DESC:DESCRIPTION
A PARENTERAL COMPOSITION OF CINNAMIC ACID

TECHNICAL FIELD
The present invention relates to a novel parenteral composition comprising cinnamic acid or its isomer or pharmaceutically acceptable salt or derivative thereof for prophylaxis, treatment and management of management of neutropenia and/or thrombocytopenia.

BACKGROUND
Neutropenia and thrombocytopenia are blood conditions that sometimes occur together in a patient. Neutropenia involves a low neutrophil count, and thrombocytopenia involves a low platelet count. Certain conditions, infections, or medications may cause reduced levels of both.
Neutropenia is when a person does not have enough neutrophils. Neutrophils are white blood cells that repair wounds and destroy pathogens. Thrombocytopenia is when a person does not have enough thrombocytes or platelets. Platelets play an important role in helping blood clot. Blood needs to clot to stop bleeding when you have a wound.

Neutropenia is one of the serious adverse effects of cytotoxic chemotherapy and other cancer therapies such as radiation therapy. Cytotoxic chemotherapy, which works by seeking out and destroying fast-growing cells, induces neutropenia because of the high proliferative rate of neutrophil precursors and the rapid turnover of blood neutrophils. The most common symptoms of neutropenia in patients undergoing chemotherapy include fever, mouth sores, and ear infections. Patients with profound neutropenia often suffer from pyogenic infections such as septicemia, cutaneous cellulitis, liver abscesses, furunculosis, pneumonia, stomatitis, gingivitis, perirectal inflammation, colitis, sinusitis, and otitis media. Chemotherapy may have to be delayed until the body can produce more neutrophils or a lower dosage of anticancer may have to be given, resulting in the treatment being less effective.

Therefore, there is a need for therapeutics that can counter neutropenia and thrombocytopenia and other haematological disorders. Granulocyte CSFs (G-CSFs) and granulocyte macrophage CSFs (GM-CSFs) are currently used to prevent or treat neutropenia among patients treated with chemotherapy. There are many concerns with such proteinaceous anti-neutropenic agents like GCSF/GMCSF as they are prone to oxidation, susceptible to storage temperatures above cold, affected by freeze thaw conditions, affected by moisture, affected by humidity, affected by shaking, the effect of head space in stored containers, oligomeration due to cysteine residues within the protein, stringent protein process requirements for manufacturing etc. The other disadvantages of GCSF include moderate plasma half-life time (~4 h), and considerable production costs, and chronic effects on the hematopoietic system. Also, recombinant proteins of human or animal origin as well as proteins obtained from cell cultures bear a potential residual risk of viral contaminations which is the main reason for the requirement of stringent manufacturing controls. Other protein-like impurities that are analytically difficult to detect can also cause immunological reactions in humans due to their antigenic properties. Hence, there is a need to identify new effective treatment and management for induced neutropenia by cancer therapy, specifically chemotherapy using molecules that are devoid of such disadvantages.

Another cause of concern with the GCSF formulations particularly in the case of subcutaneous administration, they lead to local intolerances in the patients due to the low pH value (4.5-5.0) used. The product obtained can cause pain and local tissue irritation in sensitive patients since the physiological pH range of 7.0-7.5 present in tissue is not adhered to. GCSF is also sensitive towards mechanical stress such as shaking the solution can influence the liquid pharmaceutical preparations during transport in a diverse and uncontrollable manner. rhGCSF is produced by recombinant DNA technology and is therefore expensive to manufacture and a costly treatment.

3-Phenyl-2-propenoic acid, commonly referred to as cinnamic acid is a white, crystalline solid. Cinnamic acid is soluble in ethanol, methanol, petroleum ether and chloroform; it is easily soluble in benzene, ether, acetone, acetic acid, carbon disulfide and oils but insoluble in water or very low solubility in water at 0.57 mg/mL at 25°C. Cinnamic acid is found as both trans-cinnamic acid and cis-cinnamic acid. Both trans and cis-isoforms of CA exist, but trans-cinnamic acid (t-CA) is the predominant form in nature due to its high stability. It is known in the art that cinnamic acid acts as an antioxidant, an antimicrobial agent, a healing agent, and an anti-fungal agent. However, due to the inherent insolubility of cinnamic acid in water, it poses a difficulty in formulating a pharmaceutical composition of cinnamic acid. This is in particular true, in the case of, parenteral formulation which must comprise cinnamic acid in a completely in solubilized state. As a standard practice, aqueous vehicles are generally preferred for parenteral preparations over non-aqueous vehicles.

