hField of the Invention
The present invention relates to an extract of Cissampelos pareira, its pharmaceutical
compositions and its therapeutic use in the prevention and treatment of dengue. It also relates to
processes for the preparation of these extracts.
Background of the Invention
Dengue disease remains the major public health concern around the world. The
incidence of dengue has grown dramatically around the world in recent decades. Dengue occurs
in tropical and sub-tropical climates worldwide, mostly in urban and semi-urban areas. Severe
dengue is a leading cause of serious illness and death among children in most of Asian and Latin
American countries. According to World Health Organization (WHO) estimates, ~2.5 billion
people around the globe are at risk of dengue, with ~50 million infections worldwide annually.
It is spread to humans through the bite of Aedes mosquitoes which serve as carriers of the
disease-causing viruses. There are four serotypes of dengue viruses (DENV-1, -2, -3 and -4),
belonging to the family Flaviviridae. Recovery from infection by one serotype of dengue virus
provides lifelong immunity against that particular serotype. However, cross-immunity to the
other serotypes after recovery is only partial and temporary. Subsequent infections by other
serotypes increase the risk of developing severe dengue. Infection with DENVs may be
asymptomatic or result in a range of clinical symptoms from mild dengue fever (DF) to severe
and potentially fatal dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). The
clinical symptoms for mild dengue fever include high fever, severe headache, pain behind the
eyes, muscle and joint pains, nausea, vomiting, swollen glands or rash. Symptoms usually last
for 2-7 days, after an incubation period of 4-10 days after the bite from an infected mosquito.
The clinical symptoms for severe dengue appear due to plasma leaking, fluid accumulation,
respiratory distress, severe bleeding, or organ impairment. Despite the alarming impact on both
human health and global economy, there is no specific treatment of dengue yet available.
Though some of the live attenuated dengue vaccines are being developed, the challenges faced
in dengue vaccine development remain high.
Thus, there exists an urgent need for the effective dengue treatment that can shorten the
duration of illness, reduce the severity of common symptoms, prevent the development of severe
complications, and which is easy to formulate. Furthermore, it is highly desirable to develop a
dengue treatment that can reduce the viral load at an early stage such that it potentially prevent
dengue fever as well as life-threatening severe form of dengue.
The present invention fulfills the unmet need by providing an effective as well as
patient-compliant dengue treatment. Cissampelos pareira extracts help to effectively prevent as
I P O D E L H I 17 - 1 2 - 2 0 1 5 " 17 : 3f
well treat the dengue.viral disease. The present inventors have found that the extracts of
Cissampelos pariera Linn (Cipa extract) are potent inhibitors of all four DENVs in cell-based
assays, assessed in terms of viral NS1 antigen secretion using ELISA, as well as viral
replication, based on plaque assays. Virus yield reduction assays showed that the extracts of
Cissampelos pariera decrease viral titers by an order of magnitude. The extracts of Cissampelos
pariera conferred statistically significant protection against DENV infection using the AG 129
mouse model. Surprisingly, it been discovered that the potency of Cissampelos pareira extracts
extend over a wide range of viral loads including the initial stage viral load, which could
subsequently prevent the life-threatening severe form of dengue. Further, the present inventors
have determined that both Cissampelos pareira extracts and paracetamol show synergistic effect
in decreasing the body temperature. Also, the dengue disease predisposes some patients to
hemorrhagic manifestations and tends to be associated with lowered platelet counts. Therefore,
it also becomes important to assess if Cissampelos pareira extracts have any undesired effect on
erythrocytes and platelets. The present inventors have determined that Cissampelos pareira
extracts did not have any discernible effect on platelet counts or on erythrocyte viability. They
have also determined that the extracts also possessed the ability to downregulate the secretion of
pro-inflammatory cytokines, particularly TNF-a and IL-lp\ Further, extracts of Cissampelos
pariera showed no evidence of toxicity.
Summary of the Invention
The present invention provides an extract of Cissampelos pareira, its pharmaceutical
compositions and its therapeutic use in the prevention and treatment of dengue. It also relates to
processes for the preparation of these extracts. It further provides the activity of these extracts
against dengue virus in mammals. Further, it provides the synergistic antipyretic effect of
Cissampelos pareira extract in combination with paracetamol. It also provides the antiinflammatory
effect of Cissampelos pareira extracts with no adverse effect on platelet counts and
on erythrocyte viability. Further, these extracts did not show any toxic effects.
Brief Description of the Drawings
Figure 1: Schematic representation of the antiviral screening assays
Figure 2: Inhibition of DENV antigen and virus production by the treatment of methanolic extract
Figure 3: Effect of pre-incubation time on antiviral activity of methanolic extract
Figure 4: Evaluation oiin-vivo protective efficacy of methanolic extract
Figure 5: Analysis of interaction between paracetamol and methanolic extract
Figure 6: Effect of methanolic extract on platelets
Figure 7: Effect of methanolic extract on RJBCs
O D E L H I 17 - 1 2 - 2 0 1 S 1 7 : 3 3
3
Detailed Description of the Invention
A first aspect of the present invention provides an extract of Cissampelos pareira for use in
the treatment of dengue virus infection in mammals.
A second aspect of the present invention provides a pharmaceutical composition for use in
the treatment of dengue virus infection in mammals comprising an extract of Cissampelos pareira
and one or more pharmaceutically acceptable excipients.
A third aspect of the present invention provides a pharmaceutical composition for use in the
treatment of dengue virus infection in mammals, comprising:
(a) an extract of Cissampelos pareira; and
(b) paracetamol;
wherein (a) and (b) are administered together as a single pharmaceutical composition or are coadministered
simultaneously or sequentially.
