuster SV, Lahey AP, Lutz MW, Johnson MR, Leighton HJ. J Med Chem 1991; 34:2202-8. Copyright 1991 American Chemical Society. Reprinted with permission.In an effort to discover a potent ultrashort-acting µ-opioid analgetic that is capable of metabolizing to an inactive species independent of hepatic function, several classes of 4-anilidopiperidine analgetics were synthesized and evaluated. One series of compounds displayed potent µ-opioid agonist activity with a high degree of analgesic efficacy and an ultrashort to long duration of action. These analgetics, 4-(methoxycarbonyl)-4-[1-oxopropyl)phenylamino]-1-piperidinepropanoic acid alkyl esters, were evaluated in vitro in the guinea pig ileum for µ-opioid activity, in vivo in the rat tail withdrawal assay for analgesic efficacy and duration of action, and in vitro in human whole blood for their ability to be metabolized in blood. Compounds in this series were all shown to be potent µ agonists in vitro, but depending upon the alkyl ester substitution, the potency and duration of action in vivo varied substantially. The discrepancies between the in vitro and in vivo activities and variations in duration of action are probably due to different rates of ester hydrolysis by blood esterase(s). The [structure-activity relationships] with respect to analgesic activity and duration of action as a function of the various esters synthesized is discussed. It was also demonstrated that the duration of action for the ultrashort-acting analgetic, 8, does not change upon prolonged infusion or administration of multiple bolus injections.

Quality guidelines for oral drug candidates: dose, solubility and lipophilicity.

In an attempt to seek increased understanding of compound attributes that influence successful drug pipeline progression, GlaxoSmithKline's portfolio of oral candidates was compared with reference sets of marketed oral drugs. The approach differs from other attrition studies by explicitly focusing on choosing 'the right compound' by applying relevant, experimentally derived properties. The analysis led to four proposed compound quality categories, created by combining specific criteria for three measures: dose, solubility and the property forecast index, a composite measure of lipophilicity using chromatographically determined LogD and aromaticity. The 'three properties' provide benchmarked guidelines for project teams to use when seeking and selecting clinical candidates, because they reflect the property distribution of marketed oral drugs.

Gut hormone pharmacology of a novel GPR119 agonist (GSK1292263), metformin, and sitagliptin in type 2 diabetes mellitus: results from two randomized studies.

UNLABELLED: GPR119 receptor agonists improve glucose metabolism and alter gut hormone profiles in animal models and healthy subjects. We therefore investigated the pharmacology of GSK1292263 (GSK263), a selective GPR119 agonist, in two randomized, placebo-controlled studies that enrolled subjects with type 2 diabetes. Study 1 had drug-naive subjects or subjects who had stopped their diabetic medications, and Study 2 had subjects taking metformin. GSK263 was administered as single (25-800 mg; n = 45) or multiple doses (100-600 mg/day for 14 days; n = 96). Placebo and sitagliptin 100 mg/day were administered as comparators. In Study 1, sitagliptin was co-administered with GSK263 or placebo on Day 14 of dosing. Oral glucose and meal challenges were used to assess the effects on plasma glucose, insulin, C-peptide, glucagon, peptide tyrosine-tyrosine (PYY), glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). After 13 days of dosing, GSK263 significantly increased plasma total PYY levels by ∼ five-fold compared with placebo, reaching peak concentrations of ∼ 50 pM after each of the three standardized meals with the 300 mg BID dose. Co-dosing of GSK263 and metformin augmented peak concentrations to ∼ 100 pM at lunchtime. GSK263 had no effect on active or total GLP-1 or GIP, but co-dosing with metformin increased post-prandial total GLP-1, with little effect on active GLP-1. Sitagliptin increased active GLP-1, but caused a profound suppression of total PYY, GLP-1, and GIP when dosed alone or with GSK263. This suppression of peptides was reduced when sitagliptin was co-dosed with metformin. GSK263 had no significant effect on circulating glucose, insulin, C-peptide or glucagon levels. We conclude that GSK263 did not improve glucose control in type 2 diabetics, but it had profound effects on circulating PYY. The gut hormone effects of this GPR119 agonist were modulated when co-dosed with metformin and sitagliptin. Metformin may modulate negative feedback loops controlling the secretion of enteroendocrine peptides. TRIAL REGISTRATION: Clinicaltrials.gov NCT01119846 Clinicaltrials.gov NCT01128621.

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.

CNS 7056: a novel ultra-short-acting Benzodiazepine.

