Pharmaceutical compositions containing picroside

Abstract

The present invention discloses a pharmaceutical composition comprising picroside isolated from Picrorhiza kurroa. The composition contains picroside I as an active ingredient for treating drug-induced immune disorders such as neutropenia. Methods for isolating picroside I and picroside II and preparing the composition are also disclosed herein. The composition of picroside I can be a solid oral or injectable composition with a predetermined dose. The composition of picroside I has shown recovery of drug-induced immune disorders such as neutropenia through in vivo and in vitro tests. Therefore, it can be used as a herbal therapeutic agent for treating drug-induced immune disorders such as neutropenia.

Description

[Technical Field]

[0001] Technical Field The present invention relates to compositions containing purified compounds isolated from Picrorhiza kurroa. Specifically, the present invention relates to compositions containing purified picroside I isolated from Picrorhiza kurroa. More specifically, the present invention relates to compositions containing purified picroside I and uses thereof for preventing or treating immunopathic conditions such as neutropenia. [Background technology]

[0002] background Drug-induced neutropenia, an immune dysregulation, is a potentially serious and life-threatening adverse event that can occur secondary to treatment with a variety of drugs. Neutropenia, most often caused by cytotoxic chemotherapy, is characterized by a predictable, dose-dependent decrease in neutrophil counts. It is associated with a variety of chemotherapeutic and non-chemotherapeutic drugs, including, but not limited to, cyclophosphamide, thionamide, clozapine, dapsone, methimazole, penicillin, rituximab, procainamide, sulfasalazine, thiamazole, carbimazole, amoxicillin, cotrimoxazole, ticlopidine, and valganciclovir. Neutropenia not caused by chemotherapeutic drugs is less common than neutropenia secondary to chemotherapy. It can occur at any time during the course of treatment in response to primary therapy, but most commonly occurs within the first few weeks after drug administration. Affected patients typically experience severe neutropenia for weeks to months after initial exposure to the drug. Drug-induced neutropenia is characterized by decreased production or increased destruction of neutrophils. Clinical consequences of neutropenia include increased susceptibility to infections, which can be fatal, proportional to the degree of neutropenia.

[0003] Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are approved by the U.S. Food and Drug Administration (FDA) for use in treating severe febrile neutropenia. G-CSF and GM-CSF have demonstrated proven efficacy in reducing the incidence, magnitude, and duration of neutropenia following chemotherapy; therefore, they are a highly recognized and widely accepted treatment option for drug-induced neutropenia. However, no drug is without adverse effects. Some common symptoms observed after G-CSF administration include shortness of breath, chest pain, nausea, hypoxemia, sweating, anaphylaxis, syncope, and flushing. In comparison, adverse effects of GM-CSF are moderate, including fever, myalgia, fatigue, rash, and injection site reactions; however, severe effects, such as pericarditis and thrombosis, are also known to occur at high doses. This has led to an increasing need for improved treatment options, preferably those derived from natural sources with enhanced safety and efficacy.

[0004] Over the past few decades, the spectrum of disease has shifted toward complex chronic conditions requiring aggressive treatments such as chemotherapy. Synthetic drugs generally have more associated side effects and toxicities, increasing the burden on patients. The World Health Organization Drugs Strategy 2002-2005 encouraged the acceptance of herbal medicines as complementary or alternative medicines worldwide. According to WHO data, approximately 70% of the world's population has used herbal medicines as complementary medicines over the past 20 years. Because of the general perception that herbal medicines are free of unwanted side effects, herbal medicines are often administered simultaneously with therapeutic drugs to treat major illnesses.

[0005] Picrorhiza kurroa is a well-known medicinal herb traditionally popular in the Indian Ayurvedic system for the treatment of liver and respiratory disorders. Picroside I and picroside II are two of the most important constituents found in the roots and rhizomes of the plant. Picroside I and picroside II are used individually and in combination in herbal formulations. The pharmacological benefits of picroside I and picroside II have been widely reported in the literature, including antioxidant, anti-inflammatory, antiallergic, hepatoprotective, choleretic, antiasthmatic, and anticancer activities. However, no compositions containing picroside I or picroside II, or a combination thereof, for treating neutropenia have been reported.