A fourth aspect of the present invention provides a method of treating dengue virus
infection comprising administering a pharmaceutical composition comprising:
(a) an extract of Cissampelos pareira, and
(b) paracetamol
wherein (a) and (b) are administered together as a single pharmaceutical composition or are coadministered
simultaneously or sequentially.
A fifth aspect of the present invention provides an extract of Cissampelos pareira
to reduce the viral load at an early stage in the treatment of dengue virus infection.
A sixth aspect of the present invention provides an extract of Cissampelos pareira for use in
the treatment of dengue virus infection in mammals, wherein the extract exhibits a platelet
protective effect.
A seventh aspect of the present invention provides an extract of Cissampelos pareira for use
in the treatment of dengue virus infection in mammals, wherein the extract exhibits an erythrocyte
protective effect.
An eighth aspect of the present invention provides an extract of Cissampelos pareira to
reduce the viral load at an early stage in the treatment of dengue virus infection, wherein the extract
exhibits a platelet protective effect.
A ninth aspect of the present invention provides an extract of Cissampelos pareira to reduce
the viral load at an early stage in the treatment of dengue virus infection, wherein the extract
exhibits an erythrocyte protective effect.
A tenth aspect of the present invention provides a pharmaceutical composition comprising
an extract of Cissampelos pareira and one or more pharmaceutically acceptable excipients to
IP 0.- D E L H X 1 7 - 1 2. - 2 0 1 S 1 7 :-3 3
. reduce the viral load at an early stage in the treatment of dengue virus infection, wherein the extract
exhibits a platelet protective effect.
An eleventh aspect of the present invention provides a pharmaceutical composition
comprising an extract of Cissampelos pareira and one or more pharmaceutical^ acceptable
excipients to reduce the viral load at an early stage in the treatment of dengue virus infection,
wherein the extract exhibits an erythrocyte protective effect.
In one of the embodiment of the above aspects, the extract is, an alcoholic extract, a
hydroalcoholic extract, or an aqueous extract. In a preferred embodiment, the extract is an alcoholic
extract. In a more preferred embodiment, the extract is a methanolic extract. The methanol in the
methanolic extract may be removed completely by evaporation to get a dried extract. The dried
extract may be lyophilized to form a powder, which can then be filled into a capsule of suitable
size.
In another embodiment of the above aspects, the extract of Cissampelos pareira
is used for the prevention of dengue virus infection.
"Cissampelos pareira" belongs to a family Menispermaceae and is a climbing shrub
distributed throughout the warm parts of Asia, East Africa, North and South America and common
in India and Sri Lanka. It is also commonly known as Velvet Leaf or Patha or Ambasthaki. It is
common in warm and dry regions of tropical and sub-tropical parts of India up to an altitude of
about 1500 m. It is found in Himachal Pradesh, Chota Nagpur, Bihar, West Bengal, Punjab,
Rajasthan particularly in the east of Aravalli, hilly forests of Marathwada, Konkan, Deccan,
Bababuden hills of Mysore, Tamil Nadu {Ayurvedic Pharmacopoeia of India, First Edition, Part 1,
Vol 1, p. 92-93; Govt of India, Ministry of Health and Family Welfare, Dept of Indian System of
Medicine and Homoeopathy, New Delhi; The Wealth of India, A Dictionary of Indian Raw
Materials and Industrial Products, Raw Materials, Vol II, Council of Scientific and Industrial
Research, Delhi; Database on Medicinal Plants Used In Ayurveda, Vol 2, Central Council for
Research in Ayurveda and Siddha, Dept of Indian System of Medicine and Homoeopathy, New
Delhi).
"Paracetamol," chemically is N-(4-hydroxyphenyl) acetamide. It is also commonly known
as acetaminophen. It is a well-known antipyretic used for number of years. The present invention
provides the synergistic antipyretic effect of paracetamol with the extract of Cissampelos pareira.
The present invention incorporates safe and effective use of paracetamol in combination with the
extracts of Cissampelos pareira for treating or preventing dengue viral infections. Paracetamol and
the extracts of Cissampelos pareira can be administered together as a single pharmaceutical
composition or are co-administered simultaneously or sequentially.
P O D E L H I 1 7 - 1 2 - 2 0 1 S 1 1 • 3 3
5
The term "alcoholic extract," as used herein includes any extract, for example, methanolic,
ethanolic, n-propanolic, isopropanolic, n-butanolic, iso-butanolic or /-butanolic extract of
Cissampelos pareira. In particular, the "alcoholic extract" is a methanolic extract.
The term "hydroalcoholic extract," as used herein includes an extract prepared by using a
mixture of alcohol and purified water. Examples of alcohols are methanol, ethanol, n-propanol,
isopropanol, n-butanol, iso-butanol and ^-butanol. In particular, 1:1 mixture of alcohol and purified
water is used.
The term "aqueous extract," as used herein includes a purified water extract of Cissampelos
pareira.
The extracts of Cissampelos pareira are prepared by extracting the plant mass of
Cissampelos pareira with one or more solvents selected from methanol, ethanol, n-propanol,
isopropanol, n-butanol, iso-butanol or f-butanol, purified water or a mixture thereof, concentrating
the extract, and drying the extract.
The term "plant mass of Cissampelos pareira" as used herein refers to whole plant, which
includes, aerial parts, for example, fruits, flowers, leaves, branches, stem bark, stem, seeds or
heartwood and root.
