Investigational New Drug Enabling Nonclinical Safety Pharmacology Assessment of the Iminosugar UV-4, a Broad-Spectrum Host-Targeted Antiviral Agent

Abstract

UV-4 (N-(9-methoxynonyl)-1-deoxynojirimycin) is a broad-spectrum antiviral drug candidate with demonstrated activity in vitro and in vivo against multiple, diverse viruses. Nonclinical safety pharmacology studies were conducted to support the filing of an Investigational New Drug (IND) application. Preliminary in vitro pharmacology testing evaluating potential for binding to “off-target” receptors and enzymes indicated no significant liability for advanced development of UV-4. The safety pharmacology of UV-4 was evaluated in the in vitro human ether-à-go-go-related gene (hERG) assay, in a central nervous system (CNS) study in the mouse (modified Irwin test), in a respiratory safety study in conscious mice using whole body plethysmography, and in a cardiovascular safety study in conscious, radiotelemetry-instrumented beagle dogs. There were no observed adverse treatment-related effects following administration of UV-4 as the hydrochloride salt in the hERG potassium channel assay, on respiratory function, in the CNS study, or in the cardiovascular assessment. Treatment-related cardiovascular effect of decreased arterial pulse pressure after 50 or 200 mg of UV-4/kg was the only change outside the normal range, and all hemodynamic parameters returned to control levels by the end of the telemetry recording period. These nonclinical safety pharmacology assessments support the evaluation of this host-targeted broad-spectrum antiviral drug candidate in clinical studies.

Introduction

Drug candidates targeting host proteins critical for viral replication often have broad-spectrum activity as multiple viruses are dependent on the same host factors for replication. The recent coronavirus pandemic has highlighted the importance of broadly active, host-targeted, and orally bioavailable antiviral drugs as an important strategy to combat future viral pandemics¹.

The iminosugar UV-4 (N-(9-methoxynonyl)-1-deoxynojirimycin, or (2R,3R,4R,5S)-2-(hydroxymethyl)-1-(9-methoxynonyl)piperidine-3,4,5-triol, also called MON-DNJ) inhibits the activity of both endoplasmic reticulum (ER) α-glucosidases I and II and is a potent, host-targeted antiviral (HTAV) candidate with demonstrated in vitro activity across a diverse set of enveloped viruses². UV-4, and its hydrochloride salt form known as UV-4B, is potent against diverse dengue virus strains in vitro and promotes complete survival in a lethal dengue virus mouse model^2,3. UV-4 has also been shown to provide a survival benefit in lethal and sub-lethal mouse models of influenza A and B infection⁴. UV-4 was shown to be highly efficacious via oral gavage against both oseltamivir - sensitive and -resistant influenza A (H1N1) infections in mice, even when treatment was initiated as late as 48-72 hours after infection^4,5. One of the attractive aspects of HTAV drugs is low risk for development of viral resistance to the therapy, as virally encoded processes are not targeted by the therapy. Lack of viral resistance following treatment with UV-4 has been demonstrated for dengue virus in vivo and influenza virus in vitro^6,7.

The safety of antiviral drug candidates which target host cellular pathways has not been well established, as most approved antiviral therapies act on essential viral components (direct-acting antivirals). Only a few HTAVs have been approved by the FDA and most are based on interferons targeting chronic infections^1,8. The α-glucosidase inhibitor celgosivir has been shown to be generally safe and well tolerated; however, it was not effective in reducing viral load or fever in patients with dengue infections⁹. Five iminosugar drugs have been approved, supporting the safety of this chemical class for chronic use for several diseases other than viral infection: miglustat (Zavesca®) for the treatment of Gaucher disease and Niemann-Pick Type C¹⁰; migalastat (Galafold™) for the treatment of Fabry disease¹¹; and miglitol (Glyset®), acarbose (Precose®), and voglibose (Basen®) for the treatment or prevention of type II diabetes mellitus¹². Nonclinical studies conducted to evaluate the potential general toxicology of UV-4 prior to initial clinical safety testing have been previously reported¹³. The toxicological and toxicokinetic findings in the nonclinical safety studies¹³ were used to inform dose level selection for nonclinical safety pharmacology studies reported here.

The safety of UV-4, dosed as the hydrochloride salt, was evaluated in a preliminary in vitro screening assay of “off-target” binding to receptors and enzymes and in two in vitro hERG potassium channel assays, in a GLP central nervous system study in the mouse (a modified Irwin test conducted as part of a 14-day toxicology study), in a GLP respiratory safety study in conscious mice using whole body plethysmography, and in a GLP cardiovascular safety studies in conscious, radiotelemetry-instrumented beagle dogs. The nonclinical safety pharmacology studies for UV-4 reported here conformed to ICH Guidance S7A Safety Pharmacology Studies for Human Pharmaceuticals¹⁴ and S7B: The Nonclinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals¹⁵.

