Form 2
(39 of 1970)
&
The Patent Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. Title of the Invention
PHARMACEUTICAL COMPOSITIONS COMPRISING PICROSIDE
2. Applicant (s)
Name Nationality Address
SAVA HEALTHCARE
LIMITED Indian
SAVA House, Off New Airport Road,
Viman Nagar, Pune-411014,
Maharashtra, India
3. Preamble to the description
COMPLETE SPECIFICATION
The following specification describes the invention and the manner in which it is to
be performed.
5
10
2
RELATED APPLICATION
The present application claims the benefit of priority to International Patent
application No. PCT/IB2021/053392 filed on April 24, 2021 and the entire
specification.
5
FIELD OF THE INVENTION
The present invention relates to a composition comprising a purified compound
isolated from Picrorrhizakurroa. Specifically, the present invention relates to a
composition comprising purified Picroside isolated from Picrorrhizakurroa. More
10 specifically, the present invention relates to a composition comprising purified
Picroside I for preventing or treating immunopathic condition such as neutropenia
and the use thereof.
BACKGROUND OF THE INVENTION
15 Drug-induced neutropenia, an immunopathic condition, is a potentially serious and
life-threatening adverse event that may occur secondary to therapy with a variety of
agents. Mostly caused by cytotoxic chemotherapy, neutropenia is marked by a
predictable and dose-related decrease in neutrophil count. It has been associated with
a variety of chemotherapeutic and non-chemotherapeutic medications including, but
20 not limited to, cyclophosphamide, thionamides, clozapine, dapsone, methimazole,
penicillin, rituximab, procainamide, sulfasalazine, thiamazole, carbimazole,
amoxicillin, cotrimoxazole, ticlopidine, and valganciclovir. Neutropenia from
nonchemotherapy drugs is less common than neutropenia secondary to chemotherapy.
While the condition can occur at any time during the course of treatment, depending
25 on the primary therapy, it most commonly occurs within the first few weeks after
administration of drug. Affected patients typically experience severe neutropenia for
several weeks to several months after first exposure to a drug. Drug-induced
neutropenia is characterized by decreased production or increased destruction of
3
neutrophils. The clinical consequence of neutropenia is susceptibility to infection
proportional to the degree of neutropenia and can even lead to mortality.
Granulocyte Colony Stimulating Factor (G-CSF) and Granulocyte-macrophage
5 colony-stimulating factor (GM-CSF) are approved by the United States’ Food and
Drug Administration (FDA) for use treat severe neutropenia with fever. G-CSFs and
GM-CSFs have demonstrated proven efficacy in their ability to reduce the incidence,
magnitude, and duration of neutropenia following chemotherapy administration; and
therefore, are largely and widely accepted treatment options for drug-induced
10 neutropenia. However, neither drugs are not without adverse effects. Some common
symptoms observed after administration of G-CSF are dyspnea, chest pain, nausea,
hypoxemia, diaphoresis, anaphylaxis, syncope and flushing. In comparison, the
adverse effects of GM-CSFs are moderate such as fever, myalgia, malaise, rash and
injection site reaction; but severe effects such as pericarditis and thrombosis at high
15 doses are also known to happen. This raises the need for improved therapeutical
options, preferably derived from natural resources with enhanced safety and efficacy.
In recent decades, spectrum of disease has shifted to complex chronic diseases
requiring aggressive therapies such as chemotherapy. Synthetic drugs generally have
20 more associated adverse effects and toxicities which add on to patients’ burden.
World Health Organization Drugs Strategy 2002–2005 encouraged acceptance of
herbal medicine as complimentary or alternate medicine across the globe. As per
WHO data approximately 70% of the world’s population has been using herbal
medicine as a complementary medicine over the past 2 decades. Herbal medicines are
25 often administered simultaneously with therapeutic drugs for the treatment of major
ailments owing to the general perception that they are free of undesirable side effects.
Picrorhizakurroa is a well-known medicinal herb, traditionally popular in Indian
ayurvedic system for the treatment of liver and respiratory disorders. Picroside I and
4
Picroside II are two of the most important constituents found in the roots and
rhizomes of the plant. Picroside I and Picroside II have been used in herbal
formulations individually and in combination. Pharmacological benefits of PicrosideI
and Picroside II have been widely reported in literature as an antioxidant, anti5 inflammatory, anti-allergic, hepatoprotective, choleretic, anti-asthmatic and anticancerous activity. However, there has not been reported a composition comprising
Picroside I, or Picroside II, or a combination thereof for treating neutropenia.
Accordingly, the present inventors report a novel pharmacological benefit of
10 Picroside in the treatment of neutropenia. The disclosed invention is focused on
developing a composition comprising active ingredient derived from natural
resources for treating neutropenia. According to the present invention, there is
therefore provided a composition comprised of Picroside for treating neutropenia.
