Summary The most recent Ebola virus outbreak in West Africa – unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected – highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae1. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we describe the discovery of a novel anti-EBOV small molecule antiviral, GS-5734, a monophosphoramidate prodrug of an adenosine analog. GS-5734 exhibits antiviral activity against multiple variants of EBOV in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternate substrate and RNA-chain terminator in primer-extension assays utilizing a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life = 14 h) and distribution to sanctuary sites for viral replication including testes, eye, and brain. In a rhesus monkey model of EVD, once daily intravenous administration of 10 mg/kg GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two of six treated animals. These results provide the first substantive, post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses – including filoviruses, arenaviruses, and coronaviruses – suggests the potential for expanded indications. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.

A review of commercially available oral and injectable solution formulations reveals that the solubilizing excipients include water-soluble organic solvents (polyethylene glycol 300, polyethylene glycol 400, ethanol, propylene glycol, glycerin, N-methyl-2-pyrrolidone, dimethylacetamide, and dimethylsulfoxide), non-ionic surfactants (Cremophor EL, Cremophor RH 40, Cremophor RH 60, d-alpha-tocopherol polyethylene glycol 1000 succinate, polysorbate 20, polysorbate 80, Solutol HS 15, sorbitan monooleate, poloxamer 407, Labrafil M-1944CS, Labrafil M-2125CS, Labrasol, Gellucire 44/14, Softigen 767, and mono- and di-fatty acid esters of PEG 300, 400, or 1750), water-insoluble lipids (castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, and medium-chain triglycerides of coconut oil and palm seed oil), organic liquids/semi-solids (beeswax, d-alpha-tocopherol, oleic acid, medium-chain mono- and diglycerides), various cyclodextrins (alpha-cyclodextrin, beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, and sulfobutylether-beta-cyclodextrin), and phospholipids (hydrogenated soy phosphatidylcholine, distearoylphosphatidylglycerol, L-alpha-dimyristoylphosphatidylcholine, L-alpha-dimyristoylphosphatidylglycerol). The chemical techniques to solubilize water-insoluble drugs for oral and injection administration include pH adjustment, cosolvents, complexation, microemulsions, self-emulsifying drug delivery systems, micelles, liposomes, and emulsions.

The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC 50 = 86 nM in macrophages as the clinical candidate. Structure activity relationships established that the 1′-CN group and C-linked nucleobase were critical for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclinical efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3−14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [Nature 2016, 531, 381−385]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.

Cobicistat (3, GS-9350) is a newly discovered, potent, and selective inhibitor of human cytochrome P450 3A (CYP3A) enzymes. In contrast to ritonavir, 3 is devoid of anti-HIV activity and is thus more suitable for use in boosting anti-HIV drugs without risking selection of potential drug-resistant HIV variants. Compound 3 shows reduced liability for drug interactions and may have potential improvements in tolerability over ritonavir. In addition, 3 has high aqueous solubility and can be readily coformulated with other agents.

the most at 17 each. A total of 136 products were identified of which 36 are lyophilized powders and 100 are solutions. The routes of administration are mainly subcutaneous or intravenous infusion with three intravenous bolus, two intravitreal, and one intramuscular. The subcutaneous products are ready-to-use solutions or reconstituted lyophilized powders that do not require dilution while most intravenous products are concentrates that require dilution into saline or another intravenous fluid prior to infusion. Most are packaged in single-dose units and the exception of multi-use is Herceptin Ò and its biosimilars. The package configurations are vials, prefilled autoinjectors, or prefilled syringes. A typical antibody formulation contains an antibody, an excipient to adjust tonicity or osmolality for solutions or a lyoprotectant for lyophilized powders, a buffer, and a surfactant. The ionic tonicity-adjusting excipient is mainly sodium chloride and the non-ionic osmolality-adjusting excipients include sucrose, trehalose, mannitol, maltose, and sorbitol. The lyoprotectants are trehalose and sucrose. The pH range is 4.8-8.0 and the buffers or pHmodifying agents include histidine, citrate, succinate, acetate, phosphate, glutamate, adipic acid, aspartic acid, lactic acid, tromethamine, and 2-(N-morpholino)-ethanesulfonic acid. The surfactants include mostly polysorbate 20 or polysorbate 80, with four containing poloxamer 188, and one that does not contain a surfactant but contains PEG 3350. One product does not contain a buffer, and 12 do not contain a surfactant. The viscosity-lowering excipients are sodium chloride and the amino acids arginine, glycine, proline, and lysine. Arginine may also function to adjust ionic strength and minimize aggregation. Human serum albumin is used in 2 products for intravenous infusion. Other excipients include methionine as an anti-oxidant, and EDTA or DTPA as chelating agents. The maximum volume of subcutaneous injection is 15 mL administered over 3-5 minutes, but the typically volume is 0.5-2 mL. Five fixed-dose combinations have recently been approved and four contain hyaluronidase to assist the large volume subcutaneous injection of up to 15 mL, while one is a fixed-dose combination for intravenous with three antibodies. Prefilled autoinjectors and syringes are becoming more common and many come affixed with a needle of 27-gauge or 29-gauge, while a few have a 26-gauge or a 30-gauge needle. Recent advancements include hyaluronidase to assist the large subcutaneous injection volume of 5-15 mL, fixed-dose combinations, buffer-free formulation, and smaller subcutaneous injection volume (0.1 mL).