Dual Effects of Nanoviricides Platform Technology Based NV-CoV-2 Biomimetic Polymer Against COVID-19

Remdesivir (RDV) is the only antiviral drug so far approved for COVID-19 therapy by the FDA. However its efficacy is limited in vivo due to its low stability in presence of plasma. This paper compared the stability of RDV encapsulated with our platform technology based polymer NV-387 (NV-CoV-2), in presence of plasma in vitro and in vivo. Furthermore, a non- clinical pharmacology studies of NV-CoV-2 (Polymer) and NV-CoV-2-R (Polymer encapsulated Remdesivir) in both NL-63 infected and uninfected rats were done. In an in vitro cell culture model experiment, antiviral activity of NV-CoV-2 and NV-CoV-2-R are also compared with RDV. The results are (i) NV-CoV-2 polymer encapsulation protects RDV from plasma- mediated catabolism in vitro and in vivo, too. (ii) Body weight measurements of the normal (uninfected) rats after administration of the test materials (NV-CoV-2, and NV-CoV-2-R) show no toxic effects on them. (iii) NL-63 infected rats body weights and their survival length were like uninfected rats after treatment with NV-CoV-2 and NV-CoV-2-R, and the efficacy as an antiviral regimen were found in the order as below: NV-CoV-2-R > NV-CoV-2 > RDV. In brief, our platform technology based NV-387-encapsulated-RDV (NV-CoV-2-R) drug has a dual effect on coronaviruses. First, NV-CoV-2 itself as an antiviral regimen. Secondly, RDV is protected from plasma-mediated degradation in transit, rendering altogether the safest and an efficient regimen against COVID-19.


Introduction
There are seven coronaviruses, so far, were identified, that infect humans, and only 4 of them belong to the beta family of coronavirus (HCoV-HKU1, SARS-CoV-2, MERS-CoV and SARS-CoV) [1]. SARS family were known to cause severe respiratory disease in humans. In fact, SARS-CoV-2 infection caused a pandemic COVID-19 disease with high morbidity and mortality [2]. Nanoviricide is a platform-technology-derived biomimetic polymer that can bind to a virus-specific ligands [3]. The flexible polymer backbone is comprised of a polyethylene glycol (PEG) and alkyl pendants. Different virus-specific ligands can be conjugated to this PEGbased polymer backbone to produce different individual drug substances that specifically target different viral pathogens.
Viruses bind to specific cell surface ligands in order to attach to cells and internalize. A nanoviricide is designed to act like a decoy of a human cell. When the virus sees the appropriate ligand displayed on a nanoviricide micelle, the virus is believed to bind to it. The nanoviricide, being flexible, could allow maximization of binding by spreading onto the virus particle, and potentially leading to fusion with the lipid-coated virus surface by a phase-inversion, wherein the fatty core of the nanoviricide could merge with the viral lipid coat and the hydrophilic shell of the nanoviricide could become the exterior of the particle, thus potentially engulfing the virus [4]. In the process, the coat proteins that the virus uses for binding to cells would be expected to become unavailable. This highly targeted attack may lead to loss of the coat proteins and the nanoviricide may further dismantle the engulfed virus capsid. The loss of virus particle integrity could render the virus non-infectious. This proposed mechanism of action is shown graphically in Figure 1. Support for this mechanism is shown in the electron photomicrographs in Figure 2 in which murine cytomegalovirus (MCMV) was incubated with a nanoviricide. As can be seen in the resulting CMV naked capsids are non-infectious. Thus, nanoviricide binding renders the CMV not only inactive but appears to result in the disruption of capsid organization [5]. Given this proposed mechanism of action, nanoviricides are not expected to interfere with the intracellular replication of the virus to any appreciable extent. [ Here we will be reporting the various aspects of this drug: (i) It's antiviral activity in vitro cell culture model and in vivo rat animal model; (ii) It's capability of increasing the half-life of RDV in presence of plasma in vitro and in vivo; (iii) and some safety aspects of this drug.

Methods:
Materials: The drug substance, NV-CoV-2 was made in our lab. Based on polyethylene glycol of 22 repeat units (m) substituted by 2 hexadecylamine (R 1 ) the theoretical molecular formula for the repeat unit of the polymeric drug substance (NV-CoV-2) is C 104 H 188 N 2 O 44 S 4 (Much details of the chemistry of the drug can not be disclosed at this point due to patent right obligation) . It is an off-white, waxy non-crystalline semi-solid at room temperature, has no noticeable smell and it has a slight sour test.