Materials and Methods

Test article

UV-4 has a chemical structure consisting of a 1-deoxynojiriimycin (DNJ) piperidine ring and a 9-carbon alkyl methoxy side chain attached to the nitrogen of the DNJ ring. The hydrochloride salt form of UV-4 (also referred to as UV-4B) has higher aqueous solubility than the free base (>2.4 g/mL), which is likely to impart improved dissolution characteristics for an eventual drug product and provided reliable and high bioavailability in prior nonclinical studies. The UV-4 hydrochloride salt used in these studies was manufactured by SAI Life Sciences Ltd (Karnataka, India) using current Good Manufacturing Practices. The concentrations and doses used in all studies were calculated and expressed as the active free base form, UV-4. This methodology is consistent with USP<1121>, which recommends that the strength of a drug product or preparation isexpressed in terms of the active moiety.

Test item preparation

For in vitro hERG testing in HEK293 cells stably expressing hERG, stock solutions of UV-4 hydrochloride and the positive control terfenadine were prepared in dimethyl sulfoxide (DMSO) and stored frozen. UV-4 and terfenadine working concentrations for testing were prepared fresh daily by diluting stock solutions into a HEPES-buffered physiological saline (HB-PS) solution (composition in mM): NaCl, 137; KCl, 4.0; CaCl₂, 1.8; MgCl₂, 1; HEPES, 10; glucose, 10; pH adjusted to 7.4 with NaOH. Since previous results showed that ≤ 0.3% DMSO does not affect potassium channel current, all test and control solutions contained 0.3% DMSO. For in vitro hERG testing in Chinese Hamster Ovary (CHO) cells, stock solutions of UV-4 were prepared in 120 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 10 mM HEPES, 10 mM glucose, pH 7.4 ± 0.05 (EC NaCl). In the GLP-compliant study UV-4 formulations were analyzed to confirm stability, homogeneity and concentration verification.

For animal studies, UV-4 hydrochloride formulations were prepared according to the mixing procedure by dissolving in deionized water to the desired concentration and apportioned for daily use. Correction factors for calculation of UV-4 dose where based on salt weight and assayed purity of each batch of UV-4 hydrochloride. The batch-specific correction factor was 1.178 for the mouse respiratory and nervous system safety studies. A correction factor of 1.13 was used for the dog cardiovascular safety study. All dose formulations were stored in a refrigerator, set to maintain 2 to 8°C (unless made on the day of dosing), until removed for dosing.

Dose formulations prepared on the day of use were stored at room temperature prior to dosing. Formulations were stored protected from light.

Animals

Mice [Crl:CD1(ICR)] for the modified Irwin screening and CD-1 for respiratory function assessments were obtained from Charles River Laboratories, (Portage, MI) and Harlan (UK), respectively. Beagle dogs for cardiovascular safety assessments were obtained from Covance Research Product Inc. (Cumberland, VA). The Institutional Animal Care and Use Committee at each testing facility approved the use of animals prior to study initiation. All work was performed in compliance with the Animal Welfare Act, the requirements set forth by the United States Department of Agriculture (USDA),the principles outlined in the National Research Council’s Guide for the Care and Use of Laboratory Animals¹⁶ and the United Kingdom Animals (Scientific Procedures) Act¹⁷.

Preliminary in vitro Pharmacology

Potential off-target binding of UV-4 was evaluated in a standard high throughput screening-compatible panel of targets (SafetyScreen 44) at Eurofins Cerep Inc. (Redmond, WA). The targets included within the SafetyScreen 44 include G-protein coupled receptors, transporters, ion channels, nuclear receptors, a sentinel kinase (Lck kinase) and several non-kinase enzymes (COX-1 and COX-2, acetylcholinesterase, and two phosphodiesterases). The standard concentration of test article recommended by Cerep for this screening assay (10 μM) was used for these studies on UV-4. Compound binding was calculated as a % inhibition of the binding of a radioactively labeled ligand specific for each target. Compound enzyme inhibition effect was calculated as a % inhibition of control enzyme activity. In each experiment and if applicable, the respective reference compound was tested concurrently with UV-4, and the data were compared with historical values determined at Cerep.

