Embodiment of the present invention relates to therapeutic formulation
and more particularly it relates to a formulation for inhibition of SARS-CoV-2
infection and a process for preparation thereof.
BACKGROUND
[0002] Coronavirus disease (COVID-19) is an infectious disease caused by a
virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The
worldwide impact of this pandemic is frightening. Several therapeutic regimens and
vaccine products are in developmental phase to treat the infected patients. The first
mass vaccination programme to tackle COVID-19 menace started in early
December 2020. At least 13 different vaccines have been administered and the
campaigns have started in 206 economies.
[0003] Among the vaccines administered, Pfizer/BioNTech Comirnaty
vaccine, SII/Covishield and AstraZeneca/AZD1222 vaccines (developed by
AstraZeneca/Oxford and manufactured by the Serum Institute of India and SK Bio
respectively), Janssen/Ad26.COV 2.S developed by Johnson & Johnson, Moderna
COVID-19 vaccine (mRNA 1273), and Sinopharm COVID-19 vaccine produced
by Beijing Bio-Institute of Biological Products Co Ltd, subsidiary of China
National Biotec Group (CNBG) are listed for the World Health Organization (WHO)
Emergency Use Listing (EUL).
[0004] Equitable access to safe and effective vaccines is critical to ending the
COVID-19 spread, so it is hugely encouraging to see vaccines proving and going
into development. Many countries are working tirelessly to develop, manufacture
and deploy safe and effective vaccines. However, only a small percentage of people
have access to vaccines, especially the most vulnerable lack a fair share. The
healthcare workers, doctors, civil servants, and administrators are the most
3
vulnerable group of people who are at the constant risk of exposure to the COVID19 infection and have higher infection probability. No direct medical intervention
available has shown to be really effective in protecting them from the COVID-19
infection beyond use of PPE kits. This brings to attention to a non-vaccine medical
product that can provide prevention, resistance, reduction to spread in already
infected population and co-care against microbial infections.
[0005] Moreover, Shafran, N. et al., 2021, in their paper, Secondary bacterial
infection in COVID-19 patients is a stronger predictor for death compared to
influenza patients, disclosed that the secondary bacterial infections e.g., infection
of Staphylococcus aureus is corelated with the COVID-19 complications and thus
lead to worse outcome. Feng, Y. et al., 2020, in their paper, disclosed that COVID19 with different severities: a multicenter study of clinical features, the higher
bacterial co-infections are also shown to be present in critical COVID-19 infected
patients with a higher mortality rate.
[0006] Hence, the current global health threat by the novel coronavirus disease
(COVID-19) requires an urgent deployment of advanced therapeutic options
available. The therapeutic and prophylactic strategies to deal with existing and
potentially upcoming coronavirus infections are under development. The
symptomatic treatment approach is presently followed in the absence of an
exclusive antiviral treatment against SARS-CoV-2. Artificial intelligence and other
computation tools are currently in use to assist the lengthy process of drug discovery
and development against this pathogen. Using recently available genetic
information and protein structure modeling, several therapeutic strategies are long
and time taking procedure. Recent structure-function studies have provided an
important insight into molecular details of SARS-CoV-2 entry into the respiratory
tract and other organs, including kidney and GI tract. These studies were
instrumental in understanding the mechanism of infection; however, they did not
provide any therapeutic formulation specific for prevention of SARS-CoV-2.
4
[0007] Additionally, hydroxychloroquine (HCQ) a known anti-inflammatory
drug, which represents an advanced version of chloroquine (CQ), exhibits great
potential to block the SARS-COV-2 infection and subsequently resulted in an
efficient reduction of the viral loads in host cells. There are few reports of using
tetrandrine, verapamil, and HCQ alone or repurposing their usage in combinatorial
therapy to combat devastating COVID-19. Meng-Yuan Li et.al., 2020, in their
paper, Expression of the SARS-CoV-2 cell receptor gene ACE2 in a wide variety
of human tissues, disclosed a combination of hydroxychloroquine sulfate, zinc
sulfate and azithromycin has proven to be useful in increased recovery of COVID19 infected patients. However, this combination lacks the specific targeting of
angiotensin-converting enzyme 2 (ACE-2) expressing cells and sustained delivery
of salt content with facilitated delivery of HCQ in intracellular space.