[0006] As a result, the inventors report a novel pharmacological benefit of picroside in the treatment of neutropenia. The disclosed invention is dedicated to developing a composition containing an active ingredient derived from natural resources for treating neutropenia. Thus, according to the present invention, a composition comprising picroside for treating neutropenia is provided. More specifically, a composition containing picroside I for treating neutropenia is provided. Also disclosed is a novel, industrialized method for isolating picroside I and picroside II. The present invention also discloses a method for making the novel pharmaceutical composition containing picroside. The present invention also provides a method for treating or preventing neutropenia in a subject, comprising administering to the subject a therapeutically effective amount of a novel composition containing an active ingredient, such as picroside I. Summary of the Invention

[0007] SUMMARY OF THE INVENTION In view of the above and in order to overcome the disadvantages of the prior art, it is an object of the present invention to provide a pharmaceutical composition comprising a therapeutically effective amount of picroside I for the treatment or prevention of neutropenia. To achieve the above, the present invention encompasses a composition comprising purified picroside I and a pharmaceutically acceptable excipient for treating or preventing neutropenia in a subject in need thereof, which may be in oral or injectable form.

[0008] According to one embodiment, a pharmaceutical composition for treating neutropenia is provided, comprising a therapeutically effective amount of purified picroside I and an acceptable pharmaceutical carrier or excipient. According to one embodiment, the pharmaceutical composition comprises 100-400 mg of purified picroside I and an acceptable pharmaceutical carrier or excipient.

[0009] According to a preferred embodiment, the amount of purified picroside I in the pharmaceutical composition is 150 to 400 mg. According to one embodiment, the pharmaceutical composition is an oral or injectable composition. According to a preferred embodiment, the oral pharmaceutical composition is in the form of a tablet.

[0010] According to one embodiment, the pharmaceutical composition further comprises a filler, a solvent, and a lubricant. According to another embodiment, the filler is F melt Type C, the solvent is ethanol, and the lubricant is magnesium stearate. According to a preferred embodiment, the amount of the filler is about 98 weight percent, and the amount of the lubricant is about 1 weight percent.

[0011] According to one important embodiment, there is provided a pharmaceutical composition for treating neutropenia, comprising 390 mg of purified picroside I, a filler, a solvent, and a lubricant; wherein the composition is ready for oral administration. According to an alternative embodiment, the injectable pharmaceutical composition is in the form of an injection. According to one embodiment, the pharmaceutical composition further comprises a solvent. According to a preferred embodiment, the solvent is 2.5% PEG 400.

[0012] According to one important embodiment, there is provided a pharmaceutical composition for treating neutropenia, comprising 195 mg of purified picroside I and a solvent; wherein the composition is sterile, has a pH in the range of 4.0 to 7.0, and is ready for subcutaneous administration. According to one embodiment, the injectable pharmaceutical composition is stored in a pre-filled sterile syringe or vial or cartridge.

[0013] According to one important aspect, there is provided a method for treating neutropenia in a subject, the method comprising administering a therapeutically effective amount of picroside I contained in the composition to a subject in need thereof. According to one embodiment, the composition is administered to the subject once daily. According to another embodiment, the composition is administered to the subject for up to five consecutive days. DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description In order to clarify the above and other objects, features, and advantages of the present invention, certain embodiments of the present invention have been particularly enumerated and described in detail, accompanied by examples, as follows. The principles and modes of operation of the present invention have been described and explained in the embodiments. Those skilled in the art will readily appreciate that the present invention can be practiced otherwise than as specifically described and explained. The present invention is not limited by the above-described embodiments, methods, and examples, but by all embodiments and methods that fall within the scope and spirit of the present invention. Also, in the following description of the present invention, certain terminology may be used for reference purposes only and is not intended to be limiting.

[0015] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limits of that range is also specifically disclosed, to the tenth of the unit of the lower limit. Each smaller range between any stated and / or intervening value in a stated range and 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, and each range in which either or both of the limits are included in the smaller range, or neither of the limits are included, is also encompassed within the invention, subject to any specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0016] The present invention provides pharmaceutical compositions containing picroside compounds derived from P. kurroa for the treatment or prevention of immune abnormalities such as neutropenia. More specifically, the present invention provides pharmaceutical compositions containing picroside I for the treatment or prevention of neutropenia. Furthermore, the present invention provides a novel, industrialized method for isolating picroside I and picroside II from P. kurroa. The present invention also provides a method for treating or preventing neutropenia in a subject, comprising administering to the subject a therapeutically effective amount of a novel composition containing the active ingredient picroside I together with a pharmaceutically acceptable carrier or excipient thereof.

[0017] Accordingly, it is an object of the present invention to provide a composition comprising picroside I for treating or preventing neutropenia in a subject by administering to the subject suffering from neutropenia a therapeutically effective amount of the pharmaceutical composition comprising the active ingredient picroside I together with a pharmaceutically acceptable carrier or excipient thereof.

