key: cord-0960591-61gljdcm authors: Chakraborty, Ashok; Diwan, Anil; Arora, Vinod; Thakur, Yogesh; Chiniga, Vijetha; Tatake, Jay; Holkar, Preetam; Holkar, Neelam; Pond, Bethany title: Nanoviricide’s platform technology based NV-CoV-2 polymer increases the half-life of Remdesivir in vivo date: 2021-11-19 journal: bioRxiv DOI: 10.1101/2021.11.17.468980 sha: c1de21cb90d37bf5e414c39fe0c59e59b55e2914 doc_id: 960591 cord_uid: 61gljdcm So far, there are seven coronaviruses 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). SARS family are 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. Remdesivir (RDV) is the only antiviral drug so far approved for COVID-19 therapy by the FDA. However, the efficacy of RDV in vivo is limited due to its low stability in presence of plasma. This is the report of analysis of the non-clinical pharmacology study of NV-CoV-2 (Polymer) and NV-CoV-2-R (Polymer encapsulated Remdesivir) in both infected and uninfected rats with SARS-CoV-2. Detection and quantification of NV-CoV-2-R in plasma samples was done by MS-HPLC chromatography analyses of precipitated plasma samples from rat subjects. NV-CoV-2-R show RDV peak in MS-HPLC chromatography, whereas only NV-CoV-2 does not show any RDV-Peak, as expected. NV-CoV-2 polymer encapsulation protects RDV in vivo from plasma-mediated catabolism. 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. 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. chromatography analyses of precipitated plasma samples from rat subjects. (i) NV-CoV-2-R show RDV peak in MS-HPLC chromatography, whereas only NV-CoV-2 does not show any RDV-Peak, as expected. (ii) NV-CoV-2 polymer encapsulation protects RDV in vivo from plasma-mediated catabolism. (iii) 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. 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. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes was declared a global pandemic on March 11 th , 2020 by the WHO [1]. However, the evidence for therapies against this virus is as yet inadequate. SARS-CoV-2 virus enters host cells by binding to and fusing with the cell membrane receptor, ACE-2, followed by membrane fusion. Once inside, the virus uses the host cell's machinery to replicate by using the virus's RNA dependent RNA polymerase (RdRp) for making genome and transcript copies. Among the different strains of the coronavirus, this non-structural protein is unique in structure which makes it a potentially useful drug target. Sofosbuvir, a synthetic analogue of nucleosides and nucleotides, inhibits RdRp has become a successful treatment for hepatitis C infection [2] . Remdesivir (RDV) , formerly known as GS-5734, is a nucleotide analogue that is claimed to have been originally developed as a treatment against Ebola [3] . This drug can also inhibit coronavirus replication by inhibiting RNA polymerases (RdRp4). This compound has shown broad antiviral activity in vitro against Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and SARS-CoV-2 [4-6]. Based on these facts, RDV was approved by the FDA in various clinical trials for the treatment of COVID-19 [7] . In animal studies, RDV has been found effective in protecting rhesus monkeys from MERS-CoV infection, when given prior to infection [8] . It also protected African green monkeys from Nipah virus, the cause of fatal encephalitis and rhesus monkeys from Ebola virus [9, 10]. A randomized, well marked, controlled animal study with 12 rhesus monkeys infected with SARS-CoV-2 reported that an attenuation of respiratory symptoms and reduction in lung damage with RDV administered 12 hours after virus infection [11] . However, efficacy of RDV in vitro or in animals does not match with the clinical outcomes in humans. Further, RDV has some side effects. In the Ebola trial, the side effects of RDV were possible liver damage from an increase in liver enzyme levels in the plasma. Similar increases in liver enzymes were found in three U.S. COVID-19 patients were also documented after RDV treatment. Other typical antiviral drug side effects include Nausea and Vomiting [12] ; and also affects kidney and mitochondria [13, 14] . The efficacy of RDV in vivo is limited due to the low stability in the plasma. We have tested the stability of RDV encapsulated with our platform technology based polymer NV-CoV-2-R, in presence of plasma in vitro, and the result is very supportive that polymer protects RDV from plasma-mediated catabolism [15] . In this paper, we wanted to extend our in vitro experiments in an in vivo rat model of systemic exposure of NV-CoV-2 and NV-CoV-2-R once per day for 5 days (0, 1, 3, 5, and 7) over a 7-day time period. We compared our results with commercially available Gilead RDV and used DMSO as a negative control. Here we reported our results. 14/17ARB. The study design are shown below in Table-1 ISTD ( 1 uL Auto-sampler was used in external rinsing mode, with R0 = 95/5 Water / Acetonitrile and R3 = 1 /1 IPA / Methanol. (v) Normalization of the value using the dilution factor from the original plasma sample (Table-5 (i) A standard curve for RDV from a representative experiment were shown in Table-6 and Fig. 1. (ii) RDV values in male rats plasma obtained (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) and shown in Fig. 2 and Fig. 3 . (iv) RDV values in female rats plasma obtained (mg/mL) after 1 st. and 5 th injection of the drugs were normalized by dividing with the amount administered (mg/kg of rat body weight) and shown in Fig. 4 and Fig. 5 . (v) Comparative analysis of RDV level in male rats plasma after 1 st and 5 th injection of the Drugs were shown in (Fig. 6 ). Values were normalized as a ratio of RDV found in blood (mg/mL) and amount of RDV was administered (mg/Kg of rat body weight). (vi) Comparative analysis of RDV level in female rats plasma after 1 st and 5 th injection of the Drugs were shown in Fig. 7 . Values were normalized as a ratio of RDV found in blood (mg/mL) and amount of RDV was administered (mg/Kg of rat body weight). (vi) Comparative analysis of RDV level in male and female rats plasma after 1 st injection of the drugs were shown in Fig. 8 . (vii) Comparison of RDV level in male and female rats plasma after 5 th injection of the drugs were shown in Fig. 9 . (viii) Toxicity/Tolerability study of NV-CoV-2-R: Loss of body weight analysis of male and female rats after drug administration i.v. (Fig. 10) : From all the above figures it appears that: 1) After the 1 st injection of 387-R-Low, the accumulation of RDV is better in female rats than in male rats. However, after 5 th injection, the differences between male and female rats are insignificant. 