[0008] Moreover, the major environmental hazard from different
pharmaceutical formulations comes from the unused excreted number of
pharmaceutical formulations by the subject. It generally reaches to hospital or home
waste and eventually ends up in water resources, thereby contributing to water
pollution and creating anti-biotic resistance. The easiest method to control this
ecological hazard could be the reduction of the dose of the intended pharmaceutics.
[0009] Hence, there is a need for a formulation, a process for preparing the
formulation and a method of use for inhibition of SARS-CoV-2 infection and post
bacterial infections.
SUMMARY
[0010] In accordance with an embodiment of the present invention, a
formulation for inhibition of SARS-CoV-2 infection is provided. The formulation
includes a nasal spray-based TriC4CoV formulation. The nasal spray-based
TriC4CoV formulation includes a combination of hydroxychloroquine sulfate
(HCQ) and zinc sulfate (ZnSO4) loaded inside poly-styrene-block-ploy-acrylic acid
5
(PS-b-PAA) shell cored with carbon nanoparticle (CNP). The nasal spray-based
TriC4CoV formulation is configured to inhibit entry of SARS-CoV-2 inside cells
by competitive binding to ACE-2 receptor.
[0011] In accordance with another embodiment of the present invention, a
process for preparing a nasal spray-based TriC4CoV formulation is provided. The
process includes preparing 10 wt/vol% aqueous suspension of carbon nanoparticle
(CNP). The process also includes stirring the aqueous suspension of CNP at 60°C
for a duration of 10 minutes at 1100 rpm. The process also includes mixing
hydroxychloroquine sulfate (HCQ) and zinc sulfate (ZnSO4) with the aqueous
suspension of CNP to obtain a first mixture. The process includes stirring the first
mixture at 60°C for a duration of 10 minutes at 1100 rpm. The process also includes
dissolving 0.5 wt/vol% of poly-styrene-block-ploy-acrylic acid (PS-b-PAA) in
tetrahydrofuran (THF) to obtain a THF solution. The process also includes adding
the THF solution to the first mixture by drop wise method to obtain a second
mixture. The further includes probe sonicating the second mixture for two times to
obtain the nasal spray-based TriC4CoV formulation.
[0012] To further clarify the advantages and features of the present invention,
a more particular description of the invention will follow by reference to specific
embodiments thereof, which are illustrated in the appended figures. It is to be
appreciated that these figures depict only typical embodiments of the invention and
are therefore not to be considered limiting in scope. The invention will be described
and explained with additional specificity and detail with the appended figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The disclosure will be described and explained with additional
specificity and detail with the accompanying figures in which:
6
[0014] FIG. 1 represents a pictorial view of mechanism of action of a nasal
spray-based TriC4CoV formulation, in accordance with an embodiment of the
present invention;
[0015] FIG. 2 illustrates a flow diagram representing steps involved in a
process for preparing a nasal spray-based TriC4CoV formulation, in accordance
with an embodiment of the present invention;
[0016] FIG. 3 represents application of the nasal spray-based TriC4CoV
formulation using a spray dispenser bottle, in accordance with an embodiment of
the present invention;
[0017] FIG. 4 represents TEM images of a) CNP core and b) the nasal spraybased TriC4CoV formulation, in accordance with an embodiment of the present
invention;
[0018] FIG. 5 represents results of in-vitro safety assay for the nasal spraybased TriC4CoV formulation, in accordance with an embodiment of the present
invention;
[0019] FIG. 6 represents results of in-vitro safety assay at level of DNA
degradation for the nasal spray-based TriC4CoV formulation, in accordance with
an embodiment of the present invention; and
[0020] FIG. 7 represents results of anti-bacterial efficacy assay of the nasal
spray-based TriC4CoV formulation, in accordance with an embodiment of the
present invention.
[0021] Further, those skilled in the art will appreciate that elements in the
figures are illustrated for simplicity and may not have necessarily been drawn to
scale. Furthermore, in terms of the preparation of the formulation, one or more
components of the formulation may have been represented in the figures by
conventional symbols, and the figures may show only those specific details that are
7
pertinent to understand the embodiments of the present disclosure so as not to
obscure the figures with details that will be readily apparent to those skilled in the
art having the benefit of the description herein.