Cardiovascular System Safety

In Vitro Evaluation of the Effect of UV-4 on hERG Potassium Channel Over-Expressed in Chinese Hamster Ovary (CHO) Cells

The in vitro inhibitory effects of UV-4 on the potassium-selective rapidly activating, delayed rectifier cardiac potassium (I_Kr) current was evaluated using CHO cells stably transfected with hERG, employing the whole cell patch clamp technique¹⁸. CHO cells stably transfected with hERG were treated with UV-4 at final concentrations of 0, 30, 100 and 300 μM. Propafenone hydrochloride (10 μM), an inhibitor of hERG current and other voltage dependent channels, was used as a positive control. The EC NaCl solution was used as the vehicle control. Patch clamp experiments were carried out using a Flyscreen® 8500 automated patch clamp (Flyion®, Tubingen, Germany) with an EPC-10 amplifier (HEKA Electronik, Lambrecht/Pfalz, Germany) and Flyion’s (Flyscreen® suite) software. The effect of each test article on I_Kr current was monitored continuously for approximately 600 seconds. The system performed on-line analysis for several parameters which included functions that acquired data from traces and functions that performed calculations on the results of other functions. Percent inhibition was calculated by comparing the I_Kr current before and after addition of test compound once a steady-state current had been attained. The study was conducted at Advinus Therapeutics Limited, (Bangalore, India) in compliance with OECD Principles of Good Laboratory Practice for the testing of chemicals as specified by International [C (97) 186/Final] Legislation¹⁹

Effect of UV-4 on Cloned hERG Potassium Channels Expressed in HEK293 Cells (GLP)

The in vitro effect of UV-4 on the hERG I_Kr current in stably transfected HEK293 cells was measured at near-physiological temperature in a GLP-compliant study. Cells were transferred to the recording chamber and superfused with vehicle control solution. Pipette solution for whole cell recordings was (composition in mM): potassium aspartate, 130; MgCl₂, 5; EGTA, 5; ATP, 4; HEPES, 10; pH adjusted to 7.2 with KOH. Pipette solution was prepared in batches, aliquoted, stored frozen, and a fresh aliquot thawed each day. The recording chamber and bathing solution was maintained at room temperature. Patch pipettes were made from glass capillary tubing using a micropipette puller (Sutter Instruments, CA). A commercial patch clamp amplifier was used for whole cell recordings. Before digitization, current records were low-pass filtered at one-fifth of the sampling frequency. Three concentrations of UV-4 (10, 100 and 1000 μM) were evaluated in this study. Each concentration was tested in three cells (n = 3). The positive control terfenadine was tested at 60 nM in two cells (n = 2). HEPES-buffered physiological saline was used as the vehicle control. The cells were held at −80 mV. Onset and steady state inhibition of hERG potassium current due to UV-4 were measured using a pulse pattern with fixed amplitudes (conditioning prepulse +20 mV for 1 s; repolarizing test ramp to −80 mV (−0.5 V/s) repeated at 5 s intervals). Each recording ended with a final application of a supramaximal concentration of the reference substance (E-4031, 500 nM) to assess the contribution of endogenous currents. The remaining uninhibited current was subtracted off-line digitally from the data to determine the potency of the test substance for hERG inhibition. Data acquisition and analyses were performed using the suite of pCLAMP (ver. 8.2) programs (MDS-AT, Sunnyvale, CA) Steady state was defined by the limiting constant rate of change with time (linear time dependence). The steady state before and after UV-4 application was used to calculate the percentage of current inhibited at each concentration. This study was conducted at ChanTest Corporation (Cleveland, OH) according to a standard protocol and GLP regulations.

Cardiovascular Safety Pharmacology Evaluation of UV-4 Administered by Oral Gavage to Telemetry Instrumented Conscious Dogs (GLP)