[0018] The term "picroside I" disclosed herein includes picroside I isolated from P. kurroa. The term "Picrorhiza kurroa" or "P. kurroa" disclosed herein includes cultivated or naturally-grown plants and commercially available plants, but is not intended to be limited thereto. Picrorhiza kurroa Royle ex Benth., commonly known as "Kutki or Kutaki," is a perennial herb from the Scrophulariacae family that occurs in wild form in the Himalayan and Garhwal regions of northwest India. P. kurroa has been listed as a prescription drug in the Pharmacopoeia of India, 1970, since 1970. P. kurroa is commercially available in India and was sourced in this manner. Consequently, in the present invention, the plant raw materials and extracts were commercially sourced. Picrorhiza kurroa roots were procured from Noornihal, Anekal, Bangalore; batch number RM / PK / SEP-19-03. Picrorhiza kurroa water extract was procured from Noornihal, Anekal, Bangalore; batch number RDP / PK / 011.

[0019] It should be noted that herbs and plants can be processed and ingested in various ways and forms, including whole injectables, capsules, and tablets containing the raw herb in crushed or powdered form or its dried extract. Consequently, plant parts may be used as raw materials or their liquid and / or solid extracts. Suitable procedures include multiple steps, such as maceration, percolation, and extraction using supercritical fluids, where liquefied gas or a suitable solvent is used to prepare the material. Picroside I and picroside II are derived from the roots and rhizomes of P. kurroa. The constituents must be extracted from the plant material and further purified and isolated to suit their intended use in compositions for treating or preventing neutropenia. An aspect of the present invention is a method for extracting, further purifying, and isolating picroside I and picroside II from P. kurroa to obtain purified picroside I and picroside II.

[0020] Accordingly, in an embodiment of the present invention, the pharmaceutical composition of the present invention contains purified picroside I. Accordingly, a method for extracting, purifying, and isolating picroside I and picroside II from P. kurroa is provided. Disclosed herein is a method for extracting picroside I and picroside II from P. kurroa, comprising the steps of: preparing a crude powder of P. kurroa roots and rhizomes, followed by using various ratios of an extraction solvent selected from water, demineralized water, methanol, ethanol, etc., or a mixture thereof at a predetermined pH; performing at least one or more extraction cycles using the solvent at a predetermined temperature; collecting and filtering the liquid extract; and evaporating the liquid extract under vacuum at a predetermined temperature to obtain a dry powder extract. Also disclosed herein is a method for further purifying a dry powder extract of picroside I and picroside II from P. kurroa, the method comprising the steps of: dissolving the dry powder extract in water and loading it onto a resin using ion exchange column chromatography; collecting a first fraction in water; subsequently collecting at least two or more fractions in methanol; washing the column with methanol; and drying the concentrate containing picroside I and picroside II. The method for isolating picroside I and picroside II from P. kurroa comprises the steps of: using silica column chromatography; dissolving the extracted and purified extract in methanol; collecting at least one fraction containing picroside I or picroside II in methanol and chloroform; and using thin layer chromatography to elute picroside I and picroside II.

[0021] Specifically, disclosed herein is a method for extracting picroside I and picroside II from P. kurroa, comprising the steps of: extracting crude powder of P. kurroa roots and rhizomes using demineralized water as an extraction solvent selected from water having a pH ranging from 6.5 to 7.5; performing at least one or more extraction cycles using the solvent at a temperature ranging from 50 to 90°C; collecting and filtering the liquid extract; and evaporating the liquid extract under vacuum at a temperature ranging from 40 to 55°C to obtain a dry powder extract characterized by an extraction yield of approximately 28.75%. A method for further purifying picroside I and picroside II from P. kurroa comprises the steps of: using a reverse-phase chromatography method, including the use of a resin selected from Diaion-HP-20, Amberlite-XAD-4, 8, 7, 16, and the like. Preferably, a resin suitable for the pre-purification of polar compounds, such as phenolics, is selected. The loaded column is repeatedly washed with water until a colorless eluate is obtained. The column is then eluted with 30% methanol in water, and fractions are collected. This step is repeated until a colorless eluate is obtained. The column is then washed with 100% methanol, and approximately 100 ml fractions are collected. The fractions are checked by TLC under UV for the markers of interest, and selected fractions are dried to yield a concentrate containing approximately 4-6% picroside I and approximately 5-8% picroside II.