The term "minimum lethal dose (MLD)" as used herein refers to the dose that can cause
clinical symptoms and 90-100% death 3-4 weeks post challenge.
The term "pharmaceutical composition," as used herein includes any composition that can
effectively deliver the extracts of Cissampelos pareira to the desired site of action to treat or
prevent dengue viral infection. The composition can be delivered by any suitable route of
administration such as oral, nasal, pulmonary, transdermal, or rectal. The pharmaceutical
composition includes one or more pharmaceutically acceptable excipients. The oral pharmaceutical
composition can be in the form of powder, pellet, granule, spheroid, mini-tablet, caplet, tablet, or
capsule. The powder can be in the form of lyophilized powder filled with pharmaceutically
acceptable excipients into a capsule of suitable size.
The term "pharmaceutically acceptable excipients," as used herein includes diluents,
binders, disintegrants, lubricants, glidants, polymers, flavoring agents, surfactants, preservatives,
antioxidants, buffers, and tonicity modifying agents.
Examples of diluents include microcrystalline cellulose, powdered cellulose, starch, starch
pregelatinized, dextrates, lactitol, fructose, sugar compressible, sugar confectioners, dextrose,
lactose, calcium phosphate-dibasic, calcium phosphate-tribasic, calcium sulfate, and mixtures
thereof.
Examples of binders include a water-soluble starch, for example, pregelatinized starch; a
Tp n ppJysacchar4de,,fQr example,^agar| gum acacia,^di&xtrin, sodium alginate, tragacanth gum, xanthan
6
gum, hyaluronic acid, pectin, or sodium chondroitin sulfate; a synthetic polymer, for example,
polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyacrylic acid-series polymer,
polylactic acid, or polyethylene glycol; a cellulose ether, for example, methyl cellulose, ethyl
cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, or a
hydroxypropyl methyl cellulose; and mixtures thereof.
Examples of disintegrants include calcium carbonate, carboxymethyl cellulose or a salt
thereof, for example, croscarmellose sodium, crosslinked povidone, low-substituted hydroxypropyl
cellulose, and sodium starch glycolate.
Examples of lubricants/glidants include talc, magnesium stearate, hydrogenated vegetable
oils, sodium stearyl fumarate, calcium stearate, colloidal silicon dioxide, Aerosil®, stearic acid,
sodium lauryl sulphate, sodium benzoate, polyethylene glycol, hydrogenated castor oil, sucrose
esters of fatty acid, microcrystalline wax, yellow beeswax, white beeswax, and mixtures thereof.
Examples of flavoring agents include synthetic flavor oils and flavoring aromatics, or
natural oils or extracts from plants, leaves, flowers, fruits, and combinations thereof. These may
include cinnamon oil, oil of wintergreen, peppermint oils, bay oil, anise oil, eucalyptus, thyme oil,
vanilla, citrus oil, including lemon, orange, lime and grapefruit, and fruit essences including apple,
banana, grape, pear, peach, strawberry, raspberry, cherry, plum, pineapple, and apricot.
Examples of surfactants include anionic surfactants, for example, a sulfonic acid or a salt
thereof such as benzenesulfonic acid, dodecylbenzenesulfonic acid, or dodecanesulfonic acid; an
alkyl sulfate, for example, sodium dodecyl sulfate or sodium lauryl sulfate; cationic surfactants, for
example, a tetraalkylammonium salt such as a tetraalkylammonium halide, benzethonium chloride,
benzalkonium chloride, or cetylpyridinium chloride; a nonionic surfactant, for example, a (poly)
oxyethylene sorbitan long-chain fatty acid ester such as a polyoxyethylene sorbitan monolaurate,
for example, a polysorbate; amphoteric surfactants, for example, a glycin compound such as
dodecyl-di-(aminoethyl)glycin, a betaihe compound such as betaine or
dimethyldodecylcarboxybetaine, and a phosphatidic acid derivative such as lecithin; polymeric
surfactants, for example, a polyoxyethylene polyoxypropylene glycol such as Pluronic® or
Poloxamer; and mixtures thereof.
Examples of buffers include phosphate buffer such as dihydrogen sodium phosphate, citrate
buffer such as sodium citrate, meglumine, tri(hydroxymethyl) aminomethane, or mixtures thereof.
Examples of tonicity modifying agents include sodium chloride, mannitol, dextrose,
glucose, lactose, sucrose, or mixtures thereof.
Examples of solvents for the preparation of the pharmaceutical composition include water;
water miscible organic solvents, for example, isopropyl alcohol or ethanol; dipolar aprotic solvents;
I P O DELHI. 1 7 - 1.2 - 2.0 15 1 7 : 33
7
methylene chloride; acetone; polyethylene glycol; polyethyleneglycol ether; polyethyleneglycol
derivative of a mono- or di-glyceride; buffers; organic solvents;, and combinations thereof.
While the following examples are provided to certain embodiments of the invention, these
are not intended to be limiting to the scope of the invention.
Example 1: Preparation of a Methanolic Extract of Cissampelos pareira
Pulverized Cissampelos pareira aerial parts (100 kg) were charged into an extractor.
Methanol (500 liter) was added and the extraction was done at a temperature ranging from room
temperature to the boiling point of the solvent for about 16 hours. The extract was filtered and
stored in a container. The extraction and filtration steps were repeated with 300 liters of methanol
twice. The filtered extracts were stored in containers. The three methanolic extracts were combined
and concentrated to the maximum possible extent under reduced pressure at low temperature. The
extract was decanted into stainless steel trays and dried in high vacuum oven at room temperature
for about 16 hours to 18 hours.