Four male dogs were assigned to a Latin square design. Data Sciences International Surgical implantation of the telemetry devices was completed at least 2 weeks prior to study initiation. An electrocardiogram (ECG), pressure, and temperature transmitter (Model No. TL11M2-D70-PCT) was implanted into the abdomen and sutured to the abdominal wall. The ECG leads of the transmitter were arranged in an approximate Lead II configuration. The pressure catheter tip was placed in the aorta. Dataquest® OpenART® telemetry equipment was used to generate and acquire the data input. This system transferred the data to a PONEMAH [P3P (Ponemah Physiology Platform)] analysis system. On Days 1, 8, 15, and 22 of the dosing phase, each dog received either vehicle control (deionized water) or 10, 50, or 200 mg/kg. Assessment of general health was based on mortality, clinical observations, qualitative food consumption, body temperature, and body weight. For each dose period, ECG, blood pressure, and body temperature measurements were recorded for at least 90 minutes prior to dosing and continuously for at least 25 hours after dosing. Electrocardiographic, hemodynamic, and body temperature data were analyzed by a modified Latin square design analysis of covariance, with repeated measures (time) embedded within each treatment period^{20, 21}. The covariate was the mean baseline observation for each subject on each dosing day. The variance-covariance structure used in the analysis was the one providing the smallest Akaike Information Criterion out of the four: compound symmetry, heterogeneous compound symmetry, autoregressive, and heterogeneous autoregressive. Postdose telemetry data were divided into analysis blocks based on photoperiods. The between-subject factor was Treatment, with Time as the within-subject factor in the model. When the main effect, Treatment, was significant and the Treatment x Time interaction was not significant, the data were averaged and analyzed across all time points in that block; when the Treatment x Time interaction was significant, the data were analyzed at each time point in that block. For the analyses, where applicable, a Dunnett-Hsu adjusted t-test²² was used for group comparisons when indicated by a significant Treatment effect. Group comparisons were each of the treatment groups to the control group. A significance level of 0.05 was used for all testing. The arithmetic mean, standard deviation, covariate-adjusted mean, and sample size for each treatment group were calculated at each time point for all applicable parameters. This study was conducted by Covance Laboratories Inc. (Madison, WI) according to GLP regulations.

Respiratory System Safety

Measurement of Respiratory Parameters in the Freely Moving Conscious Mouse Using Whole Body Plethysmography After Dosing With UV-4 (GLP)

Four groups of male CD-1 mice (6/group) were placed in whole body plethysmograph boxes to acclimatize and set baseline levels for at least 1 hour. The animals were free to move within the box. Respiratory parameters (tidal volume, rate of respiration and minute volume) were measured, derived and recorded using the eDacq , Electro-Medical Measurement Systems (EMMS) data acquisition system (Bordon, Hampshire UK). After setting baseline levels, the animals were removed from the plethysmograph boxes, orally dosed (at doses equivalent to 0 (vehicle control), 10, 100 or 1000 mg of UV-4/kg) and returned to their designated plethysmograph boxes. Respiratory parameters were measured continuously for a 4-hour period. Treatment groups were compared to the vehicle control group. For each post-dose time period, the recorded respiratory parameters (tidal volume, rate of respiration and minute volume) were analysed using one-way analysis of variance (ANOVA). Levene’s test for equality of variances among the groups was performed. Where this showed no evidence of heterogeneity (P≤0.01), pairwise comparisons with vehicle control were made using Dunnett’s test^23,24. Where Levene’s test²⁵ showed evidence of heterogeneity (p< 0.01) a rank transformation was applied to the data prior to analysis. This study was conducted by Covance Laboratories Inc. (North Yorkshire, UK) according to GLP regulations.

Nervous System Safety

Modified Irwin Screening (GLP)

The modified Irwin screening²⁶ test was conducted as part of a GLP 14-day repeat dose toxicology study at Covance Laboratories Inc. (Madison, WI). Male and female Crl:CD1(ICR) mice received UV-4 solution via oral gavage equivalent to 0, 25, 100 or 250 mg/kg three times per day (TID; 10 mL/kg/dose; every 8 hours ± 30 minutes) for total daily doses of 0, 75, 300 or 750 mg/kg/day, respectively. Modified Irwin Screening including body temperature was conducted on 6 male and 6 female mice in each dose group once prior to dosing and approximately 15 minutes post the first daily dose on Day 2. Each animal was evaluated during handling (hand-held observations) and in an open field (open-field observations) and assessed for sensory reactivity to stimuli (elicited responses).

Home cage observations included visual assessment of respiratory rate and activity. Hand-held observations included examination of skin color, cutaneous blood flow, body tone, appearance of fur, excessive lacrimation, excessive salivation, reactivity to handling and vocalization. For open-field observations, each animal was placed into an open-field arena for approximately 1 minute. Animals were observed for the following: number of fecal boli, fecal type, number of urine pools, locomotor activity, posture, gait abnormalities, tail elevation and other unusual behavior. Elicited responses included approach response, touch response, auditory startle response, visual response, nociception, pinna response, corneal response, pupillary status, pupillary response, catalepsy, righting reflex, and grip strength.

Results

Preliminary in vitro Pharmacology

The SafetyScreen 44 screening did not identify an inhibition or stimulation higher than 50%, the level considered to represent significant effects of the test compounds. For the receptors, ion channels, and transporters, the binding assay results ranged from −42.1% to 11.3% of control specific binding. For the enzymes, inhibition results ranged from −11.5% to 22.7% of control values.