[0022] The dried fraction is resuspended in methanol and adsorbed onto silica when two volumes of silica are added to the methanol-picroside mixture and dried by rotavapor. The dried material is then loaded onto a silica column 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 the 4% methanol in chloroform fraction. Fractions containing minimal contaminants and the maximum amount of picroside are selected, dried, and weighed. The final weight is then calculated to determine the total yield. The purity is then checked by HPLC. This process results in picroside I eluting first, followed by picroside II, which are characterized by purities of approximately 96.42% and 89.02%, respectively.

[0023] We report that the inventive process described above has a shorter time of 3 hours for the purification of picroside I compared to the 6-hour process published in the prior art (Hussain et al., 2013), while nevertheless resulting in a slightly higher yield. In addition, the inventive process utilizes a single solvent, i.e., water, which is a safer medium, compared to the use of two environmentally unfriendly solvents such as petroleum ether, ethanol, etc., as reported by the prior art (Hussain et al., 2013).

[0024] The present inventors report that the purified picroside I and picroside II compounds exhibit the ability to treat and prevent immune abnormalities, i.e., neutropenia, through various in vivo studies in mice. To the best of our knowledge, this antineutropenic activity of picroside I or picroside II has not been reported before. Furthermore, it was observed that picroside I exhibited slightly higher activity, which is desirable for treating or preventing neutropenia, than picroside II. In addition, mice treated with picroside I exhibited higher WBC counts than mice treated with picroside II. Consequently, picroside I was selected as a candidate composition for treating neutropenia due to its overall effect on neutrophil and WBC counts. The core of the present invention is the objective of providing a pharmaceutical composition of picroside I for treating or preventing neutropenia. Accordingly, according to another aspect of the present invention, there is provided a pharmaceutical composition comprising purified picroside I prepared by the method described above for treating or preventing neutropenia. Pharmaceutical compositions of picroside II are also disclosed herein.

[0025] The term "treating" as disclosed herein includes any act of alleviating or favorably altering the symptoms associated with a disease or disorder disclosed herein by means of administering the composition; and the term "preventing" as disclosed herein includes any act of inhibiting or slowing the onset of a disease or disorder disclosed herein by means of administering the composition.

[0026] An embodiment of the present invention provides a pharmaceutical composition comprising purified picroside I prepared by the method described above and a pharmaceutically acceptable carrier or excipient for the treatment or prevention of neutropenia. As defined herein, the term "pharmaceutically acceptable carrier or excipient" includes pharmaceutical additives, inactive ingredients, dyes, flavors, binders, emollients, fillers, lubricants, preservatives, and many more classifications used in preparing drugs and well known in the art or previous literature.

[0027] In an embodiment of the present invention, a pharmaceutical composition containing purified picroside I may be prepared as an oral dosage form, such as a powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixir, pill, powder, sachet, or granule. In a preferred embodiment, the pharmaceutical composition of the present invention in an oral dosage form will be a tablet. In another embodiment of the present invention, the pharmaceutical composition will further comprise pharmaceutically acceptable excipients such as a filler, a solvent, and a lubricant. In a preferred embodiment, the filler will be F-MELT Type C, the solvent will be ethanol, and the lubricant will be magnesium stearate. It should be noted that the amount of excipient will be proportional to the amount of the active ingredient, i.e., picroside I. Consequently, the amount of filler will be about 98% by weight, and the amount of lubricant will be about 1% by weight.

[0028] Thus, according to an embodiment of the present invention, there is provided a pharmaceutical composition for treating neutropenia, comprising 390 mg of purified picroside I, a filler, a solvent, and a lubricant; wherein the composition is ready for oral administration.

[0029] The compositions of the present invention can also be prepared as pharmaceutical compositions containing pharmaceutically acceptable carriers, adjuvants, or diluents, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The compositions may additionally include anti-agglutinating agents, wetting agents, flavoring agents, emulsifying agents, preservatives, etc. The compositions of the present invention may be formulated so as to provide rapid, sustained, or delayed release of the active ingredient after their administration to the patient by employing any of the procedures well known in the art.

[0030] In an alternative embodiment, a pharmaceutical composition containing purified picroside I may be prepared as an injectable preparation, such as a solution, suspension, or emulsion. In a preferred embodiment, the pharmaceutical composition of the present invention will be an injectable solution. In another embodiment of the present invention, the pharmaceutical composition will further comprise a pharmaceutically acceptable solvent selected from methanol, 2.5% PEG 400, 5% PEG 400, 5% DMSO, 5% ethyl lactate, and 1.4% propylene glycol. In a preferred embodiment, the composition will comprise 2.5% PEG 400, wherein the pH of the composition will be in the range of 4.0 to 7.0. It should be noted that the injectable composition of the present invention can also be dissolved in oil or other solvents commonly used to produce injectable solutions. Suitable examples of carriers include, but are not limited to, physiological saline, ethanol, vegetable oil, isopropyl myristate, etc. In another embodiment of the present invention, the injectable composition will be ensured to be sterile and ready for use; the composition may be stored in a prefilled syringe, vial, cartridge, or any other suitable mode or medium.