More particularly, there is provided a composition comprising PicrosideI for the
15 treatment of neutropenia. Further disclosed is a novel industrialized method for
isolating Picroside I and Picroside II. The present invention also discloses a method
for making the said novel pharmaceutical composition comprising Picroside. The
present invention also provides a method of treating or preventing neutropenia in a
subject comprising administering to said subject a therapeutically effective amount of
20 the novel composition containing active ingredient such as Picroside I.
SUMMARY OF THE INVENTION
In view of the foregoing, for overcoming the disadvantages of the prior art, the object
of the invention is to provide a pharmaceutical composition comprising a
25 therapeutically effective amount of Picroside I for the treatment or prevention of
neutropenia.
To achieve the above, the present invention includes a composition comprising
purified Picroside I and pharmaceutically acceptable excipients for treating or
5
preventing neutropenia in a subject in need thereof. Further, the said composition
shall be in oral or injectable form.
According to one embodiment,is provided a neutropenia treating pharmaceutical
5 composition comprising a therapeutically effective amount of purified Picroside I and
acceptable pharmaceutical carriers or excipients.
According to one embodiment, the pharmaceutical composition comprises 100 to 400
mg of purified Picroside I and acceptable pharmaceutical carriers or excipients.
10
According to a preferred embodiment, theamount of purified Picroside I in thesaid
pharmaceutical composition is 150 to 400 mg.
According to one embodiment, the pharmaceutical composition is an oral or
15 injectable composition.
According to a preferred embodiment, the oral pharmaceutical composition is in form
of a tablet.
20 According to one embodiment, the pharmaceutical composition further comprising a
filler, a solvent, and a lubricant. According to another embodiment, the filler is F melt
Type C, the solvent is ethanol, the lubricant is magnesium stearate.According to a
preferred embodiment the amount of filler is about 98 percent by weight, the amount
of lubricant is about 1 percent by weight.
25
According to one important embodiment, is provided a neutropenia treating
pharmaceutical composition comprising 390 mg of purified Picroside I, a filler, a
solvent, a lubricant; wherein the said composition is ready for oral administration.
6
According to an alternate embodiment, the injectable pharmaceutical composition is
in form of an injection.
According to one embodiment, the pharmaceutical composition further comprising a
5 solvent. According to a preferred embodiment, the solvent is 2.5% PEG400.
According to one important embodiment, is provided a neutropenia treating
pharmaceutical composition comprising 195 mg of purified Picroside I, and a solvent;
wherein the said composition is sterile and ready for subcutaneous having pH in the
10 range of 4.0 to 7.0.
According to one embodiment, the injectable pharmaceutical composition is stored in
a pre -filled sterile syringe or vial or cartridge.
15 According to one important embodiment, is provided a method of treating
neutropenia in a subject comprising administering to a subject in need thereof a
therapeutically effective amount of Picroside I comprised in the said composition.
According to one embodiment, the composition is administered to the subject once a
20 day. According to another embodiment, the composition is administered to the
subject for up to 5 consecutive days.
DETAILED DESCRIPTION OF THE INVENTION
To clarify the above and other purposes, features, and advantages of this invention,
25 specific embodiment of this invention is especially listed and described in detail with
the examples as follows. The principal and mode of operation of this invention have
been described and illustrated in its embodiment. At the outset, a person skilled in the
art will appreciate that this invention may be practiced otherwise than is specifically
described and illustrated. The invention should not be limited by the above-described
7
embodiment, method, and examples, but by all embodiments and methods within the
scope and spirit of the invention. Also, in the following description of the invention,
certain terminology may be used for the purpose of reference only, and is not
intended to be limiting.
5
Where a range of values is provided, it is understood that each intervening value, to
the tenth of the unit of the lower limit unless the context clearly dictates otherwise,
between the upper and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value and/or intervening value in a stated range and
10 any other stated or intervening value in that stated range is encompassed within the
invention. The upper and lower limits of these smaller ranges may independently be
included or excluded in the range, and each range where either, neither or both limits
are included in the smaller ranges is also encompassed within the invention, subject to
any specifically excluded limit in the stated range. Where the stated range includes
15 one or both of the limits, ranges excluding either or both of those included limits are
also included in the invention.
The present invention provides a pharmaceutical composition comprising a Picroside
compound, derived from P.kurroa for the treatment or prevention of immunopathic
20 conditions such as neutropenia. More particularly, the present invention provides a
pharmaceutical composition comprising Picroside I for the treatment or prevention of
neutropenia. Further, the present invention provides a novel industrialized method for
isolating Picroside I and Picroside IIfromP. kurroa. The present invention also
provides a method of treating or preventing neutropenia in a subject comprising
25 administering to said subject a therapeutically effective amount of the novel
composition comprising active ingredient Picroside I, together with pharmaceutically
acceptable carrier or additives thereof.
8
Accordingly, it is an object of the present invention to provide a pharmaceutical
composition comprising Picroside I for treating or preventing neutropenia in a
subjectsuffering from neutropenia by administering to said subject a therapeutically
effective amount of the said composition comprising active ingredient Picroside I,
5 together with a pharmaceutically acceptable carrier or additive thereof.
