DESC: Drotaverine: Use as a broad-spectrum antiviral agent

RELATED APPLICATION
The present application is related to takes priority from Provisional Application 202241033883 dated 14/6/2022 and is incorporated herein in its entirety.
FIELD OF THE INVENTION
The present invention relates to a compound Drotaverine for use in treating and/or preventing a coronavirus infection in a subject.
BACKGROUND OF THE INVENTION
The emergence of the SARS-CoV-2 virus has led to the global health care crisis and revealed our limitations to control the COVID-19 pandemic globally. Currently, no specific treatment option is available for the SARS-CoV-2 virus, except Remdesivir. Though Remdesivir was discovered for Ebola, it has been repurposed for treating SARS-CoV-2 due to broad-spectrum activity and got FDA approval on compassionate grounds (Richard et al., 2020). Hence, the search for effective therapeutic agents to tackle the pandemic remains crucial and urgent. The discovery of a new drug takes a long-gestation period of 10-15 years, and this process costs more than a billion dollars with a success rate of only 2.01%. This generates a considerable gap between therapeutic needs and the availability of new treatments. Thus, in the interest of time and urgency to control the ongoing global crisis, repurposing the existing drugs with a proven safety track record and history of clinical use remains a cost-effective and viable option to stem the tide of pandemic (Khan et al., 2021).
Emergency response to the SARS-CoV-2 pandemic generated a massive hunt for repurposed drugs and biologicals like antibodies. Several clinical trials were initiated and even completed in 2020 (www.clinical trial.gov) with potential candidates including some existing antivirals (Park et al., 2020), anti-bacterial (Yacouba, et al., 2021; Kelleni, 2021) and Steroids (Ghosh et al., 2020) with limited to no success. Most drugs like Hydroxychloroquine (HCQ), Azithromycin, Ivermectin, Favipiravir, Lopinavir-Ritonavir and Remdesivir were widely used to treat COVID-19 patients. Unfortunately, none showed significant improvements in COVID-19 patients preventing disease and severity with SARS-CoV-2 and reducing ICU stay and survival of patients. Hence there is a need to expedite the search for promising repurposable candidates or new combinations of approved drugs for more effective treatment of COVID-19 (Khan et al., 2020).

SUMMARY OF THE INVENTION
The present invention provides a compound Drotaverine of formula I
Formula I
for use in preventing and/or treating of coronavirus infection in a subject wherein the subject is primarily a human.
In one aspect of the invention, the compound of Formula I is used for preventing and/or treating infections caused by coronovirus selected from the group consisting of SARS-CoV-2, SARS-CoV-1, or Middle East respiratory syndrome coronavirus (MERS-CoV) and any new strains that is detected in the same class.
In another aspect, the invention provides a compound of formula I with an IC50 value ranging from 8.0 to 12.0 µ? against Wuhan strain and 45 to 55 µ? against delta variant.
In yet another aspect, the invention provides a compound of formula I with an IC50 value ranging from 0.5-2.0 µM against Human Respiratory Syncytial virus (HRSV), >4.0 µM against Influenza A and B, > 5.0 µM against Chikungunya and > 13 µM against ZIKA virus.
The invention also provides a compound of Formula I for use in treating and /or preventing infections caused by respiratory virus selected from Human Respiratory Syncytial virus (HRSV), Influenza A or Influenza B and non-respiratory virus selected from Chikungunya or ZIKA virus.

BRIEF DESCRIPTION OF DRAWINGS
Figure 1: Drotaverine In-vitro efficacy in Vero E6 cells.
(A). Cytotoxicity and IC50 against Wuhan strain
(B). Cytotoxicity and IC50 against Delta strain
Figure 2A: In vivo efficacy (Syrian golden hamster model), Drotaverine 10mpk UID dosing.
Figure 2B: In vivo efficacy (Syrian golden hamster model), Drotaverine 10mpk BID dosing.
Figure 3A: Body weights n = 3 for Drotaverine 10mpk UID dosing.
Figure 3B: Body weights n = 3 for Drotaverine 10mpk BID dosing

BRIEF DESCRIPTION OF THE INVENTION
The present invention relates to a compound Drotaverine for use in treating and/or preventing a coronavirus infection (Wuhan strain, Alfa, Beta, Delta, Gamma, Kappa, BavPat1, Denmark strain and related strains) in a subject. The compound Drotaverine as provided herein is also useful in treating and /or preventing the infections caused by Human respiratory syncytial virus (HRSV), Influenza A, Influenza B, Chikungunya and ZIKA virus.
The comprised compound having the following Formula I and present invention further relates to methods and uses related thereto.