[0031] Thus, according to an embodiment of the present invention, there is provided a pharmaceutical composition for treating neutropenia, comprising 195 mg of purified picroside I and a solvent; wherein the composition is sterile, has a pH in the range of 4.0 to 7.0, and is ready for subcutaneous administration.

[0032] The formulation methods and types of excipients will be described below, but the present invention is not limited thereto. A representative preparation example is described as follows.

[0033] According to another aspect of the present invention, there is also provided a method for treating or preventing neutropenia in a subject, comprising administering to a subject suffering from neutropenia a composition comprising a therapeutically effective amount of purified picroside I prepared by the method described above.

[0034] Accordingly, in an embodiment of the present invention, there is provided a method for treating or preventing neutropenia in a subject, the method comprising administering to a subject suffering from neutropenia a composition comprising a therapeutically effective amount of purified picroside I prepared by the method described above.

[0035] In a preferred embodiment, a method for treating neutropenia in a subject is provided, comprising administering a therapeutically effective amount of picroside I to a subject in need thereof as an oral or injectable composition. In a preferred embodiment, the composition is administered to the subject once daily. In another preferred embodiment, the composition is administered to the subject for up to five consecutive days.

[0036] According to another aspect of the present invention, there is also provided use of a composition comprising purified picroside I prepared by the method described above and a pharmaceutically acceptable carrier or excipient for the manufacture of a medicament employed for treating or preventing neutropenia.

[0037] According to another aspect of the present invention, the pharmaceutical compositions of the present invention can be administered to a subject via various routes. According to this embodiment of the present invention, the term "subject" as defined herein includes an animal subject, such as a rat, a mouse, a domestic animal, or a human. Several modes of administration are contemplated; for example, administration can be orally or by intravenous, intramuscular, subcutaneous, or intradermal injection. It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, uses, and preparations of the present invention without departing from the spirit or scope of the present invention.

[0038] According to aspects of the present invention, the desired dose of picroside I may vary depending on the condition and weight of the subject, severity, drug form, route and duration of administration, and may be selected by those skilled in the art. It should be noted that the extrapolation of doses from animals to humans using allometric scaling is made taking into account differences in pharmacokinetics and pharmacodynamics between species to assess the initial dose for humans. For example, a 1 mg dose and a 2 mg dose for administration in mice are extrapolated to a 195 mg dose and a 390 mg dose for administration in humans, respectively.

[0039] Consequently, this embodiment of the present invention provides that to achieve the desired effect, it is generally recommended to administer the purified picroside I compound of the present invention in an amount ranging from 100 mg / day to 200 mg / day, preferably 150-200 mg / day, more preferably 190-200 mg / day, to humans. The dose may be administered in a single dose or in several divided doses per day. Such administration may be for 5 consecutive days.

[0040] Because neutropenia is directly related to neutrophil counts, the effect of the composition on disease is specifically reported via neutrophil counts and also via WBC counts. Consequently, in vivo studies were conducted on mice to determine the effect of picroside I on neutropenia. An animal test model was created by inducing neutropenia in mice by administering cyclophosphamide. Neutropenic mice were administered a composition containing picroside I and observed for changes in body weight, blood analysis to monitor neutrophil counts, and clinical symptoms such as toxicity and death. The research model was repeated in neutropenic mice by administering a composition containing picroside II. We report that both picroside I and picroside II significantly reversed cyclophosphamide-induced neutropenia in mice. Furthermore, the efficacy and activity of picroside I for the treatment of neutropenia was compared with that of G-CSF, an FDA-approved drug for treating the disease. We report that the results of picroside I treatment in subject mice were comparable to those of G-CSF treatment.

[0041] As described in the present invention, the inventive composition of purified picroside I derived from P. kurroa has shown potent anti-neutropenic activity through in vivo testing using Swiss albino mice with drug-induced neutropenia (e.g., testing against neutrophil proliferation and activity caused by the development of neutropenia); therefore, it can be used as a therapeutic agent for treating and preventing neutropenia.

[0042] Incorporation by Reference All publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0043] example The following examples are provided for illustrative purposes only and are intended to be purely exemplary of the present disclosure and are not intended to limit the scope of the claims provided herein.