Quantitative composition of Drug Product NV-CoV-2 Solution for Injection:
The drug product NV-CoV-2 solution for injection is an injectable dosage form that will be supplied in a single vial as 10 ml of a 50 mg/ml solution of the drug substance and 40mg/ml D-mannitol (or quantity needed for osmolality) in water for injection. This solution is ready for injection to deliver 450 mg of drug substance NV-CoV-2 in 9 ml.

Pharmacokinetics:
Since NV-CoV-2 is composed of a flexible PEG-based polymer backbone, conventional methodologies to measure the pharmacokinetic properties of small molecules are not suitable for use with NV-CoV-2. We have developed a Direct ELISA to quantitatively detect NV-CoV-2 in biological matrices in order to measure its pharmacokinetic properties.
We have performed a non-GLP study to determine the levels of systemic exposure of NV-CoV-2 when NV-CoV-2 drug is administered to rats once daily for 5 days (0,1,3,5 and 7) over a 8-day period. Detection and Quantification of NV-CoV-2 in plasma samples was done by [ Direct ELISA. This assay method is a colorimetric ELISA to measure the levels of NV-CoV-2 in processed plasma samples. As described in the Table 1, thirty-six animals (three/sex/group) were administered NV-CoV-2, at three different doses each, or vehicle once daily, 5 times over an eight-day period on days 0,1,3,5 and 7. Injection on Day 0 is the 1st injection and on Day 7 is the 5th injection. Each compound was delivered via slow-push IV injection. Blood Samples for systemic exposure were collected at 0, 0.08, 0.5, 1, 2, 4, 8 and 24 hours after 1st and 5th injection of drug administration. Blood samples were taken from one animal per sex in each test article treated group at each time point. The same animal was used to draw blood across all the time points after Day 0 and Day 7 injections. Detection and Quantification of NV-CoV-2 in the plasma samples from each group was done by using a direct ELISA. The biotinylated detection antibody, 5G8-Biotin, used in this assay has been shown to be specific to NV-CoV-2. The Direct ELISA assay for NV-CoV-2 was previously characterized for linearity and detection limit. The limit of detection (LOD) of this assay is 0.25 μg/ml.

In vitro antiviral activities of NV-CoV-2:
The antiviral efficacy and potency, and the cytotoxicity of the antiviral compound NV-

In vivo antiviral activities of NV-CoV-2: Dose-response of NV-CoV-2 in rats infected with CoV-NL63
Male and female Sprague Dawley rats, 8 to 9 weeks old, were infected with 10 4 Cov-NL63 viral particles directly into the lungs. All untreated rats infected in this manner succumb to the disease in 5 to 6 days and, hence, used as a model for evaluation of the efficacy of NV-CoV-2. NV-CoV-2 and Vehicle were administered by tail vein I.V. injection (10 ml/kg), once per day, on Days 0, 1, 3, 5, and 7. RDV was administered by tail vein I.V. injection as per the standard administration protocol of twice for the first day and once daily after that for seven days. A group of infected, untreated rats was included as an additional control. The groups of animals and the treatments were shown in the  The rats were observed daily for clinical and behavior changes and body weight; moribund rats were sacrificed. The primary end points were survival and body weight loss.

Effect of Number of Days of Dosing of NV-CoV-2 in Rats Infected with CoV-NL63
Male and female Sprague-Dawley rats, 8 to 9 weeks old, were infected with 2 x 10 4 Cov-NL63 viral particles directly into the lungs. All untreated rats infected in this manner succumb to the disease in 5 to 6 days and, hence, used as a model for evaluation of the efficacy of NV-[ CoV-2. NV-CoV-2 was administered by tail vein I.V. injection, once per day, for different numbers of days of treatment, 1, 3 or 5 days beginning on Day 0. The vehicle control treated rats were treated for 5 days on Days 0, 1, 3, 5,and 7. A group of infected, untreated rats was included as an additional control. The groups of animals and the treatments were shown in the Table-3 below. The infected rats were treated with the same dose per injection, 600 mg/kg of NV-CoV-2 for the NV-CoV-2 treatment groups. Only the number of treatment days varied. The rats were observed daily for clinical and behavior changes, body weight; moribund rats were sacrificed.
The primary end points were survival and body weight loss.