DETAILED DESCRIPTION OF THE INVENTION
[0022] For the purpose of promoting an understanding of the principles of the
disclosure, reference will now be made to the embodiment illustrated in the figures
and specific language will be used to describe them. It will nevertheless be
understood that no limitation of the scope of the disclosure is thereby intended.
Such alterations and further modifications in the illustrated system, and such further
applications of the principles of the disclosure as would normally occur to those
skilled in the art are to be construed as being within the scope of the present
disclosure.
[0023] The terms "comprises", "comprising", or any other variations thereof,
are intended to cover a non-exclusive inclusion, such that a process or method that
comprises a list of steps does not include only those steps but may include other
steps not expressly listed or inherent to such a process or method. Appearances of
the phrase "in an embodiment", "in another embodiment" and similar language
throughout this specification may, but not necessarily do, all refer to the same
embodiment.
[0024] Unless otherwise defined, all technical and scientific terms used herein
have the same meaning as commonly understood by those skilled in the art to which
this disclosure belongs. The kit and examples provided herein are only illustrative
and not intended to be limiting.
[0025] In the following specification and the claims, reference will be made to
a number of terms, which shall be defined to have the following meanings. The
singular forms “a”, “an”, and “the” include plural references unless the context
clearly dictates otherwise.
8
[0026] Embodiments of the present invention relates to a formulation for
inhibition of SARS-CoV-2 infection. The formulation mainly includes a polymeric
shell-carbon core biodegradable nanoscale particles for mist based drug delivery.
The formulation is based on triggerable, repurposed, and combinatorial composites
for prevention, resistance, reduction in further spread of the SARS-CoV-2 infection
in already infected population and co-care against microbial infections. Moreover,
the present invention provides a non-vaccine biomedical product for inhibition of
viral infection (SARS-CoV-2 infection). The present invention also relates to a
process for preparing a nasal spray-based TriC4CoV formulation.
[0027] In an embodiment, the formulation for inhibition of SARS-CoV-2
infection is provided. The formulation includes the nasal spray-based TriC4CoV
formulation. The purpose of delivery of the formulation through nasal cavity is to
act on mucous membrane area where first contact of viral particle takes place with
human subjects and the infection occurs. The lungs are an important target for the
formulation delivery for treating SARS-CoV-2 infection, which infects primarily
through the respiratory tract. Therefore, use of inhaled aerosols is an effective noninvasive mode of administration. Besides, the delivery of inhalable nanoparticles to
the lungs overcomes disadvantages such as side effects caused by high drug
concentrations in the serum with conventional oral or intravenous drug
administration methods.
[0028] The nasal spray-based TriC4CoV formulation includes a combination
of hydroxychloroquine sulfate (HCQ) and zinc sulfate (ZnSO4) loaded inside polystyrene-block-ploy-acrylic acid (PS-b-PAA) shell cored with carbon nanoparticle
(CNP). The nasal spray-based TriC4CoV formulation comprises HCQ and ZnSO4
in concentration of 500 µM and 5 mM, respectively. The nasal spray-based
TriC4CoV formulation comprises PS-b-PAA and CNP in concentration of 0.5
wt/vol% and 10 wt/vol%, respectively. The PS-b-PAA shell cored with CNP is
biodegradable and enables release of drugs in extra-cellular space selectively to
9
inhibit microbial infection. The formulation includes FDA approved materials in
the nasal spray-based TriC4CoV formulation.
[0029] The nasal spray-based TriC4CoV formulation comprises size of 100nm.
The nasal spray-based TriC4CoV formulation is in mist form. The nasal spraybased TriC4CoV formulation is configured to inhibit entry of SARS-CoV-2 inside
cells by competitive binding to angiotensin-converting enzyme 2 (ACE-2) receptor.
[0030] Mechanism of action of the nasal spray-based TriC4CoV
formulation:
The nasal spray-based TriC4CoV formulation proposed to work by inhibiting
bacterial infection in extracellular space, inhibition of SARS-CoV-2 entry by
competitive binding to angiotensin-converting enzyme 2 (ACE-2) receptor and
inhibition of viral genetic material entry by raising the pH of endosomal
compartment.