[0044] Example 1. Preparation of crude extract of P. kurroa 400 gm of crude powder of Picrorhiza kurroa rhizome was extracted with demineralized water at pH 6.5-7.5. Approximately 2000 ml of water was added for each cycle. A total of three cycles were performed at 80°C. The liquid extract was collected and filtered through a PP filter cloth to remove debris. The filtered liquid extract was then evaporated under vacuum at 50-55°C using a rotary evaporator to obtain a dry powder of the extract. Component analysis was performed using HPLC. HPLC results confirmed that the crude extract of Picrorhiza kurroa contained approximately 4-6% picroside I and approximately 5-8% picroside II.

[0045] Example 2. Preparation of Picroside I and Picroside II from purified extracts of P. kurroa A crude extract of P. kurroa was prepared by the extraction method according to Example 1. 100 ml of HP20 resin was loaded onto a column and equilibrated with water. 13 gm of aqueous extract of P. kurroa was completely dissolved in 100 ml of water and adsorbed onto the resin. The first fraction was collected with 300 ml of water. The second fraction was collected with 300 ml of 30% methanol in water. The third fraction was collected with 400 ml of 60% methanol in water. The column was washed with methanol. It was observed that the third fraction and the methanol washing contained both picroside-I and picroside-II. Both fractions were mixed together and dried to obtain a concentrate.

[0046] Component analysis was performed using HPLC. HPLC results confirmed that the purified extract of P. kurroa contained approximately 14.41% picroside I and approximately 16.60% picroside II.

[0047] Example 3. Isolation of picroside I and picroside II from P. kurroa A purified extract of P. kurroa was prepared by the extraction method according to Example 2. Isolation was carried out using conventional silica column chromatography. 90 gm of silica (100-200#) was packed into a glass column with chloroform. 6 gm of the purified extract was dissolved in sufficient methanol. Further, 12 gm of silica (60-120#) was added for adsorption. The mixture was dried and loaded onto the silica bed. Multiple fraction collection was started with 5% methanol in chloroform. Fractions were pooled together according to the TLC pattern.

[0048] Component analysis was performed using HPLC. The HPLC results confirmed that picroside I with a purity of about 96.42% and picroside II with a purity of about 89.02% were obtained.

[0049] Example 4. Preparation of picroside I tablets Tablet preparation Purified picroside I prepared by the method according to Examples 1, 2, and 3 was used in the oral composition. Tablets were prepared by dissolving picroside I in a sufficient amount of ethanol (5 mL for a batch size of 50 tablets). Previously, F-Melt Type C was sieved through a #40 sieve and granulated for use in the next step. This resulted in a granulated wet mass, which was dried at 60°C. The dried granules were then sieved using an ASTM #40 sieve and lubricated with an ASTM #60 sieve. Magnesium stearate was manually passed through. Tablets were compressed using the parameters mentioned below: Punch Dimension: 5.5mm Standard Concave; Target Weight: 70mg; Hardness: 30~50N; DT: 1~2Min; Thickness: 2.6±0.2mm; Friability: NMT 1%

[0050] Table 1: Composition of Picroside I tablets (1 mg) [Table 1]

[0051] The above method, including process parameters and ingredient ratios, was repeated to prepare a tablet formulation of picroside II. Instead of 1 mg picroside I, 1 mg picroside II was added to the composition.

[0052] Example 5. Preparation and stability test of picroside I injection formulation Preparation of injection solutions Purified picroside I prepared by the methods according to Examples 1, 2, and 3 was used in the injectable compositions. The injectable preparations were prepared by dissolving 1 mg of picroside I in a solvent, controlling the pH to about 4-7, and then filling into ampoules, followed by sterilization by conventional injectable preparation methods. For the comparative compositions, various solvents, such as methanol, 2.5% PEG400, 5% PEG400, 5% DMSO, 5% ethyl lactate, and 1.4% propylene glycol, were used to solubilize picroside I.

[0053] The solution was kept sonicated for 15 min, and then allowed to stand at room temperature for 1 hour. HPLC was used for quantitative analysis. The composition prepared with 2.5% PEG400 was found to be the most suitable and was further analyzed for stability.

[0054] The above method, including process parameters and ingredient ratios, was repeated to prepare an injectable formulation of picroside II. 1 mg picroside II was added to the composition in place of 1 mg picroside I.

[0055] Stability Data Three compositions containing 1 mg picroside I and 2.5% PEG400 were sonicated for 15 min, stored at room temperature (RT), 2-8°C, and 40°C at 75% RH for 1 month, and then injected (20 μL) into HPLC at 220 nm for quantitative analysis. All compositions showed excellent stability over the 1-month period.