The term "Picroside I" disclosed herein comprises Picroside I isolated from
P.kurroa.The term "Picrorhizakurroa" or "P.kurroa" disclosed herein comprises the
cultivated or naturally grown plant and commercially available plant, but not intended
10 to limit thereto herein.PicrorhizakurroaRoyle ex Benth., commonly known as 'Kutki
or Kutaki', is a perennial herb from the Scrophulariacae family, present in wild form
in the north-western Himalayan regions and Garhwal regions of India. P. kurroais
listed as an official drug since 1970 in The Pharmacopoeia of India, 1970.P. kurroa is
commercially available in India was thus procured.Accordingly, in the present
15 invention, the plant raw material and extract were commercially procured. The roots
of Picrorhizakurroai were procured from Noornihal, Anekal, Bangalore; batch
number RM/PK/SEP-19-03. Water extract of Picrorhizakurroaiwereprocured from
Noornihal, Anekal, Bangalore; batch number RDP/PK/011.
20 It should be noted that herbs and plants can be processed and can be taken in different
ways and forms, and this may include whole injectables, capsules, and tablets that
contain a ground or powdered form of a raw herb or its dried extract. Accordingly, a
part of the plant may be used as a raw material, or a liquid and/or solid extract
thereof. Suitable procedures include multiple steps, for example maceration,
25 percolation, and extraction using supercritical fluids, liquefied gases or suitable
solvents are used for the preparation of the material. Picroside I and Picroside II are
derived from roots and rhizomes of P.kurroa. The constituents need to be extracted
from the plant material, further purified and isolated in order to make it fit for the
designated use in a composition for treating or preventing neutropenia. An aspect of
9
the present invention is a method for extracting, further purifying, and isolating
Picroside I and Picroside II from P. kurroato obtain purified Picroside I and Picroside
II.
5 Accordingly, in an embodiment of the present invention, pharmaceutical composition
of the present invention comprises of purified Picroside I. And accordingly, there is
provided a method for extracting, further purifying, and isolating Picroside I and
Picroside II from P. kurroato obtain purified Picroside I and Picroside II. Disclosed
herein is the method for extracting Picroside I and Picroside II from P.
10 kurroa comprising the steps of: making a coarse powder of P. kurroaroots and
rhizomes and then using an extracting solvent selected from water, demineralized
water, methanol, ethanol in different ratios. etc. or the mixtures thereof at a predetermined pH; performing at least one more extraction cycle using the said solvent
at a pre-determined temperature; collecting and filtering the liquid extract;
15 evaporation of liquid extract under vacuum at pre-determined temperature to obtain a
dry powder extract. Disclosed herein is also the method for further purifying
Picroside I and Picroside II from P. kurroa dry powder extract comprising the steps
of: dissolving the dry powder extract in water, loading on using ion exchange column
chromatography methods using resins; collection of the first fraction in water;
20 collection of at least two more subsequent fractions in a methanol; washing the
column with methanol; and drying the concentrate comprising of Picroside I and
Picroside II. The method of isolating Picroside I and Picroside II from P. kurroa
comprises the steps of: using silica column chromatography; dissolving the said
extracted and purified extract in methanol; collecting at least one fraction comprising
25 Picroside I or Picroside II in methanol and chloroform; using thin-layer
chromatography for eluting Picroside I and Picroside II.
Specifically, disclosed herein is the method for extracting Picroside I and Picroside
II from P. kurroa comprising the steps of: extracting coarse powder of P.kurroaroots
10
and rhizomes using demineralized water as the extracting solvent selected from
water,wherein the pH ranges from 6.5-7.5; performing at least one more extraction
cycle using the said solvent at temperature ranging between 50 -90ºC; collecting and
filtering the liquid extract; evaporation of liquid extract under vacuum at temperature
5 ranging between 40 -55ºC to obtain a dry powder extract characterized by about
28.75 % extract yield. The method for further purifying Picroside I and Picroside II
from P. kurroa comprises the steps of: using reverse phase chromatography methods
including using resins selected from Diaion-HP-20, Amberlite-XAD-4,8,7,16 etc.
Preferably, resins that are suitable for preliminary purification of polar compounds
10 like phenolics.etc. are selected. The loaded column is repeatedly washed with water
till the colourless eluates are obtained. Further, the column is eluted with 30%
methanol in water and fractions collected. This step is repeated till colourless eluates
are obtained. Subsequently, the column is washed with 100% methanol and fractions
of about 100 ml are collected. The fractions are checked by TLC for markers of
15 interest under UV and selected fractions are dried to obtain the concentrate
comprising of about 4 to 6% Picroside I and about 5 to 8% Picroside II.
The dried fraction is resuspended in methanol, adsorbed on silica where 2 volumes of
silica is added to the methanol Picroside mixture, and dried by rotavapor. The dried
20 material is then loaded onto a silica column that is packed with chloroform. The
column is washed with chloroform, 2% methanol in chloroform followed by 3%
methanol in chloroform and finally 4% methanol in chloroform. This method
provides pure Picroside I and Picroside II in 4% methanol in chloroform fraction.