Cardiovascular System Safety

In Vitro Evaluation of the Effect of UV-4 on hERG Channel (IKr), Over-Expressed in Chinese Hamster Ovary (CHO) Cells

UV-4, at concentrations of 30, 100 and 300 μM, demonstrated hERG current inhibition values of 4.05, 6.85 and 3.45 %, respectively. The vehicle control (0.1% DMSO) showed an inhibition of 8.85%. The result of the positive control, propafenone hydrochloride, confirmed the sensitivity of the test system to hERG inhibition (96.25%). The 50% inhibitory effect (IC₅₀) of UV-4 on hERG potassium current was not determined but is estimated to be greater than 300 μM.

Effect of UV-4 on Cloned hERG Potassium Channels Expressed in Human Embryonic Kidney Cells

UV-4, at concentrations of 10, 100 and 1000 μM (n=3 for each) demonstrated hERG current inhibition values of 0.2 ± 0.4%, 1.1 ± 1.1% and 21.2 ± 1.0% respectively. Under similar conditions, the positive control (60 nM terfenadine) inhibited hERG potassium current by 80.9 ± 2.5% (n=2). The hERG inhibition at 1000 μM was statistically significant (P < 0.05) when compared to vehicle control values. The IC₅₀ for the inhibitory effect of UV-4B on hERG potassium current was not determined but is estimated to be greater than 1000 μM.

Cardiovascular Safety Pharmacology Evaluation of UV-4 Administered by Oral Gavage to Telemetry Instrumented Conscious Dogs (GLP)

All four dogs survived to the end of the study. All four dogs had liquid feces at 50 or 200 mg/kg UV-4 dose level and one dog each at the 50 and 200 mg/kg dose level also had non-formed feces. No other UV-4-related clinical observations or effects on body weight or food consumption occurred during the dosing phase. UV-4 had no effect on abdominal body temperature.

There were no drug-related abnormal ECG waveforms or arrhythmias, and no drug-related effects on ventricular depolarization (QRS duration) or the time from the depolarization of the sinus node to the onset of ventricular depolarization (PR interval).

Systolic pressure (Figure 1 A) decreased in a dose-dependent manner from 1 to 2 hours post dose (maximum −11%) and increased from 10 to 19 hours post dose (+5%). At the 200 mg/kg dose, diastolic pressure (Figure 1 B) increased from 2 to 4 hours (maximum +15%) and from 10 to 19 hours post dose (+7%). At 50 mg/kg, UV-4 increased diastolic pressure at 3 hours (+8%) and 10 to 19 hours post dose (+5%). Arterial pulse pressure was decreased from 1 to 5 hours post dose (maximum −25%); at 50 mg/kg, UV-4 decreased arterial pulse pressure (Figure 1 C) from 1 to 3 hours post dose (maximum −17%) and at 200 mg/kg it also increased mean arterial pressure (Figure 1 D) from 10 to 19 hours post dose (+5%). The overall effects of UV-4 administration in dogs resulted in substantially decreased arterial pulse pressure, increased diastolic pressure, and decreased then increased systolic pressure.

Figure 1.

GLP Dog Study cardiovascular parameters and ECG data After Dosing with UV-4 Cardiovascular parameters were measured in male dogs with implanted telemetry devices. The dogs were dosed using a Latin square design with 0 (control), 10, 50, or 200 of UV-4/kg via oral gavage. The time course for potential changes for (A) systolic pressure, (B) diastolic pressure, (C) arterial pressure, (D) mean arterial pressure (E) QT interval, (F) QTc interval and (G) heart rate was monitored for 25 hours post-dosing.

At 200 mg/kg, the time from the beginning of the QRS complex to the end of the T wave (QT interval) (Figure 1 E) was shortened by as much as 6% from 1 to 7 hours post dose, but the effect was considered secondary to increased heart rate and physiologically normal. Also at 200 mg/kg, heart rate-corrected QT (QTc) interval (Figure 1 F) was 4% longer than controls at 3 hours post dose, and all dose levels were associated with shorter QTc intervals from 10 up to 25 hours post dose (up to 4, 5, and 7% at doses equivalent to 10, 50, and 200 mg/kg, respectively). These small QT and QTc interval differences were considered normal physiological consequences of changes in heart rate and physiologically unimportant by the study toxicologist. Heart rate (Figure 1 G) increased from 1 to 7 hours post dose (+17%) post dose with 50 and 200 mg UV-4/kg, and decreased from 10 to 19 hours post dose at 10, 50 and 200 mg/kg (−9, −6, and −14%, respectively). Heart rate appeared to reflexively increase then decrease, most likely in two phases, driven initially by lower pulse pressure from 1 to 7 hours post dose then subsequently by what may have been a residual increase in diastolic pressure from 10 to 19 hours post dose. Although statistical significance was reached compared to the vehicle control for the above-noted parameters, most of these parameters remained within the normal physiological range. After dosing with UV-4 at 10 mg/kg, all parameters remained within normal physiological ranges for healthy dogs²⁷. After the 50 or 200 mg/kg doses, with the exception of decreased arterial pulse pressure, all parameters remained within normal physiological ranges for healthy dogs²⁷. In addition, all hemodynamic parameters returned to control levels by the end of the telemetry recording at 25 hours post dose.