Yield =6%- 15%w/w
The dried extract was lyophilized to form a powder. This powder was then filled into a
capsule of suitable size.
Example 2: Preparation of a hydroalcoholic (1:1 Methanol: Purified Water) extract of Cissampelos
pareira
Pulverized Cissampelos pareira aerial parts (100 kg) were charged into an extractor. A
mixture of methanol and purified water (250 liter: 250 liter) was added and the extraction was done
at a temperature ranging from room temperature to the boiling point of the solvent for about 16
hours. The extract was filtered and stored in a container. The extraction and filtration steps were
repeated with methanol: purified water (150 liter: 150 liter) twice. The filtered extracts were stored
in containers. The three hydro alcoholic extracts were combined and concentrated to the maximum
possible extent under reduced pressure at low temperature. The extract was decanted into stainless
steel trays and dried in high vacuum oven at room temperature for about 16 hours to 18 hours.
Yield =10%-25% w/w.
Example 3: Preparation of an Aqueous Extract of Cissampelos pareira
Pulverized Cissampelos pareira aerial parts (100 kg) were charged into an extractor.
Purified water (500 liter) was added and extraction was done at temperature ranging from room
temperature to the boiling point of the solvent for about 16 hours. The extract was filtered and
stored in a container. The extraction and filtration steps were repeated with 300 liters of purified
water twice. The filtered extracts were stored in containers. The three aqueous extracts were
combined and concentrated to maximum under reduced pressure at low temperature. The extract
IPO DELHI 17-12-2015 17:33
8
was decanted into stainless steel trays and dried in high vacuum oven at room temperature for about
16 hours to 18 hours.
Yield =15%-30% w/w.
Example 4: BioloRical activity
(a) Plaque assay
LLCMK2 monolayers in 6 well plates were infected in duplicate with serial 10-fold
dilutions (prepared in Dulbecco's Modified Eagles Medium (DMEM) +2% heat inactivated Fetal
calf serum (AFCS) of the virus-containing samples (250ul/well). Mock-infections were performed
in parallel using an equivalent volume of virus diluent alone. Two hours later, the infected
monolayers (after aspirating off the virus inoculum) were overlaid with DMEM+6%AFCS
containing 1% methyl cellulose (2 ml/well), and incubated for 6 days (37°C, 5% CO2). On day 6
post-infection, the overlay was removed and the cells were fixed with 4% formaldehyde solution (1
ml/well). Fixed cells were washed and stained with 0.05% (w/v) crystal violet solution in 20%
ethanol. Revealed plaques were counted to determine the virus titre, expressed as plaque-forming
units (PFUs)/ml.
(b) Cell-based bioassays for antiviral screening
(i) Tvpe-1 assay: In the initial antiviral screening assay, designated as the type-1 assay,
LLCMK2 cells were seeded in 24-well plates (5xl05 cells/well), a day in advance. DENV-1, -2, -3
and -4 (100 PFU each) were separately pre-incubated with serial dilutions of the extracts of the
present invention (corresponding to 0-100 ug/ml final concentration) in 300 ul volume, at 4°C
overnight. The pre-incubation mixture was diluted with an equal volume of medium (DMEM+2 %
AFCS) and used to infect LLCMK2 cells (3 wells for each concentration at 200 ul/well) in the 24-
well plate. After 2 hours of adsorption in the incubator (37°C, 5% CO2), infected cells were
overlaid with methylcellulose-containing growth medium and processed thereafter as described for
the plaque assay (a). Cells were exposed to the extracts of the present invention (in the same
concentration range) in the absence of DENV infection to assess any potential cytotoxicity.
Additional control experiments were run in parallel, which included cells, which were either mockinfected
(negative control) or infected with DENV in the absence of the extracts of the present
invention (positive control). The half-maximal inhibitory concentration (IC50 value) for each extract
against each DENV serotype, with reference to the positive control, which represented 100%
infection (or 0% inhibition), was defined as the concentration of the extract, in ug/ml, resulting in
50% inhibition of the plaque count.
(ii) Type-2 assay: LLCMK2 cells in 24-well plates were infected with DENVs (a
multiplicity of infection (MOI) =0.002) without pre-incubating with the extracts. After 2 hours of
the adsorntipn, the^virus inqculumjwgs aspirated^.the monolayer rinsed, and then fed with complete
medium. containing the extracts of the present invention (corresponding to 0-200 ug/ml final
concentration). After 24 hours of the exposure to the extracts of the present invention, the
monolayer was aspirated and overlaid with growth medium containing methyl cellulose and
plaques developed 6 days later.
(iii) Type-3 assay: Type-3 assay was performed using Vero cells. The assay design was
similar to the type-2 assay, except that following the sequential exposure of cells to DENV and the
extracts of the present invention, cells were fed with liquid growth medium, instead of the
methylcellulose overlay. Aliquots of the culture supernatant were withdrawn at periodic intervals
up to 9 days for estimation of NS1 antigen levels (using a commercial ELISA kit) and virus titres
(by plaque assay, as described in (a)).
Figure 1 provides a schematic representation of the antiviral screening assays. An outline of
the three types of screening assays (indicated by Arabic numbers 1, 2 and 3) is shown. The multicoloured
sphere represents DENV and the Eppendorf tube with green liquid represents the extract
of the present invention. These two were pre-incubated (1) before addition to the monolayer or
added sequentially (2, 3) to the monolayer. In assays 1 and 2, the treated-monolayers were overlaid
with methyl cellulose containing growth medium. Shown at the bottom are the possible outcomes
of the type 1 and 2 assays. The 'x' mark denotes failure of entry into cells. In assay 3, liquid growth
medium was added instead of the methyl cellulose overlay, and followed by analysis of NS 1 and
virus released into the culture supernatant.