Protection Efficiency of NV-CoV-2 encapsulation of RDV (NV-CoV-2-R) from plasma in vitro:
First, a standard curve of RDV was determined by using LC-MS at different concentrations of the standard solutions ranging from 0-5 ng/uL in DMSO:MeOH (1:9). As an internal standard (ISTD), 13 C 6 -RDV (2.5 ng/uL) was used. Extraction of RDV for LC-MS assay [ was done by using acetonitrile. Concentrations of RDV compared to their ratio with the ISTDs ( 13 C 6 -RDV) was used to generate a standard curve for RDV.
Secondly, ten microliter of the test materials (20 ng/uL) was incubated with 30 uL rat plasma for different time points (as indicated in the Result section), and then extracted with acetonitrile (100 uL). The mixture was centrifuged for 10 mins at 10,000 x g to separate the supernatants from the precipitates. The supernatants containing either RDV was determined by LC-MS chromatography as described Elsewhere [9].
The ratio of RDV and its isotope, 13 C 6 -ISTD (as an internal standard) was calculated. The amount of RDV was determined using the linear equation derived from their respective standard curve. The values were normalized using the dilution factor used for the original plasma sample.

Protection Efficiency of NV-CoV-2 encapsulated RDV (NV-CoV-2-R) in vivo:
Thirty-six Sprague Dawley male rats (Taconic Biosciences, USA) (three/ in control and in treatment groups) were administered with NV-CoV-2 or NV-CoV-2-R, once per day for 5 days (0, 1, 3, 5, and 7) over a 7-day time period. NV-376 (RDV-in-SBECD; Gilead) was given as two doses on day 1 followed by a daily doses through day 7. DMSO was used as a vehicle for the control group. Injection on day 0 is considered as a 1 st injection and on day 7 is the 5 th injection. Each compound was delivered via slow-push IV injection. Blood samples for systemic exposure assay were collected at 0, 0.08, 0.5, 1, 2, 4, 8 and 24 hours after 1 st and 5 th injection of the drugs.
Blood samples were taken from one animal in each test article treated group at each time point. Same animal was used to draw blood across all the time points after "day 0" and "day 7" injections. The detailed procedures and study design were stated in elsewhere [9]. RDV values obtained in rat plasma (mg/mL) after 1 st. and 5 th injection of the drugs were normalized by dividing with the amount of RDV administered (mg/kg of rat body weight). [

Safety pharmacology studies:
We have conducted safety pharmacology studies; core battery tests as defined in the ICH S7A guidelines on the respiratory and central nervous systems in the rat and the cardiovascular system in the monkey after intravenous administration of NV-COV-2.  and after the completion of the pulmonary evaluation, animals were placed (in groups) in a fixed environment consisting of a Plexiglas enclosure, fitted with a lid. The enclosure was placed on absorbent paper that detects excretions. In this environment, the rats were free to move about.

Central Nervous System and Respiratory System:
Observances for the presence of the following symptoms were made as a group in the Plexiglas enclosure. Animal number was documented when any of the following symptoms were noted except for excretion that were documented as a group observation: Increased or decreased nociceptive response was tested by tail pinch and corneal reflex and pinnal reflex was checked by a cotton swab and a blunt probe respectively. Immobility and grip strength for each animal was tested on an inclined wire screen.   NV-CoV-2 formulations were infused intravenously for only one time using a calibrated infusion pump into a saphenous vein over a period of 30 (± 2) minutes using a percutaneous i.v. catheter.

Rectal body temperatures
Animals were restrained during dosing. Animals were trained to the restrainers prior to dosing.