Respiratory System Safety

Measurement of Respiratory Parameters in the Freely Moving Conscious Mouse Using Whole Body Plethysmography After Dosing With UV-4

Oral administration of UV-4 at a dose of 10 mg/kg had no statistically significant effect on respiratory parameters when compared to the vehicle-treated group. Following oral administration of UV-4 at 100 mg/kg, statistically significant decreases (P<0.05) in tidal volume (Figure 2 A) were evident at 0 to 60, 76 to 90, 121 to 150, 166 to 180, and 211 to 225 minutes post-dose and (P<0.01) at 91 to 120, 151 to 165, and 181 to 210 minute post-dose. This was accompanied by statistically significant increases (P<0.05) in respiratory rate volume (Figure 2 B) at 106 to 120 and 166 to 180 and (P<0.001) at 91 to 105 minutes, and a statistically significant increase (P<0.05) in minute volume (Figure 2 C) at 91 to 105 minutes post-dose when compared to the vehicle-treated group. Overall the baseline value for tidal volume for this group (100 mg/kg UV-4) was slightly less than the baseline value for the vehicle group, and the individual values for tidal volume were within the range of values for the vehicle group. Following oral administration of UV-4 at 1000 mg/kg, statistically significant increases (P<0.05) in rate of respiration and minute volume were evident at 91 to105 minutes post-dose when compared to the vehicle-treated group. Due to a lack of a dose response and the small magnitude of the changes seen, these changes were not judged to be adverse within the context of this study.

Figure 2.

Respiratory Parameters After Dosing with UV-4 Respiratory parameters [(A) tidal volume, (B) respiratory rate, (C) minute volume] were measured by whole body plethysmograph in mice orally dosed with 0 (control), 10, 100, or 1000 mg of UV-4/kg. Respiratory parameters were measured continuously for a 4-hour period.

Nervous System Safety

Modified Irwin Test in the Mouse

Results for qualitative (yes/no) or quantitative parameters for testing are shown in Tables 1 and 2, respectively. UV-4, at doses of 75, 300, or 750 mg/kg/day, did not have any effect on neurobehavioral parameters in mice.

Table 1.

Qualitative (Yes/No) Parameter Results for Modified Irwin Testing

Parameter	Dose mg/kg/day
	0		75		300		750
Sex	Male	Female	Male	Female	Male	Female	Male	Female
N	6	6	6	6	6	6	6	6
Home Cage Observations
Respiratory Rate, Appropriate	6	6	6	6	6	6	6	6
Activity, Moderate	6	6	6	6	6	6	6	6
Hand Held Observations
Skin Color, Normal	6	6	6	6	6	6	6	6
Cutaneous Blood Flow, No Blanching	6	6	6	6	6	6	6	6
Body Tone, Moderate	6	6	6	6	6	6	6	6
Appearance of Fur, Groomed	6	6	6	6	6	6	6	6
Piloerection, Absent	6	6	6	6	6	6	6	6
Excessive Lacrimation, None, Eyes	6	6	6	6	6	6	6	6
Excessive Salivation, None	6	6	6	6	6	6	6	6
Reactivity to Handling, Moderate	6	6	6	6	6	6	6	6
Reactivity to Handling, High	0	0	1	0	0	0	1	0
Vocalization, None	6	6	6	6	6	6	6	6
Vocalization, During Handling	0	0	1	0	0	0	1	0
Open Field Observations
Locomotor Activity, Moderate	6	6	6	6	6	6	6	6
Posture, Typical	6	6	6	6	6	6	6	6
Gait Abnormalities, None	6	6	6	6	6	6	6	6
Gait Abnormalities, Limited Use Right Hind Leg	0	0	0	1	0	0	0	0
Other Unusual Behavior, Absent	6	6	6	6	6	6	6	6
Fecal Type, Normal	6	6	6	6	6	6	6	6
Fecal Type, Non-formed	0	0	0	0	0	0	1	0
Tail Elevation, Horizontally Extended	6	6	6	6	6	6	6	6
Elicited Responses
Approach Response, Approaches	6	6	6	6	6	6	6	6
Approach Response, Avoidance	1	0	1	0	1	0	0	0
Touch Response, Normal	6	6	6	6	6	6	6	6
Auditory Startle Response, Visible Flinch	6	6	6	6	6	6	6	6
Visual Response, Before Whisker Contact	6	6	6	6	6	6	6	6
Pinna Response, No Response	1	0	0	0	1	0	0	0
Pinna Response, Ear Flattened/Head Shake	6	6	6	6	6	6	6	6
Corneal Response, Positive	6	6	6	6	6	6	6	6
Pupillary Status, Normal, Eyes	6	6	6	6	6	6	6	6
Pupillary Response, Both Pupils Constrict	6	6	6	6	6	6	6	6
Catalepsy, Absent	6	6	6	6	6	6	6	6
Righting Reflex, Positive	6	6	6	6	6	6	6	6
Nociceptive Reflex, Normal	6	6	6	6	6	6	6	6