(c) Determination of cytotoxicity
Cytotoxicity was evaluated in two cell lines, LLCMK2 (in which the antiviral activity of the
extracts were assayed) and HepG2, a commonly used liver cell surrogate for in-vitro cytotoxicity
testing. Cells seeded in 96-well plates were exposed to a wide concentration range of the extracts of
the invention (1-200 ug/ml) for 3 days. Cell viability was assessed using a commercial MTT (3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay kit with reference to control
cells that were not exposed to the extracts of the invention. The half-maximal cytotoxic
concentration (CC50 value) for the extract, with reference to the positive control (untreated cells)
which represented 100% cell viability (or 0% cytotoxicity), was defined as the concentration of the
extract, in ug/ml, resulting in 50% cytotoxicity. Selectivity index (SI) of an extract is defined as the
ratio of CC50 to IC50 values obtained using the LLCMK2 cell line.
(d) Inhibition of secretion of viral antigen and infectious virus
The kinetics of virus inhibition by the extracts of the present invention was analysed in a
type-3 assay. Aliquots of the culture supernatant were withdrawn at regular intervals over a period
of several days and analyzed for the presence of viral NSl antigen and infectious virus, as shown in
Figure 2 J n c o n t o l experiment, wieremJrrfecled cells were not exposed to the extract, NSl
10
antigen was detectable after day 2 onwards and rising thereafter during the course of the
experiment. In parallel experiments, the exposure of cells to the extract had a dose-dependent
inhibitory effect on NS1 antigen secretion. While the inhibition resulting from exposure to a low
dose of the extract was manifested after day 4 post-infection, inhibition at higher doses was evident
earlier and at relatively higher magnitudes (Figure 2A). In fact, at the highest dose of the extract
tested in this experiment (lOOug/ml), the inhibition of NS1 antigen was near total for the entire
duration of the experiment. The inhibition of viral antigen synthesis evident from this experiment
shows that virus production would also be similarly affected. This notion was substantiated by
determination of viral titers in the culture supernatants during the course of the above experiment,
as shown in Figure 2B. In the control experiment, viral titers increased steadily reaching a plateau
at day 3 post-infection. The extracts of the present invention lowered viral titers in a dosedependent
manner as seen for NS 1 secretion. Thus, at the lowest concentration of the extract used,
reduction in viral titers became apparent from day 4 onward. Significantly, a small increase in the
extract dosage resulted in >1 log reduction in viral titer as early as day 3 post infection. At the
highest dose of the extract tested (lOOug/ml), the drop in viral titers was ~2 logs. Importantly, the
reduction in viral titers was sustained over a period of several days. Surprisingly, the magnitude of
inhibition appeared to be greater based on NS 1 levels compared to viral titers. The data shows that
the extract may have effects on NS1 antigen synthesis and release that are distinct from its effects
on virus replication.
Figure 2 depicts the kinetics of NS1 antigen (A) and infectious virus (B) released into the
culture supernatant in the absence (empty black circles) and presence of the methanolic extract at
22 ug/ml (filled blue circles), 66 ug/ml (empty red squares) and 200 ug/ml (filled green squares)
concentrations.
(e) Determination of the effect of pre-incubation time and virus dose on the anti-DENV activity
To assess the influence of the duration of pre-incubation of the extracts of the present
invention with DENV antiviral activity in the type-1 assay format, pre-incubation. times ranging
from 0-24 hours were tested using -50 PFUs of DENV-3.
To assess the effect of the size of the DENV dose on the anti-DENV efficacy of the extracts
of the present invention in the pre-incubation step (at 4°C, overnight), type-1 assays were
performed using DENV-3 ranging from 50 to 5000 PFUs. Each dose of DENV-3 was assayed
against the extract ranging in concentration from 0-200 ug/ml.
DENV-3 was pre-incubated with increasing concentrations of the extracts of the present
invention for different periods of time before infection (type-1 assay) and overlay. Plaque counts
obtained at the end of the experiment revealed a dose- and time-dependent virucidal effect of the
extracts of the present mv^ntiqjvonDENY-f as depicted in Figure 3. A converse experiment, again
11
in type-1 format, was carried out to determine the inhibitory efficacy of the extract against DENV-3
stocks whose titers varied over 2 logs. The IC50 values of methanolic extract corresponding to
DENV-3 dosage of 50, 500 and 5000 PFUs were, respectively, 9.92, 12.5 and 44.45 ug/ml. This
leads to the conclusion that the antiviral potency of the methanolic extract extends over a wide
range of viral loads.
Figure 3 provides the effect of pre-incubation time on the antiviral activity of the
methanolic extract of the present invention. DENV-3 (50 PFU) was pre-incubated with the extract
at 11 ug/ml (filled red circles), 33 ug/ml (empty blue squares) and 100 ug/ml (filled black squares)
for different durations (2-24 hours) followed by assay of antiviral activity in a type-1 assay.
(f) Determination of in-vivo protective efficacy
To determine the in-vivo efficacy, DENV-2 (NGC) was alternately passaged between
AG 129 (intracranial inoculation of 106 PFU) and C6/36 cells in tissue culture. After 4-5 such cycles
of passaging, the virus was tested in AG 129 mice to determine the minimum lethal dose (MLD) by
i.p. injections. The challenge virus stock thus obtained was titrated, aliquotted and stored in liquid
N2 until use. To test protective efficacy (effect) of the extracts of the present invention, AG 129
mice (9-12 weeks old, 20-24 g body weight) were challenged with 106 PFU (per mouse, 0.4 ml, i.p)
of the challenge DENV-2 stock.