Fractions containing minimal contaminants and maximum Picroside are chosen and
25 dried and weighed. The final weight is then calculated for determining total yield.
The purity is then checked by HPLC. By this process, the Picroside I elutes first
followed by Picroside II, characterized by containing about 96.42% and about
89.02% purity respectively.
11
The present inventors report that the above-described process of the present invention
has a shorter time of 3 hours for purification of Picroside I as compared to the process
published in the prior art (Hussain A et al., Pharmacognosy Research, 2013; 5:30-36)
requiring 6 hours; while still resulting in a marginally higher yield. Additionally, the
5 process of the present invention utilizes a single solvent i.e.water, a safer medium, as
compared to the use of two environmental non-friendly solvents such as petroleum
ether, ethanol etc. as reported by the prior art (Hussain et al., 2013)
The present inventors report that the said purified Picroside I and Picroside II
10 compounds exhibit the ability to treat and prevent immunopathic conditions namely
neutropenia through various in vivo tests in mice. To the best of knowledge, this antineutropenia activity of Picroside I or Picroside II has not been reported before. It was
further observed that Picroside I exhibit marginally more desirable activity for
treating or preventing neutropenia as compared to Picroside II. Additionally, mice
15 treated with Picroside I exhibited higher amount of WBC as compared to those
treated with Picroside II. Accordingly, Picroside I was selected as the candidate for
composition for treating neutropenia owing to its overall effect on neutrophil and
WBC amounts. Central to this invention is the object to provide a pharmaceutical
composition of Picroside I to treat or prevent neutropenia. Accordingly, in accordance
20 with the other aspect of the present invention, present invention provides a
pharmaceutical composition comprising the purified Picroside I prepared by the
above-described methods to treat or prevent neutropenia. A pharmaceutical
composition of Picroside II is also disclosed herein.
25 The term "treat" disclosed herein comprises any act to alleviate or favorably changing
the symptom associated with certain disease or disorder disclosed herein by way of
administrating the said composition; and the term "prevent" disclosed herein
comprises any act to inhibit or postpone the occurrence of certain disease or disorder
disclosed herein by way of administrating the said composition.
12
An embodiment of the present invention provides a pharmaceutical composition
comprising purified Picroside I prepared by the above-described methods and
pharmaceutically acceptable carriers or excipients, for the treatment or prevention of
neutropenia.The term "pharmaceutically acceptable carriers or excipients" defined
5 herein comprises pharmaceutical additives, inactive ingredients, dyes, flavors,
binders, emollients, fillers, lubricants, preservatives, and many more classifications
used to prepare medications and which are well-known in the art or previous
literature.
10 In an embodiment of the present invention, the pharmaceutical composition
comprising purified Picroside I may be prepared as an oral dosage form for example,
powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder,
sachet, granules. In a preferred embodiment, the pharmaceutical composition of the
present invention in an oral dosage shall be a tablet. In another embodiment of the
15 present invention, the pharmaceutical composition shall further comprise of
pharmaceutically acceptable excipients such as fillers, solvent, and lubricant. In a
preferred embodiment, the filler shall be F melt Type C, the solvent shall be ethanol,
and the lubricant shall be magnesium stearate. It should be noted that the amounts of
excipients shall be in proportion to the amount of active ingredient i.e., Picroside I.
20 Accordingly, the amount of filler shall be about 98 % by weight, the amount of
lubricant shall be about 1% by weight.
Thus, according to an embodiment of the present invention is provided a neutropenia
treating pharmaceutical composition comprising 390 mg of purified Picroside I, a
25 filler, a solvent, a lubricant; wherein the said composition is ready for oral
administration.
The composition according to the present invention can also be provided as a
pharmaceutical composition comprising pharmaceutically acceptable carriers,
13
adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol,
erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate,
calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water,
methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and
5 mineral oil. The composition may additionally include anti-agglutinating agents,
wetting agents, flavoring agents, emulsifiers, preservatives, etc. The compositions of
the invention may be formulated so as to provide quick, sustained or delayed release
of the active ingredient after their administration to a patient by employing any of the
procedures well known in the art.
10
In an alternate embodiment, the pharmaceutical composition comprising purified
Picroside I may be preparedas an injectable preparation for example, solution,
suspension, or emulsion. In a preferred embodiment, the pharmaceutical composition
of the present invention shall be an injection. In another embodiment of the present
15 invention, the pharmaceutical composition shall further comprise of pharmaceutically
acceptable solvent selected from methanol, 2.5% PEG400, 5% PEG400, 5% DMSO,
5% ethyl lactate, and 1.4% propylene glycol. In a preferred embodiment, the said
composition shall comprise of 2.5% PEG400 wherein the pH of the composition shall
be in the range of 4.0 to 7.0. It should be noted that the injectable compositions of the
20 present invention can also be dissolved in oils, or other solvents that are commonly
used to produce an injection. Suitable examples of the carriers include physiological
saline, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them.In
another embodiment of the present invention, the injectable composition shall be
ensured for sterility and ready for use; wherein the composition can be stored in a
25 pre-filled syringe or vial or cartridge or any other suitable mode or medium.