Amphiphilic nature of the PS-b-PAA and floccular morphology in the nasal spraybased TRiC4CoV formulation helps in avoiding the surface interaction with
cationic extracellular proteins which otherwise can coagulate the nasal spray-based
TRiC4CoV formulations and avoid its cellular interaction.
Spike protein moiety on the carbon nanoparticles of the nasal spray-based
TRiC4CoV formulation will direct it to ACE-2 receptors on cells and nanoscale
size may allow the nasal spray-based TRiC4CoV formulation to bind to the ACE2 receptor. This may avoid anchorage of the SARS-CoV-2 on the ACE-2 receptor
expressing cells as receptors are acquired by the nasal spray-based TRiC4CoV
formulation. During surface interaction, the PS-b-PAA shell re-arranges itself and
leach out zinc sulfate on cell surface and in extra-cellular space. This acts as
deterrent for microbial interaction to cells and kill the bacterial population present
in vicinity. After avoiding the cellular entry to the SARS-CoV-2, polymeric nature
of the PS-b-PAA shell allows it to enter the cells with efficient endocytosis.
10
In endosomal vesicle, the un-wrapping of the PS-b-PAA shell will allow HCQ to
get released, which increases the pH from acidic to basic. In condition of basic pH,
a fusion of endosome and lysosome is not to be possible, which is very important
step in mechanism of genetic information transfer of the SARS-CoV-2 for further
multiplication of viral genome and spread of infection in a body of person infected
with the SARS-CoV-2. Thus, the nasal spray-based TriC4CoV formulation may
inhibit the SARS-CoV-2 spread at multiple levels of not allowing it to bind to new
ACE-2 expressing cells, entering the cell, delivering the genetic material to already
infected cells and containing the infection to limited cell population only. At the
same time, the nasal spray-based TriC4CoV formulation controls bacterial infection
to virally infected cell populations.
[0031] FIG. 1 represents a pictorial view of mechanism of action of a nasal
spray-based TriC4CoV formulation, in accordance with an embodiment of the
present invention.
[0032] In another embodiment of the present invention, a process for preparing
a nasal spray-based TriC4CoV formulation is provided. The process includes FDA
approved materials in the nasal spray-based TriC4CoV formulation.
[0033] FIG. 2 illustrates a flow diagram representing steps involved in the
process for preparing the nasal spray-based TriC4CoV formulation, in accordance
with an embodiment of the present invention.
[0034] The process for preparing the nasal spray-based TriC4CoV formulation
begins with preparing 10 wt/vol% aqueous suspension of carbon nanoparticle (CNP)
at step 202. The aqueous suspension of CNP is prepared using a microwave
synthesis method. The carbon nanoparticles are biodegradable nanoscale particles
for mist-based drug delivery.
[0035] In an embodiment, the aqueous suspension of CNP is stirred at 60°C
for a duration of 10 minutes at 1100 rpm at step 204. As used herein the term ‘rpm’
11
relates to the number of turns in one minute. The rpm is a unit of rotational speed
or the frequency of rotation around a fixed axis.
[0036] In an embodiment, hydroxychloroquine sulfate (HCQ) and zinc sulfate
(ZnSO4) are mixed with the aqueous suspension of CNP to obtain a first mixture at
step 206. The first mixture comprises 500 µM and 5 mM concentrations of the HCQ
and the ZnSO4, respectively. The HCQ exhibits great potential to block the SARSCOV-2 infection and subsequently results in an efficient reduction of the viral loads
in host cells. The ZnSO4 acts as deterrent for microbial interaction to cells and kills
the bacterial population present in vicinity.
[0037] In an embodiment, the first mixture is stirred at 60°C for a duration of
10 minutes at 1100 rpm at step 208. Stirring the first mixture at 60°C ensures
uniform mixing of the HCQ and ZnSO4 with the aqueous suspension of CNP.
[0038] In an embodiment, 0.5 wt/vol% of poly-styrene-block-ploy-acrylic acid
(PS-b-PAA) is dissolved in tetrahydrofuran (THF) to obtain a THF solution at step
210. Anionic natures of the PS-b-PAA helps in avoiding surface interaction with
cationic extracellular proteins.
[0039] In an embodiment, the THF solution is added to the first mixture by
drop wise method to obtain a second mixture at step 212.