Challenged mice were divided into groups (n=6) and treated orally with vehicle alone
(0.25% methyl cellulose) or with two different doses of the extracts of the present invention (at 125
mg and 250 mg/kg body weight). The methanol in the methanolic extract administered to mice was
removed completely by evaporation. The resultant methanol-free paste was thoroughly resuspended
in 0.25% methyl cellulose water and administered orally to infected mice. The volume
of the oral dose was adjusted in accordance with the body weight of each animal (10 ml/Kg/dose)
and administered by a trained veterinarian using a specially designed mouse feeder needle fitted
with a graduated 1 ml disposable syringe. The treatment was initiated 2 hours post-infection and
continued twice daily for 5 consecutive days. Animals were monitored twice daily for a period of
35 days for clinical symptoms and mortality. A control (sham) group that was not virus-challenged,
but which received the extract of the present invention (250 mg/kg), was also tested in parallel. At
the end of the experiment, the survival data was used to plot Kaplan Meier survival curves and
analysed by the log rank test (Mantel-Cox) test for statistical significance using GraphPad Prism 5
software.
The present inventors have found that the median survival time (MST) of the challenged
mice treated orally with extract (methanol-free) twice a day for 5 days post-challenge increased in a
I P O : ' D E L H I 1 7 ' - 1 2 - 2 0 - 1 5 1T • 53
dose-dependent manner. The survival data are present in Figure 4. The MST of challenged mice
was 19 days under the experimental conditions. At 125 mg dose, given twice a day for 5 days,
survival was 50% and MST was 28 days (p=0.1). This increased to -67% when the dosage was
doubled. Compared to the placebo-treated.(0.25% methyl cellulose) group, the level of protection
afforded by 250 mg/Kg dose was statistically significant (p=0.021).
Figure 4 provides evaluation of protective efficacy of methanolic extract in- vivo. AG 129
mice (9-12 weeks old) were injected i.p. with 106 PFU brain-passaged DENV-2.
Infected mice were treated orally with 0.25% methyl cellulose (solid red squares) or methanolic
extract at 125 mg (empty blue circles) and 250 mg/kg body weight (solid blue circles). Treatment
was twice daily for the first 5 days. A sham control group that was not virus-infected, but which
received the higher dose of methanolic extract orally (solid green squares), was tested in parallel.
The mice were monitored daily for mortality and the resultant data plotted as Kaplan-Meier
survival curves. The p values to assess the statistical significance in the survival rates on day 35
between the methanolic extract-treated and placebo-treated (0.25% methyl cellulose) groups were
determined using the Log-rank test.
(g) Determination of effect of Paracetamol
Interaction between paracetamol and the extracts of the present invention was assessed invitro
using type-1 assay format. DENV-3 (-50 PFUs) and the extracts of the present invention
(ranging in concentration from 0-50 ug/ml) were pre-incubated overnight at 4°C in a volume of 100
ul, and used to infect LLCMK2 cells in 24-well plates. Parallel infections were set up using preincubation
mixtures containing paracetamol (1-100 ug/ml), in addition to DENV and the extracts of
the present invention. Mock-infections and DENV only infections (in the absence of the extract
and paracetamol) were also set up and analysed in parallel.
The in-vivo effect of the extract of the present invention in the presence and absence of
paracetamol was assessed using the Wistar rat pyrexia model. Wistar rats (weighing 180-220g) of
either sex were used. Basal temperature of the rats was measured using a digital rectal thermometer
(Experimetria Ltd., Hungary) and then injected subcutaneously (in the intra-scapular region) with
20%> brewer's yeast (10 ml/kg body weight) and allowed to fast overnight with free access\to water.
At 18 hours post-injection., rectal temperatures were recorded again to identify animals that
registered >0.7°C rise in body temperature for inclusion in the study. Groups (n=7-9) of febrile rats
were orally administered paracetamol (200 mg/kg), or the extracts of the present invention (200
mg/kg) or both. Rats in the control group received just the vehicle (0.5% methyl cellulose). This
was followed by recording of rectal temperature for 3 hours at 30 minute intervals.
I P O D E L H I :. 1.7 - 1 2 - 2O 1 5 1 7 : 3 2 . "
.13
The data from the studies on the methanolic extract and paracetamol are depicted in Figure
5. A type-1 assay was carried out in which DENV-3 was pre-incubated with serial dilutions of
methanolic extract. It was observed that DENV-3 infectivity was inhibited progressively as the
methanolic extract concentration increased, with an IC50 value of 6. lug/ml. The addition of up to
lOOug/ml paracetamol into the DENV-3/methanolic extract pre-incubation mix did not
significantly affect the inhibitory profile of the extact. The calculated IC50 values in the presence of
paracetamol at 1, 10 and lOOug/ml were, respectively, 8.4, 7.4 and 8.5ug/ml (Figure 5A).
Paracetamol by itself at all concentrations tested did not have any effect on DENV infectivity
(plaque counts obtained with DENV-3 alone and DENV-3 plus paracetamol at lOOug/ml were,
43±3 and 45±4, respectively; n=3). The next experiment examined the effect of methanolic extract
on the antipyretic activity of paracetamol using the Wistar rat pyrexia model. Surprisingly, this
experiment revealed that methanolic extract possessed intrinsic antipyretic effect (Figure 5B).
