Table 7.3.

In vitro and in vivo PK, tolerability, and efficacy studies of antivirals against biothreat viral agents.

Therapeutic	Target	Efficacy data	PK and tolerability	Additional information and comments	References
EBOV
BCX4430	Viral polymerase	In vitro—Kikwit EC50: 11.8 μM; EC90: 25.4 μM	Well tolerated in all efficacy studies. Very short plasma half-life in mouse and NHP (T1/2 = 2–10 min). Mouse, 150 mg/kg, IM: liver, Cmax (triphosphate) = 65 μM, Tmax = 8 h, T1/2 = 4.3 h. Conversion to triphosphate in hepatocytes: mouse>human ∼NHP	BCX4430-TP levels in mouse liver at 150 mg/kg IM are ∼2.5× above EC90 value. Distribution into other tissues/cells not reported	[89], [90], [91]
		In vivo—mice: 150 mg/kg BID PO; 90% survival; 150 mg/kg BID IM; 100% survival
		NHP: 25 mg/kg BID D0–14; 100% survival; 100 mg/kg BID IM D2, then 25 mg/kg BID D3–11, 100% survival (67% with same regimen starting on D3)
		In vivo—HP: 10 mg/kg iv D3–15; 100% protection
Favipiravir (T-705)	Viral polymerase	In vitro—IC50: 67 μM; EC50 = 281 μM (Kikwit), 223 μM (Makona), 51 μM (Marburg Ci67)	Favipirivir-ribose-TP concentrations were measured in various cell lines and mouse tissues	Intracellular ribosylation required prior to triphosphorylation to active drug	[92], [93], [94], [95], [96]
		In vivo—mice: 100% survival at 300 mg/kg D0–7 against aerosol challenge; 100% survival at 300 mg/kg beginning D6 following intranasal challenge [92]; 90% survival observed at 8 mg/kg [41]. NHP: Marburg (Angola), 83% survival at 250 mg/kg, iv loading dose + 150 mg/kg, BID for 13 days	Biodistribution in mice assessed by PET imaging. Plasma favipirivir-ribose-TP levels in NHPs following 200 mg/kg, iv loading dose and 150 mg/kg, iv, BID daily dose were 4–34 μM—below the MARV EC50 of 51 μM
GS-5734	Viral polymerase	In vitro—replication: EC50: 0.086–0.14 μM	NHP PK 10 mg/kg, iv, short plasma T1/2 of GS-5734, rapid intracellular conversion to triphosphate with persistent levels >EBOV EC50 for 24 h, intracellular triphosphate T1/2 = 14 h	NHP tissue distribution plasma∼testes> eye>brain	[97]
Clomiphene	CAD, entry inhibitor	In vitro—IC50: 11.1 μM (Kikwit) IC50: 3.83 μM (Mayinga)	Mouse PK unavailable. Human PK 50 mg QD: Cmax = 37 nM. Protein binding not reported, but likely high due to structure similarity to toremifene	Estrogen receptor modulator, human-free drug exposure = Ebola EC50	[98], [99]
		In vivo—Mice: 60 mg/kg IP QD on days 0, 1, 3, 5, 7, and 9; 90% survival
Bepridil	CAD, entry inhibitor	In vitro—IC50: 5.08 μM (Vero) IC50: 3.21 μM (HepG2)	Mouse PK unavailable; human PK 300 mg PO QD: Cmax ∼ 6.3 μM, PPB > 99%	Calcium channel blocker, human-free drug plasma exposure = Ebola EC50, QT prolongation issues	[100]
		In vivo—Mice: 12 mg/kg; 100% survival
Brincidofovir	Unknown	In vitro—EC50: 120 nm–1.3 μM		Interferes with viral DNA replication	[101], [102]
		In vivo—no preclinical efficacy reported
Type I IFN	NA	In vitro—IFN-α IC50: 0.038 μM IFN-β IC50: 0.016 μM			[103], [104], [105]
		In vivo—IFN-α2b in NHP: delayed time to death
		IFN-β in NHP: 10.5 μg/kg at 18 h and days 1, 3, 5, 7, and 9; delayed time to death
TKM-100802, TKM-130803	L, VP35, VP24	In vitro—not available		Lipid nanoparticle formulation of siRNAs	[106], [107]
		In vivo—NHP: 100% survival against Kikwit and Makona
AVI-6002	VP24/VP35	In vitro—not available		Combination of AVI-7537 and AVI-7539 phosphorodiamidate morpholino oligomers	[108], [109], [110]
		In vivo—NHP: up to 63% survival at 40 mg/kg
AVI-7537	VP24	In vitro—0.585 μM		Phosphorodiamidate morpholino oligomer	[108], [110]
		In vivo—NHP: 40 mg/kg; 75% survival
LASV