A PCT application, WO 2023/099968 discloses a non-aqueous parenteral composition of cinnamic acid comprising ethanol, PEG 400, DMSO, ethyl lactate, propylene glycol and a combination of 30% ethanol and water. However, non-aqueous composition are known to have inherent administration issues. Further, the inventors of the present inventors found that the non-aqueous composition could only solubilize Cinnamic acid up to 6 mg/ml whereas the composition of the present invention could solubilize up to 20 mg/ml while maintaining the desired pH of the composition.

The present inventors have now developed a stable parenteral composition comprising cinnamic acid. Further, present inventors have unexpectedly found that the said parenteral composition of cinnamic acid can used for the prophylaxis, treatment and management of management of neutropenia associated with disease such as dengue, chemotherapy; and thrombocytopenia. Also, the present inventors have found that the said composition can be also used for treatment and management of other haematological disorders. The composition has pH of 6-7.5, hence does not cause any local pain or irritation at the pain site as was seen in the case of GCSF.

OBJECTIVE OF THE INVENTION

An important objective of the present invention is to provide an effective prophylaxis, treatment and management of neutropenia associated with dengue, chemotherapy and thrombocytopenia.

A specific objective of the present invention is to provide an effective prophylaxis, treatment and management of neutropenia and/or other haematological disorders caused by cancer therapy specifically chemotherapy.

Another important objective of the present invention is to provide a stable parenteral composition of cinnamic acid or its isomer or pharmaceutically acceptable salt or derivative thereof for the prophylaxis, treatment and management of neutropenia and/or thrombocytopenia, wherein the pH of the composition remains from about 6 to about 7.5 throughout the shelf life.

Yet another objective of the present invention is to provide a stable parenteral composition of cinnamic acid or its isomer or pharmaceutically acceptable salt or derivative thereof wherein post-administration patient does not show significant histopathological changes in kidney.

Yet another objective of the present invention is to provide a stable parenteral composition of cinnamic acid or its isomer or pharmaceutically acceptable salt or derivative thereof wherein the composition shows improved safety profile in context of histopathological changes in liver when compared with G-CSF (Granulocyte Colony Stimulating Factor) treatment.

SUMMARY OF THE INVENTION

The present invention relates to a stable pharmaceutical composition for parenteral administration, comprising cinnamic acid or its isomer or pharmaceutically acceptable salt or derivative thereof and pH adjustment agent in an amount more than 0.4% by weight of the composition.

The present invention provides a stable pharmaceutical composition for parenteral administration, comprising cinnamic acid or its isomer or pharmaceutically acceptable salt or derivative thereof and a pH adjustment agent; wherein the pH of the composition is in the range from about 6.0 to about 7.5.

The present invention provides a method for prophylaxis, treatment and management of neutropenia and thrombocytopenia, the method comprising administering to the patient a stable pharmaceutical composition for parenteral administration, comprising cinnamic acid or its isomer or pharmaceutically acceptable salt or derivative thereof and a pH adjustment agent; wherein the pH of the composition is in the range from about 6.0 to about 7.5.

The present invention provides a stable pharmaceutical composition for parenteral administration, comprising cinnamic acid or its isomer or pharmaceutically acceptable salt or derivative thereof; wherein the pH of the composition is in the range from about 6.0 to about 7.5 and the composition is buffered.

The present invention further provides a stable parenteral composition comprising cinnamic acid or its isomer or pharmaceutically acceptable salt or derivative thereof wherein parenteral composition has about 0.5mg/ml to about 30mg/ml of cinnamic acid.

The present invention also relates to a stable aqueous parenteral composition of cinnamic acid, comprising
a) cinnamic acid or its isomer or pharmaceutically acceptable salt or derivative thereof;
b) a buffer; and
c) a pH adjustment agent
wherein the pH adjustment agent is present in an amount from about 0.5% to about 5% by weight of the composition and the pH of the composition is in the range from about 6.0 to about 7.5.

The present invention also relates to a stable aqueous intravenous composition of cinnamic acid, comprising
a) cinnamic acid or its isomer or pharmaceutically acceptable salt or derivative thereof;
b) a buffer; and
c) a pH adjustment agent
wherein the pH adjustment agent is present in an amount from about 0.5% to about 5% by weight of the composition and the pH of the composition is in the range from about 6.0 to about 7.5.