[0040] Further in an embodiment, the second mixture is probe sonicated for
two times to obtain the nasal spray-based TriC4CoV formulation at step 214. The
second mixture is stirred at 60°C for a duration of 12 hours at 350 rpm before probe
sonicating. The probe sonicating the second mixture for two times is carried for a
duration of 30 minutes at an interval of a duration of 10 minutes.
[0041] As used herein the term ‘probe sonication’ relates to the process of
applying sound energy to agitate particles or discontinuous fibers in a liquid.
Ultrasonic frequencies (>20 kHz) are usually used.
12
[0042] The nasal spray-based TriC4CoV formulation comprises size of 100nm.
The nasal spray-based TriC4CoV formulation is configured to inhibit entry of
SARS-CoV-2 inside cells by competitive binding to angiotensin-converting
enzyme 2 (ACE-2) receptor.
[0043] In the present invention characterization of the prepared nasal spraybased TriC4CoV formulation is carried as follows:
Evaluation of hydrous size of particles of the nasal spray-based TriC4CoV
formulation:
A stable aqueous suspension of sub-micron size particles is the basic requirement
for the nasal spray-based TriC4CoV formulation. The hydrous diameter of the
prepared nasal spray-based TriC4CoV formulation particles is evaluated and
checked the feasibility of converting the suspension to mist. The observations
depicted that overall nasal spray-based TriC4CoV formulation in hydrous state was
around 100 nm in size and stable for long time even in storing condition of room
temperature. The prepared nasal spray-based TriC4CoV formulation was found to
be usable through mist making pump (a spray dispenser bottle) as per requirement
(shown in Figure 3).
Table 1 shows characteristics of the prepared nasal spray-based TriC4CoV
formulation.
Characteristics Sample/condition
(TriC4CoV formulation)
Time span
Hydrous size 110±25 nm -
Stability Room temperature 4 months
Stability 4˚C > 6 months (continuing)
Table 1
13
[0044] FIG. 3 represents application of the nasal spray-based TriC4CoV
formulation using the spray dispenser bottle, in accordance with an embodiment of
the present invention. A representative appearance of brownish stain on paper-wipe
(encircled in violet color, B2) after application of components of the nasal spraybased TriC4CoV formulation using the spray dispenser bottle (B1). Arrow heads
indicate the spray dispenser bottle including the nasal spray-based TriC4CoV
formulation and nozzle head.
[0045] FIG. 4 represents TEM images of a) CNP core and b) the nasal spraybased TriC4CoV formulation, in accordance with an embodiment of the present
invention. Doble headed arrows in the image show diameter position of nanoscale
morphologies present in TEM images. TEM imaging showed the sub-100 nm
morphology of the nasal spray-based TriC4CoV formulation with a sub-50 nm core
of carbon present in the nasal spray-based TriC4CoV formulation. Sizes are smaller
than hydrous size evaluated through dynamic light scattering as TEM images
represent anhydrous state of the formulations.
In-vitro safety assay for the nasal spray-based TriC4CoV formulation:
After establishing the nanoscale nature, satisfactory stability and feasibility of being
used as nasal spray, evaluating non-toxic nature of the material against cellular
system of the nasal spray-based TriC4CoV formulation was important. An in-vitro
cell culture model was used for the purpose. Vero, which is a kidney epithelial cell
line of the organism Cercopithecus aethiops was used for the experiment and was
obtained from ATCC. Cells were seeded in DMEM (Dulbecco's Modified Eagle
Medium) medium (Thermo Fisher Scientific, India) supplemented with 10% fetal
bovine serum (FBS). The cells were incubated at 37˚C and 5% CO2 in a humidified
atmosphere. Regularly cells were passaged by trypsinization using 0.05% Trypsin
EDTA (Thermo Fisher Scientific, India) and PBS. The in-vitro safety assessment
was performed by MTT assay (cell viability). Percentage of the cell viability was
14
assessed with Thiazolyl Blue Tetrazolium Bromide (MTT) assay in Vero cell line.