Thus, according to an embodiment of the present invention is provided a neutropenia
treating pharmaceutical composition comprising 195 mg of purified Picroside I, and a
14
solvent; wherein the said composition is sterile and ready for subcutaneous having pH
in the range of 4.0 to 7.0.
Hereinafter, the formulating methods and kinds of excipients will be described, but
5 the present invention is not limited to them. The representative preparation examples
were described as follows.
In accordance with another aspect of the present invention, there is also provided a
method of treating or preventing neutropenia in subject, wherein the method
10 comprises administering a composition comprising therapeutically effective amount
of the purified Picroside I prepared by the above-described methods into the subject
suffering from neutropenia.
Accordingly, in an embodiment of the present invention, there is provided a method
15 of treating or preventing neutropenia in a subject, wherein the method comprises
administering a composition comprising therapeutically effective amount of the
purified Picroside I prepared by the above-described methods into the subject
suffering from neutropenia.
20 In a preferred embodiment is provided a method of treating neutropenia in a subject
comprising administering to a subject in need thereof a therapeutically effective
amount of Picroside I as an oral composition or as an injectable composition. In a
preferred embodiment, the said composition is administered to the subject once a day.
In another preferred embodiment, the said composition is administered to the subject
25 for up to 5 consecutive days.
In accordance with another aspect of the present invention, there is also provided a
use of a composition comprising purified Picroside I prepared by the above-described
15
methods and pharmaceutically acceptable carriers or excipients, for manufacture of
medicines employed for treating or preventing neutropenia.
In accordance with another aspect of the present invention, the pharmaceutical
5 composition of present invention can be administered to a subjectvia various routes.
In accordance with an embodiment of the present invention. The term "subject"
defined herein comprisesanimal subjects such asrat, mouse, domestic animals or
human. Several modes of administration are contemplated, for example,
administration can be made orally, or by intravenous, intramuscular, subcutaneous, or
10 intracutaneous injection. It will be apparent to those skilled in the art that various
modifications and variations can be made in the compositions, use and preparations
of the present invention without departing from the spirit or scope of the invention.
In accordance with an aspect of the invention, the desirable dose of Picroside I may
15 vary depending on the condition and the weight of the subject, severity, drug form,
route and period of administration, and may be chosen by those skilled in the art. It
should be noted that extrapolation of dose from animals to humans using allometric
scaling has been done to assess the initial dose in humans taking into consideration
the difference in pharmacokinetics and pharmacodynamicsamong species. For
20 example, a 1 mg and 2 mg dose for administration in mice is extrapolated as 195 mg
and 390 mg dose for administration in humans respectively.
Accordingly, an embodiment of the invention provides that in order to obtain
desirable effects, it is generally recommended to administer, in humans, at the amount
25 ranging from 100 to 200 mg/day, preferably 150 to 200 mg/day, more preferably 190
to 200 mg/day of the purified Picroside I compound of the present invention. The
dose may be administered in single or divided into several times per day. Such
administration shall be done for 5 consecutive days.
16
Since neutropenia is directly linked to neutrophil count, effect of the composition on
the condition is reported specifically via neutrophil and also via WBC count.
Accordingly, in order to determine the effect of Picroside I on neutropenia, in vivo
tests were performed on mice. Neutropenia was induced in mice by administering
5 cyclophosphamide to create an animal test model. The neutropenia induced mice
were administered with composition comprising Picroside I and observed for changes
in body weight, blood analysis to monitor neutrophil counts, and clinical symptoms
such as toxicity and mortality. The study model was replicated in neutropenia induced
mice by administering composition comprising Picroside II. The present inventors
10 report that both Picroside I and Picroside II significantly reversed cyclophosphamide
induced neutropenia in mice. Further, the efficacy and activity of Picroside I for
treatment of neutropenia was compared with that of G-CSF which is the FDA
approved drug for treating the condition. The present inventors report that the results
of Picroside I treatment in subject mice were comparable to that of G-CSF treatment.
15
As described in the present invention, inventive composition of purified Picroside I
derived from P. kurroa showed potent anti-neutropenic activity through in vivo tests
using by swiss albino mice with drug-induced neutropenia, for example, a test on the
proliferation and activity of neutrophils caused by neutropenia occurrence; Therefore,
20 it can be used as the therapeutics for treating and preventing neutropenia.
INCORPORATION BY REFERENCE
The publications mentioned in this specification are herein incorporated by reference
to the same extent as if each individual publication was specifically and individually
25 indicated to be incorporated by reference.
17
EXAMPLES
The following examples are provided for illustrative purposes only, and are intended
to be purely exemplary of the disclosure and are not intended to limit the scope of the
5 claims provided herein.