[0056] Table 2: Stability analysis of the injectable composition of picroside I and 2.5% PEG400; analyzed by HPLC quantitative analysis. [Table 2]

[0057] Example 6. Animal model testing (mice) To determine the antineutropenic effect of picroside I, studies were performed using neutropenic-induced mice. Preparation of experimental animals In-house bred Swiss albino mice were housed and maintained under standard laboratory conditions, including air conditioning with adequate fresh air supply (12–15 air changes per hour), a room temperature of 21.1–23.2°C, and a relative humidity of 55–69%, with a 12-hour light / 12-hour dark cycle. Mice were allowed to acclimate to laboratory conditions for a minimum period of 7 days.

[0058] Cyclophosphamide to induce neutropenia Selected Swiss albino mice were randomly divided into four groups based on body weight. Each group contained six mice. Animals in groups 2, 3, and 4 were treated with 150 mg / kg cyclophosphamide via the intraperitoneal route on day 0 to initiate neutropenia. On day 3, animals in groups 2, 3, and 4 reflected cyclophosphamide-induced neutropenia when compared with mice in group 1 (control). On day 4, mice from groups 2, 3, and 4 were again injected with a single dose of 100 mg / kg cyclophosphamide. Total and differential counts (neutrophils, lymphocytes, and monocytes) were recorded on day 3. Neutrophil counts were compared between groups on day 3 using one-way ANOVA followed by Dunnett's test or Student's t test. P<0.05 was considered statistically significant.

[0059] Table 3: Mean %WBC and %Neutrophil Counts on Day 3 Following Cyclophosphamide Administration [Table 3]

[0060] Example 7. Administration of Picroside I (for injection) to cyclophosphamide-induced neutropenic mice Cyclophosphamide-associated neutropenia was induced in mouse subjects using the method described in Example 6. Injectable compositions were prepared using the method described in Example 5. Picroside I composition was administered subcutaneously to animals in Group 3 at a dose of 1 mg / mouse / day for 5 days (starting on Day 5 and ending on Day 10). Picroside II composition was administered subcutaneously to animals in Group 4 at a dose of 1 mg / mouse / day for 5 days (starting on Day 5 and ending on Day 10). Blood was collected again on Day 10, and total leucocyte counts and differential leucocyte percentages (neutrophils, lymphocytes, and monocytes) were manually counted. Neutrophil counts on Day 10 were compared between groups using one-way ANOVA followed by Dunnett's test or Student's "t" test. P<0.05 was considered statistically significant. In addition, the mice were observed for changes in weight and any clinical symptoms.

[0061] There were no treatment-related changes in body weight in either picroside I or picroside II-treated control mice, and no clinical signs of toxicity or mortality were observed in either picroside I or picroside II-treated control mice.

[0062] Results on day 10 revealed that cyclophosphamide caused neutropenia and reduced total lymphocyte counts in Group 2 (characterized as the vehicle control) compared with Group 1 (characterized as the normal control). Treatment of subjects in Groups 3 and 4, respectively, with picroside I and picroside II showed a significant increase in both total white blood cell and neutrophil counts on day 10 compared with Group 2, indicating a total reversal of neutropenia. Furthermore, picroside I and picroside II significantly (P<0.01) increased neutrophil counts more than those in Group 1 (normal control) mice.

[0063] Based on the above results, it is evident that picroside I and picroside II injections can significantly reverse cyclophosphamide-induced neutropenia in mice. Table 4: Mean %-neutrophil and WBC counts on day 10 for picroside I and picroside II. [Table 4]

[0064] Example 8. Administration of picroside I (oral administration) to cyclophosphamide-induced neutropenic mice Cyclophosphamide-associated neutropenia was induced in mouse subjects using the method according to Example 6. Oral tablet compositions were prepared using the method according to Example 4. Picroside I composition was orally administered at a dose of 2 mg / mouse / day (2 tablets) to animals in Group 1 and Group 2 for 5 days (starting on Day 5 and ending on Day 10), respectively. For comparative studies, the oral compositions were administered using distilled water and PEG. Specifically, mice in Group 1 were administered oral tablets in distilled water, and mice in Group 2 were administered oral tablets in 5% PEG. Blood was collected again on Day 10, and total leucocyte counts and differential leucocyte percentages (neutrophils, lymphocytes, and monocytes) were manually counted. Neutrophil counts on Day 10 were compared between groups using one-way ANOVA followed by Dunnett's test or Student's "t" test. P<0.05 was considered statistically significant. Additionally, mice were observed for changes in body weight and any clinical symptoms.