4×105
number of cells were plated in each well of a 6 well plate. After a duration
of 24 hours of plating cells were treated with 4µM HCQ, 0.4mM ZnSO4, 4µM
HCQ+ 0.5mM ZnSO4, 50µM Niclosamide , and 4µM HCQ containing optimized
nasal spray-based TriC4CoV formulation for a duration of 44 hours. After that all
media was aspirated and 1ml of DMSO (dimethyl sulfoxide) (Molychem, Mumbai,
India) was added in each well to dissolve formazan crystal. Once crystals were
dissolved properly, solution from one well of 6 well plate was divided into 5wells
of 96 well plate as 200µl in each well. After that absorbance of samples were
measured by EnSpire 2300 plate reader at 572nm. The percentage of cell viability
was calculated and used to conclude that the nasal spray-based TriC4CoV
formulation is safe toward the Vero cells and did not reduce the viability of any
cells to any significant level up to 48 hours of treatments (shown in Figure 5). The
niclosamide concentration used as medicinal control while mixture of 4µM HCQ+
0.5mM ZnSO4 was used as positive control. It is also established that protected
4µM HCQ+ 0.5mM ZnSO4 in the nasal spray-based TriC4CoV formulation has no
safety issue against the Vero cells up to 48 hours interactions.
[0046] FIG. 5 represents results of in-vitro safety assay for the nasal spraybased TriC4CoV formulation, in accordance with an embodiment of the present
invention. The results of in-vitro safety assay show % cells viability against use of
4µM HCQ, 0.4mM ZnSO4, 4µM HCQ+ 0.5mM ZnSO4 mixture, 50µM
Niclosamide, and 4µM HCQ containing optimized nasal spray-based TriC4CoV
formulation for a duration of 44 hours.
[0047] FIG. 6 represents results of in-vitro safety assay at level of DNA
degradation for the nasal spray-based TriC4CoV formulation, in accordance with
an embodiment of the present invention. In results of in-vitro safety assay at level
of DNA degradation for the nasal spray-based TriC4CoV formulation abbreviations
represent L: Ladder; P: Plasmid; 1: control; 2: HCQ; 3: ZnSO4; 4: HCQ+ZnSO4; 5:
TriC4CoV and 6: Niclosamide.
15
Anti-bacterial efficacy of the nasal spray-based TriC4CoV formulation:
S. aureus has been found as one of the microbes responsible for infection after virus
(SARS-CoV-2) attack. Alamar blue assay is performed to find out that same
concentration of ZnSO4 (0.4 mM) in form of the nasal spray-based TriC4CoV
formulation has around three fold inhibitory effect against S. aureus (shown in
Figure 7).
[0048] FIG. 7 represents results of anti-bacterial efficacy assay of the nasal
spray-based TriC4CoV formulation, in accordance with an embodiment of the
present invention.
[0049] The present invention is explained further in the following specific
examples which are only by way of illustration and are not to be construed as
limiting the scope of the invention.
Example 1
A 5 ml aqueous suspension of carbon nanoparticle (CNP) was heated at 60°C while
stirring at 1100 rpm for 10 minutes. An amount of HCQ (Sigma-Aldrich, India) and
ZnSO4 (Sigma-Aldrich, India) was mixed with 5ml of aqueous suspension of CNP
to achieve 500 µM and 5 mM concentration of HCQ and ZnSO4, respectively.
Mixture was allowed for further stirring at same speed and temperature.
Simultaneously, 5 mg amount of PS-b-PAA (purchased from Sigma-Aldrich, USA)
was dissolved in 1 ml of tetrahydrofuran (THF). 5mg/ml concentration of the THF
solution of PS-b-PAA was added to the aqueous suspension of CNP containing 500
µM HCQ and 5 mM ZnSO4 by drop-wise manner. The whole sample was heated at
60°C and stirred at 350 rpm for a duration of 12 hours. At the end of the preparation
method, the whole sample was probe (2mm) sonicated twice using LABMAN,
PRO-650 (Pulsed Amp, 2s; on 2s; off) for 30 minutes at an interval of 10 minutes.