The present invention also relates to a stable aqueous subcutaneous injection composition of cinnamic acid, comprising
a) cinnamic acid or its isomer or pharmaceutically acceptable salt or derivative thereof;
b) a buffer; and
c) a pH adjustment agent
wherein the pH adjustment agent is present in amount from about 0.5% to about 5% by weight of the composition and the pH of the composition is in the range from about 6.0 to about 7.5.

According to another aspect of the invention there is provided a stable pharmaceutical composition for parenteral administration comprising
a) Trans-cinnamic acid or a pharmaceutically acceptable salt or a derivative thereof;
b) About 0.2M to about 1M phosphate buffer; and
c) About 0.5 to about 5% of sodium carbonate.

The present invention further provides a stable parenteral composition of cinnamic acid or its isomer or pharmaceutically acceptable salt or derivative thereof wherein the composition shows no histopathological changes in the kidney.

The present invention further provides a stable parenteral composition of cinnamic acid or its isomer or pharmaceutically acceptable salt or derivative thereof wherein the composition shows an improved safety profile in the context of histopathological changes in the liver when compared with G-CSF (Granulocyte Colony Stimulating Factor) treatment.

In another aspect, there is provided a method for the prophylaxis, treatment, and management of haematological disorders due to cancer therapy, the method comprising administering to the patient a stable pharmaceutical composition for parenteral administration, comprising cinnamic acid or its isomer, pharmaceutically acceptable salt, or derivative thereof and pH adjustment agent in an amount more than 0.4% by weight of the composition, wherein post-administration patient does not show significant histopathological changes in kidney.

In yet another aspect of the invention, there is provided a method of treating neutropenia in a human subject comprising the step of administering to a human subject exhibiting neutropenia, thrombocytopenia or at risk of developing neutropenia or thrombocytopenia, the method comprising administering to the patient a stable pharmaceutical composition for parenteral administration, comprising cinnamic acid or its isomer, pharmaceutically acceptable salt, or derivative thereof and pH adjustment agent in an amount more than 0.4% by weight of the composition, wherein post-administration patient does not show significant histopathological changes in kidney.

Another aspect of the invention provides a method of treating neutropenia in a human subject comprising the step of administering to a human subject exhibiting neutropenia, thrombocytopenia or at risk of developing neutropenia or thrombocytopenia, the method comprising administering to the patient a stable pharmaceutical composition for parenteral administration, comprising cinnamic acid or isomer, pharmaceutically acceptable salt, or derivative thereof and pH adjustment agent in an amount more than 0.4% by weight of the composition, wherein the composition shows improved safety profile concerning histopathological changes in liver when compared with G-CSF (Granulocyte Colony Stimulating Factor).

Another aspect of the invention provides a stable pharmaceutical composition for intravenous administration, comprising cinnamic acid or its isomer or pharmaceutically acceptable salt or derivative thereof and a pH adjustment agent in an amount more than 0.4% by weight of the composition wherein the composition exhibits Cmax of around 1000 to around 4000 ng/mL when administered in a dose of 10 mg/kg to a Wistar rat.

The pharmaceutical composition for intravenous administration comprising cinnamic acid or its isomer or pharmaceutically acceptable salt or derivative thereof and a pH adjustment agent in an amount more than 0.4% by weight of the composition exhibits AUC of around 1200 to around 3000 ng*h/mL to a Wistar rat.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates a molecular docking of the Granulocyte colony-stimulating factor receptor (2D9Q) binding domain complexed with trans-cinnamic acid and shows a 3D model of the interactions and the 2D interaction patterns and H-bond interaction.
DETAILED DESCRIPTION

The details of one or more embodiments of the invention are outlined in the accompanying description below including specific details of the best mode contemplated by the inventors for carrying out the invention. The embodiments of the invention which are apparent to a person skilled in the art after reading the present disclosure and applying the common general knowledge of the technical field are within the scope of this invention.

Definitions:
The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and this detailed description are exemplary and explanatory only and are not restrictive.

Unless otherwise defined, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

In a principle embodiment, a stable pharmaceutical composition for parenteral administration, comprising cinnamic acid or pharmaceutically acceptable salts or derivatives thereof for the prophylaxis, treatment and management of neutropenia and/or other haematological disorders induced by cancer therapy; and wherein the composition is buffered and is in the range from about 6.0 to about 7.5. The pH of the composition is maintained in the range from about 6.0 to about 7.5, throughout the shelf life of the composition.