Values are the number of mice demonstrating the designate response for each parameter

Table 2.

Quantitative Parameter Results for Modified Irwin Testing

	Dose mg/kg/day
Parameter	0		75		300		750
Sex	Male	Female	Male	Female	Male	Female	Male	Female
N	6	6	6	6	6	6	6	6
Number of Urine Pools	0±0.4	0±0.0	0±0.8	0±0.0	0±0	0±0.0	0±0	0±0.4
Number of Fecal Boli	0±0.8	1±1.0	1±0.8	2±1.9	0±0.5	0±0.5	0±0.4	0±0.4
Forelimb Grip Strength (g)	168±33	167±41	155±58	143±34	169±31	153±34	178±50	158±40
Hindlimb Grip Strength (g)	161±36	152±36	162±33	143±38	161±21	145±25	138±27	146±43
Body Temperature	37.5±0.7	37.8±0.4	38.1±0.3	37.8±0.4	37.7±0.5	37.8±0.2	37.3±0.9	37.6±0.5

Values are Means ± standard deviation

Discussion

The goal of these preclinical safety pharmacology studies, in conjunction with preclinical toxicology studies¹³, was to enable the IND application, and, provide support for the dose levels toinvestigate the safety and tolerability of single ascending oral doses of UV-4 delivered orally as solution of the hydrochloride salt in healthy human subjects. The preclinical testing program conformed with ICH M3(R2) “Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals²⁸ to support the planned Phase I clinical trial of a single ascending dose. Mice were used as the rodent species for the IND-enabling GLP toxicology studies¹³, including modified Irwin testing, as in vivo antiviral activity models predominantly utilize murine strains. Mice were also chosen as the rodent species for the respiratory safety evaluation for this reason. The mouse is a common species used for whole body plethysmography assessment of respiratory safety. Dogs historically have been used in safety evaluation studies and are accepted by the appropriate regulatory agencies.

Initial screening of UV-4 for potential pharmacologic effects and cardiovascular system effects were conducted using established in vitro models. The 44 selected targets evaluated in the SafetyScreen 44 were recommended by four major pharmaceutical companies²⁹. For this testing, results showing an inhibition (or stimulation for assays run in basal conditions) higher than 50% are considered to represent significant effects of the test compounds. The SafetyScreen 44 testing indicated no significant liability for adverse pharmacological effects of UV-4 on any of the potential pharmacological targets.

The hERG channel current is a surrogate for IKr, the rapidly activating delayed rectifier cardiac potassium current, in human ventricles ²⁹. This channel was selected for evaluation because inhibition of IKr is the most common cause of cardiac action potential prolongation by non-cardiac drugs, ^{30, 31,32}. Increased action potential duration causes prolongation of the QT interval and has been associated with a dangerous ventricular arrhythmia, torsade de pointes²¹. The IC₅₀ of UV-4 in CHO and HEK293 cells stably expressing hERG was not able to be determined because 50% inhibition was not observed at the highest concentrations tested. The estimated IC₅₀ value of greater than 1000 μM (319 μg/mL) in human HEK293 cells and greater than 300 μM in CHO cells far exceeds drug level concentrations (Cmax 5.1 μg/mL) predicted in humans³⁴ and is not considered of clinical relevance.