16
[0050] The present invention provides the formulation including nasal spraybased TriC4CoV formulation. The nasal spray-based TriC4CoV formulation is the
non-vaccine biomedical product for inhibition of viral infection. The present
invention may keep a person with dose of the nasal spray-based TriC4CoV
formulation safe against the SARS-Co-V2 for at least 4 to 24 hours. The nasal
spray-based TriC4CoV formulation prevents immature release of loaded HCQ and
ZnSO4 in-vivo. The nasal spray-based TriC4CoV formulation helps in prevention,
resistance, reduction in further spread of the SARS-CoV-2 infection in already
infected population and co-care against microbial infections. The present invention
also provides the process for preparing the nasal spray-based TriC4CoV
formulation. The process provides stability of the nasal spray-based TriC4CoV
formulation 4 months even at room temperature. The process helps in converting
solution form of the formulation to mist form.
[0051] While specific language has been used to describe the invention, any
limitations arising on account of the same are not intended. As would be apparent
to a person skilled in the art, various working modifications may be made to the
method in order to implement the inventive concept as taught herein.
[0052] The figures and the foregoing description give examples of
embodiments. Those skilled in the art will appreciate that one or more of the
described elements may well be combined into a single functional element.
Alternatively, certain elements may be split into multiple functional elements.
Elementsfrom one embodiment may be added to another embodiment. For example,
order of processes described herein may be changed and are not limited to the
manner described herein. Moreover, the actions of any flow diagram need not be
implemented in the order shown; nor do all of the acts need to be necessarily
performed. Also, those acts that are not dependent on other acts may be performed
in parallel with the other acts. The scope of embodiments is by no means limited by
these specific examples.

WE CLAIM:
1. A formulation for inhibition of SARS-CoV-2 infection, comprising:
a nasal spray-based TriC4CoV formulation, characterized in that
a combination of hydroxychloroquine sulfate (HCQ) and zinc sulfate
(ZnSO4) loaded inside poly-styrene-block-polyacrylic acid (PS-b-PAA)
shell, cored with carbon nanoparticle (CNP).
2. The formulation as claimed in claim 1, wherein the nasal spray-based
TriC4CoV formulation comprises HCQ and ZnSO4 in concentration of 500
µM and 5 mM, respectively.
3. The formulation as claimed in claim 1, wherein the nasal spray-based
TriC4CoV formulation comprises PS-b-PAA and CNP in concentration of
0.5 wt/vol% and 10 wt/vol%, respectively.
4. The formulation as claimed in claim 1, wherein the nasal spray-based
TriC4CoV formulation comprises size of 100nm.
5. The formulation as claimed in claim 1, wherein the nasal spray-based
TriC4CoV formulation is configured to inhibit entry of SARS-CoV-2 inside
cells by competitive binding to angiotensin-converting enzyme 2 (ACE-2)
receptor.
6. A process for preparing a nasal spray-based TriC4CoV formulation,
comprising:
preparing 10 wt/vol% aqueous suspension of carbon nanoparticle
(CNP);
stirring the aqueous suspension of CNP at 60°C for a duration of 10
minutes at 1100 rpm;
18
mixing hydroxychloroquine sulfate (HCQ) and zinc sulfate (ZnSO4)
with the aqueous suspension of CNP to obtain a first mixture;
stirring the first mixture at 60 °C for a duration of 10 minutes at 1100
rpm;
dissolving 0.5 wt/vol% of poly-styrene-block-polyacrylic acid (PS-bPAA) in tetrahydrofuran (THF) to obtain a THF solution;
adding the THF solution to the first mixture by drop wise method to
obtain a second mixture; and
probe sonicating the second mixture for two times to obtain the nasal
spray-based TriC4CoV formulation.
7. The process as claimed in claim 6, wherein the aqueous suspension of CNP
is prepared using a microwave synthesis method.
8. The process as claimed in claim 6, wherein the first mixture comprises 500
µM and 5 mM concentrations of the HCQ and the ZnSO4, respectively.
9. The process as claimed in claim 6, wherein the second mixture is stirred at
60°C for a duration of 12 hours at 350 rpm before probe sonicating.
10. The process as claimed in claim 6, wherein the probe sonicating the second
mixture for two times is carried for a duration of 30 minutes at an interval of
a duration of 10 minutes.
11. The process as claimed in claim 6, wherein the nasal spray-based TriC4CoV
formulation comprises size of 100nm.
12. The process as claimed in claim 6, wherein the nasal spray-based TriC4CoV
formulation is configured to inhibit entry of SARS-CoV-2 inside cells by competitive binding to angiotensin-converting enzyme 2 (ACE-2) receptor.