2) After the 1 st injection of 387-R-Med, the accumulation of RDV is little greater in female rats compared to male rats, which is more evident after 5 th injections. 3) No such differences were found in the accumulation of RDV from 376-R-SBECD injections, 1 st and 5 th , both, in male and female rats. 8) For all doses, NV-CoV-2 was detected in rat plasma producing an initial increase that peaked between 4-8 hours, but decreases to below detection level between 24 to 48 hrs. 9) Last but not the least, in all the cases our encapsulated polymer is nontoxic to the animals, based on their steady level of body weight. RDV formerly known as GS-5734, is a nucleotide analogue that was originally developed as a treatment against Ebola In animal studies, RDV has been found effective in protecting rhesus monkeys from MERS-CoV infection when given prior to infection [8] . It also protected African green monkeys from Nipah virus, a cause of fatal encephalitis and rhesus monkeys from the Ebola virus [9, 10]. A randomized, well marked, controlled animal study with 12 rhesus monkeys infected with SARS-CoV-2 reported that an attenuation of respiratory symptoms and reduction in lung damage with RDV administered 12 hours after virus infection [11] . However, efficacy of RDV in vitro or in animals does not match with the clinical outcomes in humans. We searched for other compounds or drugs that could be used in conjunction with RDV to potentiate its effect against SARS-CoV-2 and minimize the side effects of RDV. Recently we have reported that our lab-made nano-polymer (NV-CoV-2) can encapsulate RDV and guards it from degradation in the blood stream [15, 16] . This nanoviricide® bio-mimetic polymer binds and engulfs a virus particle into the polymeric nanoviricide, acting like a "Venus-flytrap". Once engulfed, the virus particle gets destroyed (Figure 11 ). Using a plug-and-play approach, we can change the virus binding ligand portion of this nano-medicine to attack a different virus. We have already tested several drug candidates for broad- RDV (Veklury, Gilead) is supplied as a lyophilized solid containing 100 mg RDV and 600 mg SBECD (sulfobutylether-β-cyclodextrin), with the requirement of being re-dissolved in WFI water by the Pharmacist and injected into infusion fluid (saline) preferably through a sterile filter [20] . It is likely that re-dissolving fully may not complete and filtration may remove undissolved RDV, thus reducing the dose applied. This could explain the discrepancy between RDV controlled clinical trials and the WHO datasets. Cyclodextrins form colloids at high concentrations and allow insoluble drugs to be held in the colloid. However, the colloid dilutes out into the bloodstream quickly and would lead to falling out of the API if it is not held by a true cage-binding mechanism. Thus, protecting RDV from metabolism and keeping it in an encapsulated form is essential if its full potential is to be realized clinically. Detection and Quantification of NV-CoV-2 in rat Plasma samples from NV-1067 Toxicology study was done using a validated LC-MS spectrometry as described in the Materials and Methods section. For all doses, NV-CoV-2 was detected in Rat plasma producing an initial increase that peaked between 4-8 hours. The results show that Plasma concentrations decreased to below detection level between 24 to 48 hrs. Author, Anil Diwan, was employed by the company Nanoviricides, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Authors' contribution: All the authors contributed equally to prepare this article, read and approved the final manuscript. Legends to Figures Fig. 1 : Standard curve of RDV was determined by using LC-MS, using different concentration of the standards solution in DMSO + MeOH (1:9). Final concentrations of RDV ranged from 0-5 ug/mL, which is described in the method section. Values (Mean ± SD) are from a representative experiment done in duplicate. As in Fig. 2 . RDV values in female rat plasma after 1 st injection of the drugs. As in Fig. 2 . RDV values in female rat plasma after 5 th injection of the drugs. As in Fig. 2 . Comparison of RDV level in male and female rat plasma after 5 th injection of the drugs Fig. 10 : Toxicity/Tolerability study of NV-CoV-2-R: Loss of Body weight analysis of male and memale rats after drug administration i.v. Mechanism of Nanoviricide Action WHO Declares COVID-19 a Pandemic Nucleoside inhibitors of hepatitis C virus NS5B polymerase: a systematic review Controlled Trial of Ebola Virus Disease Therapeutics The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus Broadspectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro COVID-19 Treatment Guidelines GS-5734) treatment in the rhesus macaque model of MERS-CoV infection GS-5734) protects African green monkeys from Nipah virus challenge Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2 Remdesivir (RDV) Remdesivir in Patients with Acute or Chronic Kidney Disease and COVID-19 Mitochondria and microbiota dysfunction in COVID-19 pathogenesis Preetam Holkar, Vijetha Chinige Nanoviricides Platform Technology based NV-387 polymer Protects Remdesivir from Plasma-Mediated Catabolism in vitro: Importance of its increased lifetime for in vivo action Candidate-NV-CoV-2-was-Effective-Against-SARS-CoV-2-Further-Demonstrating-Its-Broad-Spectrum-Pan-Coronavirus-Activity We acknowledge all our colleagues, Secretaries for their help during the preparation of the manuscript by providing all the relevant information.Fundings: Fundings from Nanoviricide, Inc.