BACKGROUND: A new benzodiazepine derivative, CNS 7056, has been developed to permit a superior sedative profile to current agents, i.e., more predictable fast onset, short duration of sedative action, and rapid recovery profile. This goal has been achieved by rendering the compound susceptible to metabolism via esterases. The authors now report on the profile of CNS 7056 in vitro and in vivo. METHODS: The affinity of CNS 7056 and its carboxylic acid metabolite, CNS 7054, for benzodiazepine receptors and their selectivity profiles were evaluated using radioligand binding. The activity of CNS 7056 and midazolam at subtypes (alpha1beta2gamma2, alpha2beta2gamma2, alpha3beta2gamma2, alpha5beta2gamma2) of the gamma-aminobutyric acid type A (GABAA) receptor was evaluated using the whole cell patch clamp technique. The activity of CNS 7056 at brain benzodiazepine receptors in vivo was measured in rats using extracellular electrophysiology in the substantia nigra pars reticulata. The sedative profile was measured in rodents using the loss of righting reflex test. RESULTS: CNS 7056 bound to brain benzodiazepine sites with high affinity. The carboxylic acid metabolite, CNS 7054, showed around 300 times lower affinity. CNS 7056 and CNS 7054 (10 mum) showed no affinity for a range of other receptors. CNS 7056 enhanced GABA currents in cells stably transfected with subtypes of the GABAA receptor. CNS 7056, like midazolam and other classic benzodiazepines, did not show clear selectivity between subtypes of the GABAA receptor. CNS 7056 (intravenous) caused a dose-dependent inhibition of substantia nigra pars reticulata neuronal firing and recovery to baseline firing rates was reached rapidly. CNS 7056 (intravenous) induced loss of the righting reflex in rodents. The duration of loss of righting reflex was short (< 10 min) and was inhibited by pretreatment with flumazenil. CONCLUSIONS: CNS 7065 is a high-affinity and selective ligand for the benzodiazepine site on the GABAA receptor. CNS 7056 does not show selectivity between GABAA receptor subtypes. CNS 7056 is a potent sedative in rodents with a short duration of action. Inhibition of substantia nigra pars reticulata firing and the inhibition of the effects of CNS 7056 by flumazenil show that it acts at the brain benzodiazepine receptor.

Potent, selective, and orally efficacious antagonists of melanin-concentrating hormone receptor 1.

The high expression of MCH in the hypothalamus with the lean hypophagic phenotype coupled with increased resting metabolic rate and resistance to high fat diet-induced obesity of MCH KO mice has spurred considerable efforts to develop small molecule MCHR1 antagonists. Starting from a lead thienopyrimidinone series, structure-activity studies at the 3- and 6-positions of the thienopyrimidinone core afforded potent and selective MCHR1 antagonists with representative examples having suitable pharmacokinetic properties. Based on structure-activity relationships, a structural model for MCHR1 was constructed to explain the binding mode of these antagonists. In general, a good correlation was observed between pKas and activity in the right-hand side of the template, with Asp123 playing an important role in the enhancement of binding affinity. A representative example when evaluated chronically in diet-induced obese mice resulted in good weight loss effects. These antagonists provide a viable lead series in the discovery of new therapies for the treatment of obesity.

6-(4-chlorophenyl)-3-substituted-thieno[3,2-d]pyrimidin-4(3H)-one-based melanin-concentrating hormone receptor 1 antagonist.

Genetic manipulation studies in mice at both the MCH receptor 1 (MCHR1) as well as the MCH peptide levels have implicated MCHR1 as a key player in energy homeostasis. The phenotype exhibited by these studies, that is, increased metabolic rate, resistance to high fat diet, and subsequent weight loss, has spurred considerable efforts to develop antagonists of MCHR1. In continuation of efforts directed toward this goal, the present work capitalizes on the putative binding mode of an MCH antagonist, resulting in the identification of several novel chemotypes that are potent and selective MCHR1 antagonists. In addition, the favorable pharmacokinetics of representative examples has allowed for the evaluation of an MCHR1 antagonist in a high fat diet-induced obese rodent model of obesity. The tolerability of the right-hand side of the template for diverse chemotypes accompanied by favorable effects on weight loss enhances the attractiveness of this template in the pursuit toward development of effective anti-obesity agents.

Preclinical pharmacology of GW280430A (AV430A) in the rhesus monkey and in the cat: a comparison with mivacurium.