The term “pharmaceutically acceptable salts or derivatives” includes salts and esters such as sodium, potassium, hydrocinnamate or propenyl cinnamate, allyl cinnamate, cinnamaldehyde, zinc cinnamate, magnesium cinnamate; Ester derivatives such as ethylhexyl methoxycinnamate (octinoxate), isoamyl p-methoxycinnamate (amiloxiate), octocrylene and cinoxate, indium tetra cinnamic acid phthalocyanine complexes, 2-ethylhexyl and isoamyl esters of p-methoxycinnamic acid, rosmarinic acid, cinnamic acid derivatives containing dithioacetal moieties, 1,2,3-triazolic moiety, oleanolic acid cinnamic acid ester derivatives, glycyrrhetinic acid cinnamic acid ester derivatives, lysine cinnmate, arginine cinnmate, histidine cinnmate and cinnamic acids linked with other amino acids, cinnamic acid linked with vitamins such as ascorbic acid at the COOH group, cinnamic acid glucunorate, cinnamic acid-progenone hybrids, presence of a trimethoxyl group on the phenyl ring of cinnamic acid, an ethylenediamine linker, inserting aryl substitutes at the C-a position and the presence of a,ß-unsaturated ketone carbonyl substituent. Isomers of cinnamic acid include trans-cinnamic acid and cis-cinnamic acid. In a preferred embodiment, Cinnamic acid is trans-cinnamic acid.

The term “pharmaceutical composition” includes solution, suspension, emulsion, and powder.

In an embodiment, the pharmaceutical composition for parenteral administration is in the form of an aqueous solution.

In an embodiment, the pharmaceutical composition for parenteral administration is ready to use injection, infusion, depot injection, concentrated solution for injection or lyophilized powder composition for reconstitution.

In an embodiment, the lyophilized composition for reconstitution is prepared by the process comprising the steps of:
a) Preparing an aqueous solution a pH adjustment agent and a buffer and adjusting the pH of the mixture to 6-7.5;
b) Adding cinnamic acid or its isomer, pharmaceutically acceptable salt or derivative thereof to the above solution;
c) optionally, adding other excipients such as a solubilizer, a solvent, an antioxidant;
d) adjustment the pH of the solution to a pH of about 6 to about 7.5, if required; and
e) freeze drying the above solution and packing the powder obtained in vials under sterile conditions.

In an embodiment, the ready to use parenteral composition is prepared by the process comprising the steps of:
a) Preparing an aqueous solution a pH adjustment agent and a buffer and adjusting the pH of the mixture to 6-7.5;
b) Adding cinnamic acid or its isomer, pharmaceutically acceptable salt or derivative thereof to the above solution;
c) optionally, adding other excipients such as a solubilizer, a solvent, an antioxidant;
d) adjustment the pH of the solution to a pH of about 6 to about 7.5 by adding a pH adjustment agent, if required; and
e) adding the solution to a vial or ampoule, and closing the vial or ampoule and sterilizing the vial or ampoule.

In an embodiment, the ready to use parenteral composition is prepared by the process comprising the steps of:
f) Preparing an aqueous solution of cinnamic acid or its isomer, pharmaceutically acceptable salt or derivative thereof along with a pH adjustment agent and a buffer to obtain a clear solution;
g) optionally, adding other excipients such as a solubilizer, a pH adjustment agent, a solvent, an antioxidant;
h) adjustment the pH of the solution to a pH of about 6 to about 7.5 by adding a pH adjustment agent, if required; and
i) adding the solution to a vial or ampoule, and closing the vial or ampoule and sterilizing the vial or ampoule.

The neutropenia and thrombocytopenia may be associated with diseases such as autoimmune disease, cancer and cancer treatments, infections, and certain medications. During an autoimmune disorder such as rheumatoid arthritis or systemic lupus erythematosus, the patient’s body makes antibodies that kill certain types of blood cells leading to neutropenia and thrombocytopenia.

Leukemia and Hodgkin’s lymphoma are two types of cancer that affect the body’s ability to produce blood cells. Further cancer treatments like chemotherapy and radiation destroy stem cells that develop into neutrophils or platelets. Bacterial and viral infections like hepatitis C, HIV, dengue and sepsis can deplete the body’s stores of neutrophils and platelets.

The term “cancer therapy” includes chemotherapy or radiation therapy and includes the various chemotherapeutic agents used for the treatment of cancer.