When rats, in which fever was induced by subcutaneous injection of brewer's yeast, were treated
with methanolic extract, the fever was suppressed at an efficiency that was comparable to that of
paracetamol. Surprisingly, co-administration of methanolic extract with paracetamol had a
synergistic effect, resulting in a more pronounced decrease in body temperature.
Figure 5 provides analysis of interaction between paracetamol and methanolic extract.
(A) DENV-3 (50 PFU) was pre-incubated with methanolic extract in the absence (solid black
diamonds) or presence of 1 ng/ml (solid blue diamonds), 10 ug/ml (solid red circles), or 100 ug/ml
(solid green circles) paracetamol overnight at 4°C, followed by analysis of viral inhibition in a type-
1 assay.
(B) Febrile Wistar rats were mock-treated (empty red circles), or treated with paracetamol (solid
blue circles), methanolic extract (empty green squares) or a combination of both (solid black
squares), followed by monitoring of rectal temperature for 3 hours post-treatment at regular
intervals.
Rectal temperatures between the control (mock-treated) and treatment groups were
compared using one-way ANOVA followed by Dunnett's multiple comparison test (the single and
double stars indicate significant differences in the treatment groups with respect to the control
group, corresponding to p values of <0.05 and <0.01, respectively).
(h) Determination of effect on platelets and erythrocytes
For ex-vivo studies, erythrocytes were pelleted down in a centrifuge (1500xg, 5 minutes)
from freshly collected heparinized human blood, rinsed thoroughly with phosphate buffered saline
(PBS, pH 7.4), and used to make a 1% cell suspension in PBS. The extracts of the present invention
ranging in concentration from 12.5 to 400 mg/L were added to the erythrocyte suspension and
incubated at 37°CLfqr J hour^ Aitergthis^the sajrigles were spun down, and the absorbance of the
14
supernatant measured at 576 nm to determine the extent of erythrocyte lysis. Controls, wherein
erythrocytes were incubated with buffer alone (0% lysis), DMSO alone and 0.1% Triton X-100
(100% lysis) were processed in parallel. Basal platelet count in freshly collected heparinized blood
and in blood pre-incubated with DMSO (vehicle) or the extracts of the present invention (2-10
ug/ml) for different, durations (1-4 hours) was determined using a Beckman Coulter hematology
analyser.
For in-yivo studies, four groups (n=5) of Wistar rats were fasted overnight and administered
orally with vehicle (0.25% methyl cellulose) or the extracts of the present invention, at three
different dosages (100, 300 and 1000 mg/kg body weight). Blood was collected just before the
extract administration (0 hour) and at 1 and 4 hours post-administration. Hematology parameters
were measured using ADVIA-120 hematology analyser.
Figure 6 provides effect of methanolic extract on platelets. Whole blood from human
volunteers was collected and platelets counts were obtained before and after 1 -4 hours post-mixing
with methanolic extract. The results are shown in Figure 6A. In the control sample, blood mixed
with vehicle (saline), platelet counts declined steadily over time. Methanolic extract-treated blood
samples manifested no statistically significant change in platelet counts with respect to their
cognate controls, up to 2 hours (p>0.05). At four hours, the methanolic extract-treated samples
displayed significantly higher (p<0.05) platelet counts, compared to corresponding saline-treated
control. Similar results were observed at 2 and 10 ug/ml methanolic extract concentrations,
indicating that methanolic extract did not affect platelets adversely. The effect of methanolic extract
on platelets was also evaluated in an in- vivo experiment using Wistar rats. In this experiment,
platelet counts were determined in blood drawn from rats which had been given methanolic extract
orally. The results presented in Figure 6B show that up to 4 hours post treatment, methanolic
extract (up to 1000 mg/kg body weight), did not affect platelet counts significantly (p>0.05 at the
highest dose of methanolic extract treatment for 4 hours).
Figure 6 (A): Freshly collected human blood was incubated with saline (white bars) or
methanolic extract (at 2 ug/ml: blue bars; or 10 ug/ml: red bars) for up to 4 hours. Aliquots were
drawn at the indicated times for determination of platelet counts.
Figure 6 (B): Wistar rats were orally administered 0.25% methyl cellulose containing
methanolic extract ranging from 0-1000 mg/Kg body weight.
Fresh blood collected from these rats at 0 (white bars), 1 (blue bars) and 4 (red bars) hours
post-administration, were analysed for platelet counts. For both panels, data shown are mean values
(n=5); the vertical bars represent standard deviation (SD).
The effect of methanolic extract on erythrocytes was also assessed, both in ex- vivo and inv/
iwJissaysTas done for platelets^abo^e. Figure 7^pEovides effect of methanolic extract on RBCs. -
15
Incubation of freshly collected human erythrocytes with methanolic extract at concentrations up to
400 ug/L did not cause discernible haemolysis (Figure 7A). The blood samples, withdrawn from
the Wistar rats (given methanolic extract, described above), were also analysed for erythrocyte cell
counts. This analysis, once again revealed that methanolic extract (at concentrations as high as
1000 mg/kg body weight), did not affect erythrocyte counts in the blood of Wistar rats up to 4
hours post-administration (Figure 7B). The difference in erythrocyte counts between the treated and
untreated rats was not statistically significant (p>0.05). The inventors also analyzed total leucocyte
and differential counts in the blood of methanolic extract-treated Wistar rats ((described in Figures
6B and 7B) and no significant difference was found .
Figure 7 (A): Erythrocytes from freshly collected human blood were incubated for 1 hour at
37°C, with varying concentrations of methanolic extract (0-400 ug/ml), followed by measurement
of haemolysis at 576 nm. TX-100 represents a control in which an equivalent aliquot of
erythrocytes were treated with Triton X-100 to achieve complete lysis.