BACKGROUND: No replacement for succinylcholine is yet available. GW280430A (AV430A) is a representative of a new class of nondepolarizing neuromuscular blocking drugs called asymmetric mixed-onium chlorofumarates. It undergoes rapid degradation in plasma by chemical hydrolysis and inactivation by cysteine adduction, resulting in a very short duration of effect. The neuromuscular, cardiovascular, and autonomic pharmacology of GW280430A is compared herein with that of mivacurium. METHODS: Adult male rhesus monkeys and adult male cats were anesthetized with nitrous oxide-oxygen-halothane and chloralose-pentobarbital, respectively. The neuromuscular blocking properties of GW280430A and mivacurium were compared at a stimulation rate of 0.15 Hz in the extensor digitorum of the foot (monkey) and the tibialis anterior (cat). Sympathetic responses were assayed in the cat in the nictitating membrane preparation, and vagal effects were evaluated in the cat via observation of bradycardic responses after stimulation of the cervical right vagus nerve. RESULTS: GW280430A and mivacurium were equipotent in the monkey (ED95 was 0.06 mg/kg in each case). GW280430A was half as potent as mivacurium in the cat. The total duration of action of GW280430A was less than half that of mivacurium in the monkey; recovery slopes were more than twice as rapid. The 25-75% recovery index of GW280430A did not vary significantly after various bolus doses or infusions, averaging 1.4-1.8 min in the monkey, significantly shorter than the same time interval (4.8-5.7 min) for mivacurium. Dose ratios for autonomic versus neuromuscular blocking properties in the cat were greater than 25 for both GW280430A and mivacurium. The ratio ED Hist:ED95 Neuromuscular Block in the monkey was significantly greater (approximately 53 vs. 13) for GW280430A, indicating approximately four times less relative prominence of the side effects of skin flushing and decrease of blood pressure, which are associated with release of histamine. CONCLUSIONS: These experiments show a much shorter neuromuscular blocking effect and much-reduced side effects in the case of GW280430A vis-à-vis mivacurium. These results, together with the novel chemical degradation of GW280430A, suggest further evaluation in human subjects.

Neuromuscular blocking activity and therapeutic potential of mixed-tetrahydroisoquinolinium halofumarates and halosuccinates in rhesus monkeys.

Structure-activity relationships in rhesus monkeys for a novel mixed-onium class of ultra-short-acting nondepolarizing tetrahydroisoquinolinium neuromuscular blockers (NMBs) are described. Bis-onium chlorofumarate 20a with (1R,2S)-benzyltetrahydroisoquinolinium groups was a potent lead compound (ED(95) = 0.079 mg/kg) with an ultra-short duration of NMB effect (7.1 min) and a selectivity index (SI: defined as a ratio of the cardiovascular threshold dose to the ED(95)) similar to that of mivacurium (3). The mean threshold dose for cardiovascular effects with 20a was ca. 20 times its ED(95) value (SI = 20). A novel mixed-onium analogue of 20a was prepared by replacing the benzyltetrahydroisoquinolinium group distal to the fumarate chlorine atom with a (1S,2R)-phenyltetrahydroisoquinolinium moiety. The resulting mixed-onium chlorofumarate 24a displayed good NMB potency (ED(95) = 0.063 mg/kg), ultra-short duration of action (5.6 min) and an improved selectivity index (SI = 57). Several other mixed-onium derivatives containing octanedioate (25a; ED(95) = 0.103 mg/kg), difluorosuccinate (27c; ED(95) = 0.056 mg/kg), and fluorofumarate (28a; ED(95) = 0.137 mg/kg) linkers were also potent, ultra-short-acting NMBs with good to excellent selectivity index values (SI = 37-96). Octanedioate 25a was longer acting at higher doses compared to difluorosuccinate 27c and chlorofumarate 24a. Durations of NMB effect following a 0.4 mg/kg bolus dose (100% block) of 25a, 27c, and 24a were 16.9, 13.0, and 10.0 min, respectively. Recovery time for mixed-onium chlorofumarate 24a following a 1 h continuous infusion at 10-20 microg/kg/min (95-100% block) was ca. 5 min which is similar to that observed following a 0.2 mg/kg bolus dose of this compound and indicates a lack of cummulative effects. Preliminary studies with chlorofumarate 24a in whole human blood revealed that mixed-onium thiazolidine 29 was the major metabolite and that plasma cholinesterases do not play the primary role in duration of NMB effect. The NMB properties of 24a in rhesus monkeys led to its clinical evaluation as a possible alternative to succinylcholine.

Identification and structure-activity studies of novel ultrashort-acting benzodiazepine receptor agonists.

The synthesis and evaluation of novel ultrashort-acting benzodiazepine (USA BZD) agonists is described. A BZD scaffold was modified by incorporation of amino acids and derivatives. The propionate side chain of glutamic acid tethers an enzymatically labile functionality where the metabolite carboxylic acid displays markedly reduced BZD receptor affinity. The USA BZDs were characterized by full agonism profiles. Copyright2000 Elsevier Science Ltd.

Relating the structure, activity, and physical properties of ultrashort-acting benzodiazepine receptor agonists.

The ultrashort-acting benzodiazepine (USA BZD) agonists reported previously have been structurally modified to improve aqueous solubility. Lactam-to-amidine modifications, replacement of the C5-haloaryl ring, and annulation of heterocycles are presented. These analogues retain BZD receptor potency and full agonism profiles.