Method of Cardiovascular Evaluation:
Data were collected for approximately 24 hours before the first dosing day in order to establish acceptable baseline parameters for each animal. Baseline data were retained in the study file, but not included in the final report. Data were also collected for at least 24 hours prior [ to each subsequent dose to determine pre-dose values. Following dose administration, parameters were recorded continuously for approximately 24 hours. A washout period of at least 6 days was allowed between test article doses.
The following parameters were analyzed using the Ponemah 5.20 SP9 and ECG Analysis

Immunogenicity:
Since NV-CoV-2 is administered I.V., it is important to determine if there is any evidence of immunogenicity. A study was conducted for the detection of Anti-NV-CoV-2 antibodies in Rats with serum samples collected at 14 and 28 days following the start of 6 sequential I.V. injections of NV-CoV-2 over a period of 10 days, as described in the Table 7 below. Detection and quantification of anti-NV-CoV-2 antibodies in serum samples was performed using an Indirect ELISA assay. This assay method is a colorimetric ELISA to measure the levels Indirect ELISA method is specific and selective to the NV-CoV-2. Results:

1.Pharmacokinetics:
A typical time course of drug plasma concentration was observed over 24 hours. Results of the analyses are summarized in the Table 8 below with Observed Maximum Plasma Concentration of NV-CoV-2 (C max ), and the time to reach C max (t max ). The dose is expressed in mg/kg while the (C max ) and (t max ) are expressed in mg/ml and hours respectively.

In vivo antiviral activity of NV-CoV-2: Days and Dose-response of NV-CoV-2 in male rats infected with CoV-NL63 :
As shown in the Importantly, both treatments were also markedly better than RDV treatment alone.
Further, the survival of rats following a lethal dose of Cov-NL63 was dependent on the number of days and dosage of the drug used for the treatment (Figure 5). An increase in the number of days of treatment increased the survival days. The survival of rats administered NV-CoV-2 for 1 day was similar to that of untreated rats and administration of vehicle-treated rats, 6-7 days survival; injection for 3 days and 5 days increased survival to 13.4 days and 17.6 days, respectively.

The survival of rats after administration of NV-CoV-2-R for 1 day was slightly increased,
8.8 Days, compared to that of vehicle-treated rats, 7.7 days; injection for 3 days and 5 days increased survival to 13.9 days and 18 days, respectively ( Figure 6). Similar to treatment with NV-CoV-2, the increase in survival after 3 days and 5 days of treatment with NV-CoV-2-R was also reflected in the body weight changes over time post-infection. [

Body weight measurement:
The efficacy of NV-CoV-2, at both dose levels, on survival was also reflected in the body weight changes. Body weight loss increased significantly as the animals became moribund. As shown in the Table-  Thus, the treatment with NV-CoV-2 at both 160 and 320 mg/kg/injection of Days 0,1,3,5, and 7 had significant effect on survival in rats using a lethal model of infection with the human coronavirus CoV-NL63. Importantly, NV-CoV-2 treatment was clearly superior to daily treatment with Remdesivir.
In vitro, NV-CoV-2 polymer encapsulation protects RDV from plasma-mediated catabolism (Figure 7). In vivo study with male rats animal model, the results were shown in

Respiratory Parameter Results:
Effects of the vehicle and NV-CoV-2 (25, 50 and 100 mg/kg) on pulmonary parameters are shown in the Table 10 below. [ The intravenous administration of NV-CoV-2 at 25, 50 or 100 mg/kg did not induce any significant effect on respiratory rate, tidal volume or minute volume when compared to the vehicle control group.

Neurobehavioral Parameter Results:
As shown in the Table 11 (Table 11).
Body temperature, 1 hour after the administration of 100 mg/kg dose of NV-CoV-2 was slightly higher (p≤0.05) compared to the control group. Since the difference was marginal (<1.0°C), it may not be considered biologically relevant. Body temperature was not statistically different at 4 hours post-dose by the 100 mg/kg dose of NV-CoV-2. [

Cardiovascular Parameter Results:
Blood Pressure and Heart Rate: Random occurrence of fluctuations in blood pressure and heart rate are inherent in conscious freely moving animals. Hence consistent (2 or more time points) changes of 15% or more from vehicle controls were considered a significant (biologically relevant) change for blood pressure and heart rate.
[ No significant effects on blood pressure (systolic, diastolic and mean) or heart rate were observed after the intravenous administration of 25, 37.5 or 50 mg/kg doses of NV-CoV-2. The Figure-10 below shows the mean heart rate and the mean arterial pressure after the 50 mg/kg dose.

ECG:
All monkeys maintained sinus rhythm throughout the study. The intravenous administration of 25, 37.5 and 50 mg/kg NV-CoV-2 did not induce any significant effects on quantitative ECG parameters, as measured using the analysis software, in conscious telemetered cynomolgus monkeys.