Example 1. Preparation of the crude extract of P. kurroa
400 gm coarse powder of Picrorhizakurroarhizomes was extracted with
demineralized water at pH 6.5-7.5. About 2000 ml water was added for each cycle.A
total three cycles were performed at 800
10 C.Liquid extract was collected and filtered
through a PP-filter clothto remove the debris. The subsequent filtered liquid extract
was evaporated under vacuum at 50-55ºC by using rotary evaporator to obtain a dry
powder of extract. Component analysis was performed using HPLC. HPLC result
confirmed that the crude extract of Picrorhizakurroa contained about 4 to 6 %
15 Picroside I and about 5 to 8% Picroside II.
Example 2. Preparation of the purified extract Picroside I and Picroside II of P.
kurroa
The crude extract of P.kurroawasprepared by the extraction method according to
20 Example 1. 100 ml of HP20 resin was loaded on column and equilibrate with
water.13 gm of water extract of P.kurroa was completely dissolved in 100 ml water
and adsorbed on resin.First fraction was collected with 300 ml water.Second fraction
was collected with 30% Methanol in 300 ml water.Third fraction was collectedwith
60% Methanol in 400 ml water. The column was washed with Methanol. It was
25 observed that the third fraction and methanol washing contained both Picroside-I and
Picroside II. Both fractions were mixed together and dried to obtain a concentrate.
Component analysis was performed using HPLC. HPLC result confirmed that the
purified extract of P.kurroa contained about 14.41 % Picroside I and about 16.60%
30 Picroside II.
18
Example 3. Isolation of Picroside I and Picroside II of P. kurroa
The purified extract of P. kurroa prepared by the extraction method according to
Example 2. Isolation was performed using normal silica column chromatography. 90
5 gm of silica (100-200#) was packed with chloroform into a glass column. 6 gm of
purified extract was dissolved in sufficient amount of methanol. Further, 12 gm of
silica (60-120#) was added for adsorption. This mixture was dried and loaded on
silica bed. Multiple Fractions collection were started with 5% methanol in
chloroform.Fractions were pooled together according to TLC pattern.
10
Component analysis was performed using HPLC. HPLC result confirmed that the
Picroside I of purity of about 96.42% and Picroside II of purity of about 89.02% were
obtained.
15 Example 4. Preparation of Picroside I tablet
Preparation of tablet
The purifiedPicroside I prepared by the methods according to Example 1, 2, and 3
was used for the said oral composition. Tablet was prepared by dissolving Picroside I
in sufficient quantity of ethanol (5 mL for 50 tablets batch size). F-melt Type C was
20 previously sifted through #40 sieve, and granulated using the previous step.
Granulated wet mass, thus obtained,wasdried at 60°C. Dried granules were then
sifted using ASTM #40 and lubricated with ASTM #60. Magnesium Stearate was
passed manually. Tablets were compressed using below mentioned parameters:
Punch Dimension: 5.5 mm standard Concave; Target weight: 70 mg; Hardness: 30-50
25 N; D.T: 1-2 Min; Thickness: 2.6±0.2 mm; Friability: NMT 1%
Table 1: Composition of Picroside I Tablet (1 mg)
Ingredients mg/tablet w/w%
Picroside I 1.0 1.43
F melt Type C 68.50 97.86
Ethanol qs
Magnesium stearate 0.5 0.71
19
The above method including process parameters and ingredients ratio were replicated
to prepare tablet formulation of Picroside II. 1 mg Picroside II was added to the
composition instead of 1 mg Picroside I.
5 Example 5. Preparation and stability test of Picroside I injectable formulation
Preparation of injection
The purifiedPicroside I prepared by the methods according to Example 1, 2, and 3
was used for the said injection composition. Injection preparation was prepared by
dissolving 1 mg Picroside I in solvent, controlling pH to about 4-7and then filling in
10 ampules followed by subsequent sterilizing by conventional injection preparation
method. For comparative compositions, different solvents such as methanol, 2.5%
PEG400, 5% PEG400, 5% DMSO, 5% ethyl lactate, and 1.4% propylene glycol were
used for Picroside I solubility.
15 The solutions were kept for sonication for 15 min and left the solutions stand at room
temperature for 1 hour. HPLC was used for quantitative analysis. Composition
prepared with 2.5% PEG400 were found to be the most suitable and were further
analyzed for stability.
20 The above method including process parameters and ingredients ratio were replicated
to prepare injectable formulation of Picroside II. 1 mg Picroside II was added to the
composition instead of 1 mg Picroside I.
Stability Data
25 Three compositions of 1 mg Picroside I and 2.5% PEG400 were kept for sonication
for 15 min and left to stand at room temperature (RT), 2-8°C and 40°C, 75%RH for 1
month. and injected (20µL) into HPLC at 220nm for quantitative analysis. All
compositions exhibited excellent stability over a period of 1 month.
20
Table 2: Stability analysis of injectable composition of Picroside I and 2.5% PEG400;
analyzed by HPLC quantitative analysis.