[0065] There was no treatment-related change in body weight in any of the control mice treated with picroside I. Also, no clinical signs of toxicity or mortality were observed in any of the control mice treated with picroside I.

[0066] Results on day 10 revealed that treatment of subjects in group 1 with picroside I in distilled water and subjects in group 2 with picroside I in 5% PEG showed a significant increase in both total white blood cell and neutrophil counts on day 10 compared to those on day 3.

[0067] Based on the above results, it is evident that picroside I oral tablets can significantly reverse cyclophosphamide-induced neutropenia in mice. Table 5: Mean neutrophil and WBC counts on day 10 of picroside I [Table 5]

[0068] Example 9. Comparative administration of picroside I and G-CSF to cyclophosphamide-induced neutropenic mice Cyclophosphamide-associated neutropenia was induced in mouse subjects using the method according to Example 6. Group 1 was characterized as the vehicle control group, Group 2 was characterized as the cyclophosphamide control group, Group 3 was characterized as the picroside I treatment group, and Group 4 was characterized as the G-CSF treatment group. The picroside I composition was administered subcutaneously to the animals in Group 3 at a dose of 1 mg / mouse / day for 5 days (starting on Day 5 and ending on Day 10). The G-CSF composition was administered subcutaneously to the animals in Group 4 at a dose of 2 μg / mouse / day for 5 days (starting on Day 5 and ending on Day 10). Blood was collected again on Day 10, and the total leucocyte count and differential leucocyte percentages (neutrophils, lymphocytes, and monocytes) were manually counted. Neutrophil counts on day 10 were compared between groups using one-way ANOVA followed by Dunnett's test or Student's "t" test. P<0.05 was considered statistically significant. In addition, hepatotoxicity was observed by examining ALT and AST levels.

[0069] The results showed that treatment with picroside I mitigated the cyclophosphamide-induced decrease in percent neutrophils. The changes observed in the picroside I-treated group were comparable to those observed in the G-CSF-treated group. Administration of picroside I to control mice resulted in a significant recovery (p<0.001) of AST and ALT levels compared with Group 2. The observed decrease in AST and ALT levels was comparable to the decrease in AST and ALT levels achieved upon treatment with G-CSF.

[0070] Table 6: Mean % neutrophil counts on days 0, 3, and 10 after administration of picroside I and G-CSF. [Table 6]

[0071] Table 7: Effect of picroside I on cyclophosphamide-induced hepatotoxicity [Table 7]

Claims

1. A pharmaceutical composition for treating neutropenia comprising a therapeutically effective amount of purified picroside I and an acceptable pharmaceutical carrier or excipient.

2. 10. The pharmaceutical composition of claim 1, comprising 100 to 400 mg of purified picroside I and an acceptable pharmaceutical carrier or excipient.

3. 3. The pharmaceutical composition of claim 2, wherein the amount of purified picroside I is 150-400 mg.

4. 10. The pharmaceutical composition of claim 1, wherein the composition is an oral or injectable composition.

5. 5. The pharmaceutical composition of claim 4, wherein the oral composition is in the form of a tablet.

6. 6. The pharmaceutical composition of claim 5, further comprising a filler, a solvent, and a lubricant.

7. 1. A pharmaceutical composition for treating neutropenia, comprising 390 mg of purified picroside I, a filler, a solvent, and a lubricant, wherein the composition is ready for oral administration.

8. 8. The pharmaceutical composition of claim 7, wherein the filler is F-Melt® Type C, the solvent is ethanol, and the lubricant is magnesium stearate.

9. 8. The pharmaceutical composition of claim 7, wherein the amount of filler is about 98 weight percent and the amount of lubricant is about 1 weight percent.

10. The pharmaceutical composition according to claim 4, wherein the injectable composition is in the form of an injection solution.

11. 11. The pharmaceutical composition of claim 10, further comprising a solvent.

12. A pharmaceutical composition for treating neutropenia, comprising 195 mg of purified picroside I and a solvent, wherein the composition is sterile, has a pH in the range of 4.0 to 7.0, and is ready for subcutaneous administration.

13. 13. The pharmaceutical composition of claim 12, wherein the solvent is 2.5% PEG 400.

14. 13. The pharmaceutical composition of claim 12, wherein the composition is stored in a pre-filled sterile syringe or vial or cartridge.

15. A pharmaceutical composition for the treatment and prevention of neutropenia, comprising a therapeutically effective amount of picroside I.

16. The pharmaceutical composition of claim 15, which is administered to a subject once a day.

17. The pharmaceutical composition of claim 15, which is administered to a subject for up to 5 consecutive days.

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