Safety Pharmacology Conclusion:
The The intravenous administration of NV-CoV-2 at doses of 25, 37.5 and 50 mg/kg in conscious telemetered cynomolgus monkeys did not induce any significant, biologically relevant effects on heart rate, arterial blood pressure, cardiac rhythm, ECG parameters. All monkeys maintained sinus rhythm throughout the study.

Immunogenicity:
This assay detects all relevant Immunoglobulin (Ig) Isotypes. Since a non-mucosal route of administration was employed and there was no indication of anaphylaxis, the relevant isotypes would be IgG and IgM. The above screening assay is validated to determine lower limit of detection level of isotype IgG and IgM as a group, and is scientifically justified for quantification [ of IgG and IgM as a group, using a linear calibration curve. This ELISA has a reliable lower limit of detection of 15ng/ml at which all relevant Ig Isotypes are clearly distinguishable from negative control, and quantifiable with acceptable accuracy and precision.
Immunogenicity determination was done on all animals (4/sex/group) in duplicate following I.V. dosing of NV-CoV-2 in rats with serum samples collected at days 14 and 28 days following the start of the 6 sequential doses over 10 day period. The mean OD of all dose groups is not statistically distinguishable from the vehicle group, both sexes in both Day 14 and Day 28 animals. Under the conditions of the assay method, IgG and IgM antibodies against NV-CoV-2 were not detected and, therefore NV-CoV-2 was not shown to be immunogenic.

Discussion:
NV-CoV-2 is a nanomedicine that we call a "nanoviricide®" that is designed to attack most strains of coronaviruses in a broad-spectrum manner. It is composed of a number of antiviral small chemical ligands covalently attached to a micelle-forming polymeric chain. The polymeric chain is a homopolymer from a monomer that is composed of covalently attached polyethylene glycol (PEG), a connecting group, and alkyl pendants. This allows self-assembly of the polymeric chain in aqueous solutions with a PEG shell, a hydrophobic alkyl core, and the ligands being displayed on the outside. The ligand in this case is designed using molecular modeling to bind to the S-protein of SARS-CoV-2 at the same site as where the cognate cellular receptor, ACE2, binds. Thus the nanoviricide NV-CoV-2 was designed to neutralize ACE2binding coronaviruses such as SARS-CoV-1, SARS-CoV-2, and NL63. In practice, it also has activity against human coronavirus 229E (hCoV-229E), which uses Aminopeptidase N (APN) as its cellular receptor, and not ACE2. This appears to be because of certain conserved elements in the quaternary structures of ACE2 and APN that are both peptidases with different specificities.
The broad-spectrum activity of NV-CoV-2 against h-CoV-NL63 and hCoV-229E together indicate that it may continue to be active against a number of coronaviruses in spite of mutations.
The mechanism of action of NV-CoV-2 is not well defined but, as described above, the working hypothesis is to bind to free virion particles at multiple points and encapsulate the virus, disabling its ability to infect cells thus neutralizing the virus. This putative mechanism is [ orthogonal to the mechanism of action of most other anti-coronavirus agents in development (Fig. 1). In addition, it may be possible to achieve a stronger antiviral effect by combining NV-CoV-2 therapy with other anti-coronavirus therapies in the future.
The study exhibited no evidence of any adverse reactions in all groups of animals. All groups including the test article groups and the control groups tolerated the compounds with no clinical or behavioral changes observable, no evidence of any deviation from normality. The tolerability of NV-CoV-2 was the same as that of the vehicle control animals. There were no adverse reactions observed while and during the administration of the compounds or during the study period and at postmortem examination. The body fluids and fecal analysis showed no significant difference between the groups, except within the high dosage groups.
Histopathological examination showed no changes in any of the organs examined. Based on the results of this study, no Observable Adverse Effects on tolerability could be reported.
At present remdiesivir is the only drug that are approved by FDA to be used for COVID- [ Nonetheless, our platform technology based NV-CoV-2 encapsulated-RDV drug has a dual effect on coronavirus. Our polymer (NV-CoV-2) posses antiviral activity, and protects RDV more than Gilead-RDV, rendering RDV effectiveness against the virus. Furthermore, potential mutations in the virus are unlikely to enable it to escape these drug candidates, as it is a broad spectrum antiviral regimen.