Formula I
The active compound of the present invention, Drotaverine and method for its preparation are disclosed in patent BE621917, published on Dec.14, 1962, which is incorporated herein by reference.
The in-vitro screening of a panel of existing drugs in Vero-E6 cells by the inventors of the present invention has led to the identification of the use of Drotaverine with potent antiviral activity. The antiviral activity is also confirmed in in-vivo studies using the Syrian Golden Hamster model of SARS-CoV-2 infection. Drotaverine is an antispasmodic medicine that relieves contractions (spasms) associated with smooth muscles in the organs such as the stomach and heart.
Drotaverine works by inhibiting phosphodiesterase-4 (PDE4), an enzyme responsible for the degradation of cyclic adenosine monophosphate, cAMP. It is a benzyl iso-quinoline derivative structurally related to papaverine, a widely used drug. Several clinical studies/data demonstrate the efficacy and safety of Drotaverine in humans for alleviating pain, symptoms of irritable bowel syndrome (IBS), augmentation of labour and management of recurrent abdominal pain in children.
The present invention provides a compound Drotaverine for use in treating and/or preventing coronavirus infections in a subject wherein the subject is primarily a human. The coronavirus infection includes but not restricted to SARS-CoV-2, SARS-CoV-1, or Middle East respiratory syndrome coronavirus (MERS-CoV) and also envisages any new strains that will be detected in the future.
The compound of the invention works towards treating or preventing the infection by inhibition of coronavirus replication.
The compound of the invention works towards treating or preventing the infection by inhibition of cell lysis caused by the said virus, for example the coronavirus.
The compound of the invention works towards treating or preventing the infection by inhibition of tissue destruction, preferably lung tissue destruction caused by the said virus, for example the coronavirus.
The compound of the invention is provided for use in inhibiting the coronavirus replication wherein the inhibitor of coronavirus has IC50 values ranging from 8.0 to 12.0 µM against Wuhan strain and 45.0-55.0 µM against delta variant.
The compound Drotaverine as provided herein is also shows antiviral activity against other respiratory viruses such as, Human Respiratory Syncytial virus (HRSV) with an IC-50 value ranging from 0.5-2.0 µM, Influenza A, and Influenza B with an IC-50 >4.0 µM, and non-respiratory viruses like, Chikungunya with an IC-50 > 5.0 µM or and ZIKA virus with an IC-50 > 13 µM.

Uses
The compound disclosed as Formula I of the invention is useful for the treatment of infections in subjects, mammals in particular, including humans. In one embodiment, the compounds may be used for the treatment of infections of viral infections affecting lungs and respiratory tract.
The compound disclosed as Formula I of the invention is useful for the treatment of infections caused by microorganisms, particularly viruses.
In another embodiment, the compound of Formula I is useful for prevention or treatment of coronavirus infection in a subject, mammals in particular, including humans. The coronavirus infection is selected from the group consisting of SARS-CoV-2, SARS-CoV-1, or Middle East respiratory syndrome coronavirus (MERS-CoV) and any new strain that is detected in the same class.

Route of Administration
The compounds of the present invention are delivered to the subjects by forms suitable for each administration route. For example, the compounds are administered as tablets, capsules, injection, drops, inhaler, ointment, foams suppository. In a preferred embodiment, the route of administration is oral, parenteral, or topical. Topical or transdermal administration includes powders, sprays, ointments, pastes creams, lotions, gels, solutions, patches and inhalants.

Dosage Forms
The composition of the present invention is presented in unit dosage form generally in an amount that produces a therapeutic effect in the subject.
The compounds of the present invention are administered at a daily dose that is the lowest dose effective to produce a therapeutic effect. Generally, the dosage will affect from about 0.0001 to about 100mg per kg body weight per day. Preferably, the dosage will range from about 0.001 to 75mg per kg body weight per day and more preferably, the dosage will range from about 0.1 to about 50mg per kg body weight per day. Each unit dose may be, for example, 5, 10, 25, 50, 100, 125, 150, 200 or 250 mg of the compound of the invention. As per the requirement of the subject, the effective daily dose of the compound is administered as two, three, four or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms.