Ribavirin	Viral polymerase	In vitro—yield reduction assay, Vero cells, IC90 = 2.5 μM, Junin (Romero); 3.6 μM, Junin (Candid#1); plaque assay, Junin (Romero), Vero E6 cells, EC90 = 71 μM In vivo—NHP: 50 mg/kg, s.c. (loading dose) followed by 10 mg/kg, s.c. every 8 h through day 18; 4 animals dosed immediately after infection, 4 dosed 5 days post infection; 8/8 survived versus 4/10 in control group	Ribavirin is intracellularly triphosphorylated to the active drug. In humans, plasma concentrations of ribavirin have been correlated to virological response	Nucleoside drug; FDA approved for RSV and HCV	[111], [112], [113], [114]
Stampidine	Viral polymerase	In vivo—CBA mice, cerebral injection of Lassa (Josiah): 50 mg/kg, i.p., dosed daily for 6 days starting 24 h prior to infection, 90% survival compared to 25% vehicle survival	Rapidly converted to its active form (Ala-MP) within 5 min in plasma. No notable toxicity after daily i.p. or p.o. admin for 8 weeks (cumulative dose 6.4 g/kg)	Nucleoside monophosphate prodrug; reverse transcriptase inhibitor against HIV	[115]
Zidampidine	Viral polymerase	In vivo—CBA mice, cerebral injection of Lassa (Josiah): 25 mg/kg, i.p., dosed daily for 6 days starting 24 h prior to infection, 100% survival compared to 28% vehicle survival	Rapidly converted to its metabolites Ala-AZT-MP and AZT following iv injection. Nontoxic to mice up to 250 mg/kg	Nucleoside monophosphate prodrug of zidovudine (AZT)	[116]
LASV, JUNV
LHF-535	Entry inhibitor	In vitro—yield reduction assay, Vero cells, IC90 = 9.3 nM, Junin (Romero); 3 μM, Junin (Candid#1)	Not available	Small molecule	[111]
		In vivo—Tacaribe virus, AG129 mice: 100% survival, 10 mg/kg/day, p.o. for 14 days starting 30 min prior to infect.; 60%–90% survival when dosed 24–72 h post infection
Favipiravir (T-705)	Viral polymerase	In vitro—plaque assay, Junin (Romero), Vero E6 cells, EC90 = 21 μM	PK, biodistribution, and favipiravir-ribose-triphosphate levels discussed above	Intracellular ribosylation required prior to triphosphorylation to active drug	[93], [94], [95], [112], [117]
		In vivo—LASV (Josiah), cynomolgus macaques: 4/4 survival versus 0/4 placebo, 300 mg/kg, iv on day 4 followed by 300 mg/kg, s.c., qd days 5–17
VEEV
β-d-N4-hydroxycytidine	Viral polymerase	In vitro—VEEV EC50 = 1.2 μM (HeLa), 1.3 μM (Vero); 99% inhibition at 10 μM (astrocytes)	Rat brain nucleoside TP levels after single 50 mg/kg, p.o. dose are near VEEV EC50 values (526 ng/g at 2.5 h, 135 ng/g at 24 h)	Broad-spectrum antiviral (EEEV, WEEV, VEEV, and numerous nonbiothreat viruses such as CHIKV, MERV, influenza, and RSV)	[118]
ML366	nsP2, nsP4	In vitro—EC50 (VEEV TC-83) = 32 nM (Vero)	Mouse brain drug levels (single 10 mg/kg, i.p.) at 2 h post dose were 0.35 μM		[119], [120]
		In vivo—VEEV TrD (s.c. injection), BALB/c mice, 12.5 mg/kg, i.p. every 12 h for 8 days starting 2 h preinfection, 100% survival
BDGR-4	nsP2, nsP4	In vitro—EC50 (Vero) = 47 nM (VEEV TC-83), 150 nM (EEEV), 102 nM (WEEV)		Close-in analog to ML366	[119]
		In vivo—VEEV TrD (s.c. injection), BALB/c mice, 12.5 mg/kg, i.p. every 12 h for 8 days starting 24 h post infection, 100% survival (90% starting 48 h post infection). EEEV (s.c. injection), C57BL/6 mice, 25 mg/kg, i.p. every 12 h for 8 days starting 2 h preinfection, 90% survival

EBOV, Ebola virus; HCV, Hepatitis C virus; IFN, Interferon; JUNV, Junin virus; LASV, Lassa virus; NHP, nonhuman primate; NHP, nonhuman primate; PPB, plasma protein binding; PK, pharmacokinetic; PET, positron emission tomography; RSV, respiratory syncytial virus; TP, triphosphate; VEEV, venezuelan equine encephalitis virus.