Temperature conditions % Result (1 month)
2-8°C 96.59
RT 102.33
40°C, 75%RH 86.89
Example 6. Animal model test (mice)
5 In order to determine the anti-neutropenia effect of Picroside I test was performed by
using neutropenia induced mice.
Preparation of Experiment animal
In-house bred Swiss albino mice were housed under standard laboratory conditions,
10 such as, air-conditioned with adequate fresh air supply (air changes 12-15 per hour),
room temperature of 21.1-23.2°C and relative humidity of 55-69 %, with 12 hours
light and 12 hours dark cycle were strictly maintained in the experimental room. The
mice were acclimatized for a minimum period of seven days to laboratory conditions.
15 Cyclophosphamide administration to induce neutropenia
The selected Swiss albino mice were randomized into four groups based on body
weight, each group comprising of 6 mice. Group 2 and 3, 4 animals were treated with
150 mg/kg of cyclophosphamide through intraperitoneal route on Day 0 to initiate
inducing neutropenia. On Day 3, the animals of group 2, 3 and 4, reflected
20 thecyclophosphamide inducedneutropenication when compared with Group 1
(Control) mice.On day 4, mice from Groups 2, 3 and 4 were again injected with a
single dose of 100 mg/kg of cyclophosphamide.Total and differential count
(neutrophils, lymphocytes, and monocytes) was recorded on day 3.The neutrophil
count on day 3
21
was compared between groups using One-way ANOVA followed by Dunnett’s test or
student ‘t’ test. P<0.05 was considered statistically significant.
Table 3: mean %-WBC and %-Neutrophil count on Day 3 of administering
cyclophosphamide
Groups % WBC % Neutrophil
Group 1 6.52 25.83
Group 2 1.78 19.33
Group 3 1.53 20.83
Group 4 2.00 21.83
5
Example 7. Administration of Picroside I (injectable) to mice with
cyclophosphamide induced neutropenia
Cyclophosphamide related neutropenia was induced in mice subjects using method
according to Example 6. Injectable composition was prepared using the method
10 according to Example 5. The Picroside I composition was administered through
subcutaneous route at a dose of 1 mg/mouse/day to the animals in Group 3 for 5 days
respectively starting from day 5 ending at day 10.Picroside II composition was
administered through subcutaneous route at a dose of 1 mg/mouse/day to the animals
in Group 4 for 5 days respectively starting from day 5 ending at day 10. On day 10,
15 again blood was collected to count the total leucocyte count and differential count
(neutrophils, lymphocytes, and monocytes) manually.The neutrophil count on day 10
was compared between groups using One-way ANOVA followed by Dunnett’s test or
student ‘t’ test. P<0.05 was considered statistically significant. Additionally, the mice
were observed for changes in body weight and any clinical symptoms.
20 There were no treatment related changes in body weight in any of the subject mice
treated with Picroside I or Picroside II. There were also no clinical signs of toxicity
and mortality observed in any of the subject mice treated with Picroside I or Picroside
II.Day 10 results revealed that cyclophosphamide showed neutropenia and decreased
total lymphocyte count in Group 2 (marked Vehicle control) as compared with Group 1
25 (marked Normal control). Treatment to subjects of Group 3 and Group 4 with Picroside
22
I and Picroside IIrespectivelyshowed significant increase in both Total leukocyte count
and neutrophil count on Day 10 as compared to Group 2, indicating total reversal of
neutropenia. Moreover, Picroside I and Picroside II increased neutrophil count
significantly (P<0.01) more than that of Group 1 (Normal control) mice.
5 Based on the above results it is evident that injection of Picroside I and Picroside II
significantly reverses cyclophosphamide induced neutropenia in mice.
Table 4: mean %-Neutrophil and WBC count on Day 10 of Picroside I and Picroside
II
Groups % Neutrophil
(day 3)
% Neutrophil
(day 10)
% WBC
(day 3)
% WBC
(day10)
Group 1 25.83 26.17 6.52 6.37
Group 2 19.33 20.17 1.78 3.08
Group 3
(Picroside I)
20.83 32.67 1.53 9.23
Group 4
(Picroside II)
21.83 32.83 2.00 6.98
10
Example 8. Administration of Picroside I (oral) to mice with cyclophosphamide
induced neutropenia
Cyclophosphamide related neutropenia was induced in mice subjects using method
according to Example 6. Oral tablet composition was prepared using the method
15 according to Example 4. The Picroside I composition was administered orally at a
dose of 2 mg/mouse/day (2 tablets) to the animals in Group 1 and Group 2 for 5 days
respectively starting from day 5 ending at day 10. For a comparative study, oral
composition was administered using distilled water and PEG.Specifically, oral tablets
were administered in distilled water to mice in Group 1 and in 5% PEG to mice in
20 Group 2. On day 10, again blood was collected to count the total leucocyte count and
differential count (neutrophils, lymphocytes, and monocytes) manually.
The neutrophil count on day 10 was compared between groups using One-way
ANOVA followed by Dunnett’s test or student ‘t’ test. P<0.05 was considered
23
statistically significant. Additionally, the mice were observed for changes in body
weight and any clinical symptoms.