Figure 7 (B): Fresh blood collected from the methanolic extract-treated Wistar rats at (0
white bars), 1 (blue bars) and 4 (red bars) hours post-administration, were analysed for RBC
counts. Data shown are mean values (n=5); the vertical bars represent SD.
(i) Cytokine release assay
Freshly collected heparinized blood was diluted with an equal amount of RPMI 1640
medium and layered on Ficoll Hypaque 1077 and centrifuged at 2,500 rpm for 25 minutes at room
temperature. The upper layer was discarded and the fluffy layer at the interphase was harvested,
rinsed and re-suspended in RPMI 1640 at 5xl05 cells/ml to obtain human peripheral blood
mononuclear cells (PBMCs). Freshly collected PBMCs were seeded in 96-well plates (105
cells/well) and treated with the extracts of the present invention at different dilutions (in RPMI
1640), followed by 30 minutes incubation at room temparature on a rotary shaker (200 rpm). Next,
wells were treated with 50ul (4ug/ml) lipopolysaccharide and allowed to incubate for a further 30
minutes at room temparature. The volume per well was made up to 200 ul using RPMI+10%FCS
and the plates incubated overnight at 37°C in a CO2 incubator. Negative controls (no
lipopolysaccharide treatment) were run in parallel. The plates were centrifuged (3000 rpm, 10
minutes) to obtain clarified supernatants for TNF-a and IL-ip determinations using commercial
ELISA kits.
Methanolic extract efficiently suppressed the secretion of TNF-a and IL-ip with IC50 values
of 6.1±1.3 and 5.7±2.7 ug/ml, respectively. An MTT assay showed that at these concentrations,
methanolic extract has no discernible cytotoxicity in both cell lines tested (CC50=78.9 ug/ml in
HepG2; >200 ug/ml in LLCMK2). These data shows the anti-inflammatory activity of the
methanolic extract. .„„_.,
I P O DELHI 1 7 - 1 2 - 2 G 1 5 - 1 7 - 33
16
(j) Toxicology •
Groups of 5 adult Wistar rats were orally administered 4.ml 0.25% methyl cellulose
(vehicle)/kg or 4 ml vehicle containing 400 mg to 2000 mg of the extracts of the present invention
/kg, once daily for 7 days (in accordance with OECD guidelines- 407). During this period, food
intake, body weight, and clinical signs were monitored daily. At the end of the experiment, animals
were euthanized, followed by the determination of hematological (Hb, WBC count, RBC count,
platelet count and hematocrit) and biochemical (SGOT, .SGPT, total protein, serum albumin, total
cholesterol, urea, creatinine and random sugar) parameters. Necropsy was performed. Organ
weights were recorded and histopathology was done.
The results showed that animals treated with up to 2000 mg/kg body weight did not
manifest any significant changes in any of these parameters compared to vehicle treated controls.

WE CLAIM:
1. A pharmaceutical composition for use in the treatment of dengue virus infection in
mammals, comprising:
(a) an extract of Cissampelos pareira, and
(b) paracetamol
wherein (a) and (b) are administered together as a single pharmaceutical composition or are coadministered
simultaneously or sequentially.
2. A method of treating dengue virus infection comprising administering a pharmaceutical
composition comprising:
(a) an extract of Cissampelos pareira, and
(b) paracetamol
wherein (a) and (b) are administered together as a single pharmaceutical composition or are coadministered
simultaneously or sequentially.
3. An extract of Cissampelos pareira to reduce the viral load at an early stage in the treatment
of dengue virus infection.
4. An extract of Cissampelos pareira for use in the treatment of dengue virus infection in
mammals, wherein the extract exhibits a platelet protective effect.
5. An extract of Cissampelos pareira for use in the treatment of dengue virus infection in
mammals, wherein the extract exhibits an erythrocyte protective effect.
6. The extract of Cissampelos pareira of any of the preceding claims, wherein the extract is
selected from an alcoholic extract, a hydroalcoholic extract, or an aqueous extract.
7. The extract of Cissampelos pareira of claim 6, wherein the alcoholic extract is selected
from the group consisting of methanolic, ethanolic, n-propanolic, isopropanolic, nbutanolic,
iso-butanolic or /-butanolic extract.
8. An extract of Cissampelos pareira to reduce the viral load at an early stage in the treatment
of dengue virus infection, wherein the extract exhibits a platelet protective effect.
9. An extract of Cissampelos pareira to reduce the viral load at an early stage in the treatment
of dengue virus infection, wherein the extract exhibits an erythrocyte protective effect.
10. A pharmaceutical composition comprising the extract of claim 8 or claim 9 and one or more
pharmaceutically acceptable excipients.
. 11. The pharmaceutical composition of claim 10, wherein the pharmaceutically acceptable
excipients are selected from the group comprising diluents, binders, disintegrants,
• lubricants, glidants, polymers, flavoring agents, surfactants, preservatives, antioxidants,
buffers, and tonicity modifying agents.
I P O DELHI. 1 7 - 1 2 - 2 : 0 1 S 1 7 : 3 5 '
18
12. The extract of claim 8 or claim 9, wherein the extract is selected from an alcoholic extract, a
hydroalcoholic extract, or an aqueous extract.
13. The extract of Cissampelos pareira of claim 12, wherein the alcoholic extract is selected
from the group consisting of methanolic, ethanolic, n-propanolic, isopropanolic, nbutanolic,
iso-butanolic or /-butanolic extract.