Formulation
The compositions of the present invention may be administered by any method known in the art. Some examples of suitable modes of administration include oral, intravenous, intramuscular, topical or any other parenteral mode of administration.
In certain embodiments, the present invention is directed to a method of formulating compounds of the present invention in a pharmaceutically acceptable carrier or excipient and may be administered in a wide variety of different dosage forms e.g. tablets, capsules, sprays, creams, lotions, ointments, aqueous suspensions syrups, and the like. Such carriers may include one or more of solid diluents or fillers, sterile aqueous media, and various nontoxic organic solvents, etc.
For oral administration, tablets may contain various excipients such as one or more of microcrystalline cellulose, sodium citrate, calcium carbonate and the like, along with various disintegrants such as starch and certain complex silicates, together with granulation binders like polyvinylpyrrolidone, sucrose and the like. Solid compositions of a similar type may also be employed as fillers in gelatine capsules.
The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluents or solvent e.g., as solution in 1, 3 butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid find in the preparation of injectables. These aqueous solutions may be suitable for intravenous injection purposes. The oily solutions may be suitable for intra articular, intramuscular, and/or subcutaneous injection purposes.
In another embodiment, the compounds of the present invention may be administered topically that include transdermal, buccal, or sublingual application. For topical applications, therapeutic compounds may be suitably admixed in a pharmacologically inert topical carrier such as a gel, an ointment, a lotion, and/or a cream. Such topical carriers may include water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, and/or mineral oils.
The timing of the administration of the pharmaceutical composition may also be regulated. For example, the compounds may be administered intermittently or by controlled release.

Examples
BIOLOGICAL ACTIVITY

Example 1. Antiviral Screening of Drotaverine against SARS-CoV-2
The Drotaverine compound was tested for in-vitro antiviral activity against SARS-CoV-2 virus. SARS-CoV-2 testing was performed in Vero-E6 cell lines using test media DMEM (high glucose) supplemented with 2% FBS and antibiotic and antimycotic solutions.

The testing compound Drotaverine was solubilized in sterile water for injection to prepare a 20 mg/mL or 10 mM stock solution. The antiviral activity of the compound against SARS-CoV-2 was determined by standard plaque assay. For SARS-CoV-2 testing the Drotaverine was serially diluted (two-fold) with a top concentration of 25 µM. Each dilution was added to 2 wells of a 96-well plate with 80-100% confluent cells and infected with virus at MOI of 0.01. The cells were incubated for 3 days before processing the plates for plaque scoring.

The controls such as cell only control and virus only controls were maintained through out to determine the antiviral activity.
The cytotoxicity of the compound Drotaverine was determined in Vero-E6 cells by measuring LDH assay using Cytoquant LDH assay kit. For other viruses, test compound was serially diluted using eight half-log10 dilutions in test media so that the starting (high) test concentration was 100 µg/ml. Each dilution was added to 5 wells of a 96-well plate with 80-100% confluent cells. Three wells of each dilution were infected with virus, and two wells remained uninfected as toxicity controls. Six wells were infected and untreated as virus controls, and six wells were uninfected and untreated as cell controls. Virus was prepared to achieve a MOI=1. A positive control compound was tested in parallel for each virus. Plates were incubated at 37±2°C, 5% CO2.

On day 3 post infection the SARS-CoV-2 plate were fixed using 4% paraformaldehyde for 30 minutes and stained with crystal violet for 10-15 minutes. The plaques were scored in each well and % reduction in the virus load was calculated in comparison to virus only control wells. The IC50 of test compound was then determined using Graph Pad Prism (version-9.0). For other viruses, on day 3-7 post-infection, once untreated virus control wells reached maximum cytopathic effect (CPE), plates were stained with neutral red dye for approximately 2 hours (±15 minutes). Supernatant dye was removed, and wells rinsed with PBS, and the incorporated dye was extracted in 50:50 Sorensen citrate buffer/ethanol for >30 minutes and the optical density was read on a spectrophotometer at 540 nm. Optical densities were converted to percent of cell controls and normalized to the virus control, then the concentration of test compound required to inhibit CPE by 50% (EC50) was calculated by regression analysis. The concentration of compound that would cause 50% cell death in the absence of virus was similarly calculated (CC50).

The Drotaverine screened against a panel of virus and results are summarised in the following
Table-1 and Figure-1 contains the in-vitro efficacy in Vero E6 cells against SARS-CoV-2
Table-1: IC 50 against SARS-CoV-2, Human respiratory syncytial virus, influenza A & B types, Chikungunya virus, Zika virus and Junín virus

Compound Name SARS-CoV-2
IC 50 (µM)
HRSV
IC 50 (µM)
influenza A
IC 50 (µM)
influenza B
IC 50 (µM)
Chikungunya IC 50 (µM)
ZIKA
IC 50 (µM)
Wuhan Delta
Drotaverine 9.9 52.6 1.0 > 4.2 > 4.8 > 5.2 > 13

Example 2. In vivo efficacy:
The hamsters were procured from ACTREC, Mumbai. All experiments were performed in BSL-3 FNDR facility. The hamsters were quarantined for a period of 7 days and acclimatized in BSL-3 facility for 5 days before the experimentation. BSL3 environment was monitored for temperature and relative humidity once a daily. The temperature range was maintained at 23°C± 5°C and humidity range was 30-70%. The hamsters were provided with 12 hours light and 12 hours dark cycle. Hamsters were housed in IVC system during experiment. Feed and water were provided ad libitum.