There were no treatment related changes in body weight in any of the subject mice
treated with Picroside I. There were also no clinical signs of toxicity and mortality
5 observed in any of the subject mice treated with Picroside I.
Day 10 results revealed that treatment to subjects of Group 1with Picroside Iin
distilled water and of Group 2 with Picroside I in 5% PEG showed significant
increase in both Total leukocyte count and neutrophil count on Day 10 as compared
that on Day 3.
10
Based on the above results it is evident that Picroside Ioral tablets significantly
reverses cyclophosphamide induced neutropenia in mice.
Table 5: meanNeutrophil and WBC count on Day 10 of Picroside I
Groups Neutrophil(109
/L)
(day 3)
Neutrophil
(109
/L)
(day 10)
WBC(109
/L)
(day 3)
WBC(109
/L)
(day10)
Group 1 0.38 1.58 1.75 9.5
Group 2
0.31 1.03 2.01 8.9
15
Example 9. Comparative administration of Picroside I and G-CSF to mice with
cyclophosphamide induced neutropenia
Cyclophosphamide related neutropenia was induced in mice subjects using method
according to Example 6.Group 1 was marked vehicle control group, Group 2 was
20 marked cyclophosphamide control, Group 3 was marked treated with Picroside I and
Group 4 was marked treated with G-CSF treated. The Picroside I
compositionwasadministered through subcutaneous route at a dose of 1
mg/mouse/day to the animals in Group 3 for 5 days respectively starting from day 5
ending at day 10.The G-CSF composition was administered through subcutaneous
24
route at a dose of 2 µg/mouse/day to the animals in Group 4 for 5 days respectively
starting from day 5 ending at day 10.On day 10, again blood was collected to count
the total leucocyte count and differential count (neutrophils, lymphocytes, and
monocytes) manually. The neutrophil count on day 10 was compared between groups
5 using One-way ANOVA followed by Dunnett’s test or student ‘t’ test. P<0.05 was
considered statistically significant. Additionally, hepatotoxicity was observed by
investigating ALT and AST levels.
Results showed treatment with Picroside I caused alleviation in cyclophosphamide
10 induceddecrease in % neutrophil. The observed changes in Picroside I treatment
group were comparable to that of G-CSF treatment. Administration of Picroside I to
the subject mice caused a significant reversal (p<0.001) of AST and ALT levels when
compared to the Group 2. This observed decrease in AST and ALT was comparable
to that of AST and ALT levels attained upon treatment with G-CSF.
15
Table 6: mean %-Neutrophil count on Days 0, 3, and 10 after administration of
Picroside I and G-CSF
Groups % Neutrophil (day 0) % Neutrophil (day 3) % Neutrophil (day 10)
Group 1
(control)
47.30 48.20 47.10
Group 2
(CPH
control)
46.20 6.800
8.500
Group 3
(Picroside I)
46.00 6.600 23.90
Group 4
(G-CSF)
46.10 6.700 28.20
Table 7: Effect of Picroside I in cyclophosphamide induced hepatotoxicity
Groups AST (IU/L) ALT (IU/L)
Group 1 25.82 22.32
25
(control)
Group 2
(CPH
control)
136.70 119.30
Group 3
(Picroside I)
50.32 37.16
Group 4
(G-CSF)
50.54 36.17
26
WE CLAIM:
1. A neutropenia treating pharmaceutical composition comprising a therapeutically
effective amount of purified Picroside I and acceptable pharmaceutical carriers or
excipients.
5 2. The pharmaceutical composition of claim 1 comprising 100 to 400 mg of purified
Picroside I and acceptable pharmaceutical carriers or excipients.
3. The pharmaceutical composition of claim 2, wherein the amount of purified
Picroside I is 150 to 400 mg.
4. The pharmaceutical composition of claim 1, wherein the said composition is an
10 oral or injectable composition.
5. The pharmaceutical composition of claim 4 wherein the said oral composition is
in form of a tablet.
6. A neutropenia treating pharmaceutical composition comprising 390 mg of
purified Picroside I, a filler, a solvent, a lubricant; wherein the said composition is
15 ready for oral administration.
7. The pharmaceutical composition of claim 6, wherein the filler is F melt Type C,
the solvent is ethanol, the lubricant is magnesium stearate.
8. The pharmaceutical composition of claim 6, wherein the amount of filler is about
98 percent by weight, the amount of lubricant is about 1 percent by weight.
20 9. The pharmaceutical composition of claim 4, wherein the said injectable
composition is in form of an injection.
10. A neutropenia treating pharmaceutical composition comprising 195 mg of
purified Picroside I, and a solvent; wherein the said composition is sterile and
ready for subcutaneous having pH in the range of 4.0 to 7.0.
25 11. The pharmaceutical composition of claim 10, wherein the solvent is 2.5%
PEG400.
12. The pharmaceutical composition of claim 10, wherein the composition is stored in
a pre -filled sterile syringe or vial or cartridge.