Cardiovascular function was assessed by an evaluation of hemodynamic and ECG parameters. Isolated ECG segments were qualitatively reviewed for rhythm abnormalities and disturbances. Qualitative assessments included, but were not limited to normal sinus rhythm variations, abnormal sinus rhythms, conductance or repolarization abnormalities, bradycardia, and tachycardia^{35, 36}. Changes in heart rate, including periods of sinus tachycardia, sinus bradycardia, and sinus arrhythmia, are expected in telemetry recordings in freely moving animals and are considered normal variants^{35, 36}. Transient periods of sinus tachycardia, sinus bradycardia, and/or sinus arrhythmia were considered normal physiologic observations and were not included in the results unless considered test article-related.

The potential effects of UV-4 were evaluated at concentrations expected to be at or above the therapeutic dose level. Clinical signs including vomitus, soft feces, and liver enzyme changes were observed at the 200 mg/kg dose level in previous toxicology studies in dogs while only minimal changes were observed at 50 mg/kg. The dose of 10 mg/kg was based on the human clinical dose and the no observed adverse effect level¹³. No abnormal ECG waveforms or arrhythmias were attributed to UV-4, and administration of UV-4 had no effect on QRS duration or PR interval. Noted small differences in QT and QTc intervals were considered normal and secondary to changes in heart rate. Decreased arterial pulse pressure after 50 or 200 mg/kg was the only change outside the normal range, and all hemodynamic parameters returned to control levels by the end of the telemetry recording period. The GLP cardiovascular safety study did not include PK sampling. Results for male dogs from Day 1 of the 14-Day toxicokinetic study showed a Cmax of 34.4 μg/mL, half-life of 2.32 hr and AUC_(0-8hr) of 69,590 ng·hr/mL at a similar dose of 60 mg/kg.

The respiratory safety of UV-4 was evaluated in freely moving, conscious mice using whole body plethysmography. Oral administration of a single dose of UV-4 (at doses of 10, 100, or 1000 mg/kg) was not judged to have caused biologically significant or adverse effects on respiratory parameters in mice over a 4 hour post dose period when compared to a group of vehicle treated mice. The neurological safety of UV-4 evaluated using a modified Irwin test showed no effect of UV-4 on neurobehavioral parameters in mice at doses of 75, 300, or 750 mg/kg/day.

Iminosugars are being investigated for their potential use as broad-spectrum HTAV therapeutics, mediated via a mechanism involving inhibition of ER α-glucosidases leading to misfolding of critical viral glycoproteins^{37, 38}. The misfolding of viral glycoproteins reduces infectivity by producing defective virus particles or targeting the glycoproteins for degradation. In general, antiviral drugs targeting viral genetic sequences or viral encoded proteins quickly select for resistant viruses within a few replication cycles. By targeting the activity of a host protein such as ER α-glucosidases, iminosugars do not expose the virus to direct selective pressure, but deny the virus a critical host pathway for which there is no viral gene-encoded alternative. This has been shown to result in low likelihood of development of resistance^{6,7, 39}. However, it has been hypothesized that host-targeted drug candidates may have increased risk of toxicity due to disruption of cellular pathways in both infected and healthy cells¹.The safety pharmacology data reported here indicate this hypothesis must be evaluated on a case-by-case basis, and support the potential use of UV-4 therapeutically either up to TID for multiple days or as single dose. In summary, in vitro and in vivo safety pharmacology studies showed that there were no observed adverse treatment-related effects following administration of UV-4 in the hERG potassium channel, cardiovascular assessment in dogs, respiratory function in mice, and neurobehavioral parameters in mice.

Abbreviations:

ANOVA

analysis of variance

CHO

Chinese Hamster Ovary cells

CNS

central nervous system

DNJ

1-deoxynojiriimycin

DMSO

dimethyl sulfoxide

ECG

electrocardiogram

HB-PS

HEPES-buffered physiological saline

HTAV

host-targeted antiviral

hERG

human ether-à-go-go-related gene

I_Kr

the potassium-selective rapidly activating, delayed rectifier cardiac potassium current

IC₅₀

50% inhibitory concentration

ICH

International Conference on Harmonisation

IND

Investigational New Drug

QRS duration

ventricular depolarization

QT

interval from the beginning of the QRS complex to the end of the T wave

QTc

heart rate-corrected QT interval

PR interval

the time from the depolarization of the sinus node to the onset of ventricular depolarization

TID

three times per day

UV-4

N-(9-methoxynonyl)-1-deoxynojirimycin, or (2R,3R,4R,5S)-2-(hydroxymethyl)-1-(9-methoxynonyl)piperidine-3,4,5-triol, also called MON-DNJ

UV-4B

UV-4 hydrochloride salt