Formulation: Drotaverine is weighed and dissolved using 0.5% Hydroxy Propyl Methyl Cellulose (HPMC) and 0.1% Tween 80 in water to achieve the desired concentration for dosing in a suspension form. For parental formulation (I.V), the compound is dissolved in the mixture of 5% dimethylacetamide (DMA) and 10% solutol in 85% normal saline. Drotaverine is dissolved in normal saline for inhalation to achieve the desired concentration. Whereas, for the topical formulation the compound is dissolved in propylene glycol. Sonicate to ensure proper solubility. Post solubility, PEG400 is added slowly to the content while stirring using a magnetic stirrer. This test compound mixture is added slowly to another container that contains PEG4000 which is weighed and dissolved in a water bath. For pharmacokinetic and efficacy evaluation, a dose that ranges between 1 to 80 mg/kg is prepared and evaluated.

Procedure: Female Hamsters 6 to 10wk old were anesthetized with ketamine (150mg/kg) and xylazine (10mg/kg) by intraperitoneal injection and inoculated intranasally with 100µL containing 1× 105 PFU/hamster. A Drotaverine dose response efficacy study was initiated at 3 doses (1, 3 & 10mpk oral UID) from Day 1 post infection for 3 days continuously. All doses were prepared as an oral suspension in 0.5% HPMC + 0.1% Tween 80. Remdesivir, was used as positive control at 15mpk UID in formulation vehicle 5% DMSO/5%Ethanol/ 40%PEG300/50%NS. Mock control and infection vehicle control groups were included along with treatment groups. A second study was done with similar treatment groups with only one dose of drotaverine (10mpk BID) along with respective controls. Hamsters were daily monitored for any adverse clinical symptoms and body weight was recorded on daily basis. At day 4 pi, hamsters were euthanized by overdosage of Isoflurane. Lung tissues was collected to quantify the virus. Lungs samples were homogenized in 1:2 ratio of PBS and used for viral load determination.
The viral counts were converted to PFU/lung, which were then evaluated by a one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison (Graph Pad software program). Differences were considered as significant at 99% level of confidence. A probability level of 5% (P < 0.05) was statistically significant.
Drotaverine showed a good dose response (Figure: 2A) with lung viral load reduction when given once a day orally starting a day after infection. A significant 0.86 log PFU/lung reduction was demonstrated at 10mpk UID. Drotaverine showed improvement in lung viral load reduction (Figure: 2B) of 1.36 log PFU/lung when given twice a day (10mpk) starting a day after infection as compared to when administered once a day. The body weight trend for drotaverine was like the mock control (Figure: 3A & 3B). The control drug Remdesivir treatment showed a significant reduction (1.2 log PFU/lung) in lung viral load, that was comparable to published literature data.
Dated 5th day of June 2023

Kausalya Santhanam
digitally signed for e-filing
(Agent for the Applicant, PA/IN/1183)

References
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,CLAIMS:Claims
We Claim:
1. A compound of formula I
Formula I
for use in preventing and/or treating of coronavirus infection in a subject wherein the subject is primarily a human.
2. The compound of Formula I as claimed in claim 1 wherein the said coronavirus infection is selected from the group consisting of SARS-CoV-2, SARS-CoV-1, or Middle East respiratory syndrome coronavirus (MERS-CoV) and any new strains that is detected in the same class.
3. The compound as claimed in claim 1 wherein the said compound has an IC50 value ranging from 8.0 to 12.0 µ? against Wuhan strain and 45 to 55 µ? against delta variant.
4. The compound as claimed in claim 1 wherein the said compound has an IC50 value ranging from 0.5-2.0 µM against Human Respiratory Syncytial virus (HRSV), >4.0 µM against Influenza A and B, > 5.0 µM against Chikungunya and > 13 µM against ZIKA virus.
5. The compound of Formula I for use in treating and /or preventing infections caused by respiratory virus selected from Human Respiratory Syncytial virus (HRSV), Influenza A or Influenza B and non-respiratory virus selected from Chikungunya or ZIKA virus.