In an embodiment, the other haematological disorder includes a decrease in monocytes, WBC count, platelet count and an increase in lymphocytes.

The term “buffer” is well known in the art and refers to a chemical which, on inclusion within a composition, resists a change in pH on the addition of acid or base to the composition. Within a composition, a buffer comprises a weak acid and its conjugate base. A suitable buffer comprises an acid with a pKa value that lies within ±1 of the desired pH of the composition, i.e. about pH 6 to about 7.5. In another embodiment, the composition comprises a buffer selected from the group consisting of acetate, phosphate, citrate, histidine, TRIS, borate, carbonate or a combination thereof. The buffer may be present in a range from about 0.1M to about 10 M of the composition.

In yet another embodiment, the buffering is phosphate buffer having pH 6.8 or pH 7.2. In yet another embodiment, the buffer is about 0.2 M to 1 M sodium phosphate buffer.

In another embodiment, a stable pharmaceutical composition for parenteral administration can be administered route through intravenous, intramuscular, intraperitoneal, or subcutaneous route.

In another embodiment, a stable pharmaceutical composition for parenteral administration wherein parenteral administration is administered through a subcutaneous or intramuscular route and may provide a prolonged or depot effect.

The present invention further provides a stable parenteral composition of cinnamic acid or pharmaceutically acceptable salts or its derivatives thereof comprising cinnamic acid in a dose strength of about 0.2 mg/kg to 15 mg/kg body weight
wherein the composition shows no histopathological changes in the kidney comparable to G-CSF (Granulocyte Colony Stimulating Factor).

The present invention further provides a stable parenteral composition comprising cinnamic acid or pharmaceutically acceptable salts or its derivatives thereof comprising cinnamic acid in a dose strength of about 0.2 mg/kg to 15 mg/kg body weight
wherein the composition shows an improved safety profile in the context of when compared with G-CSF (Granulocyte Colony Stimulating Factor) with respect to histopathological changes in the liver.

In principle embodiment, a stable pharmaceutical composition for parenteral administration, comprising cinnamic acid or pharmaceutically acceptable salts or derivatives thereof comprising cinnamic acid in a dose strength of about 0.2 mg/kg to 15 mg/kg body weight and having pH in the range from about 6.0 to about 7.5;
wherein the said composition reverses the haematological changes induced by cancer therapy and shows an increase in neutrophil
and the composition shows an improved safety profile in the context of when compared with G-CSF (Granulocyte Colony Stimulating Factor) with respect to histopathological changes in the liver.

In principle embodiment, a stable pharmaceutical composition for parenteral administration, comprising cinnamic acid or pharmaceutically acceptable salts or derivatives thereof having pH in the range from about 6.0 to about 7.5;
wherein the said composition reverses the hematological changes induced by cancer therapy thereby shows
a) increase in neutrophil counts
b) increase in monocytes counts
c) increase in white blood cell counts
d) increase in platelet counts
e) decrease in lymphocyte counts and the composition shows improved safety profile in context of histopathological changes in liver when compared with G-CSF (Granulocyte Colony Stimulating Factor) treatment.

In the principle embodiment, the stable pharmaceutical composition for parenteral administration comprising cinnamic acid or pharmaceutically acceptable salts or derivatives thereof for the prophylaxis, treatment and management of neutropenia or thrombocytopenia or other haematological disorders wherein the concentration of cinnamic acid is 0.5 mg/ml to 30 mg/ml, in particular, 2 mg/ml to 25 mg/ml of cinnamic acid.

In the yet embodiment, the stable pharmaceutical composition comprising cinnamic acid or pharmaceutically acceptable salts or derivatives thereof for parenteral administration wherein cinnamic acid is administered at a dose in the range of 0.2 mg/kg body weight to 30 mg/kg body weight.

In the yet embodiment, the stable pharmaceutical composition for parenteral administration comprises trans-cinnamic acid.

In yet another embodiment, a pH adjustment agent is selected from the group consisting of potassium bicarbonate, potassium chloride, potassium citrate, sodium citrate, sodium bicarbonate, sodium carbonate, sodium borate, sodium carbonate, sodium hydroxide, sodium propionate, tromethamine and triethylamine or a combination thereof. The purpose of the pH adjustment agent is to assist the buffer in maintaining a pH throughout the shelf life to keep the cinnamic acid in the solubilized state.

In yet another embodiment, a pH adjustment agent is present in an amount more than 0.4% by weight of the composition, particularly from about 0.04 to 10% of the composition. In a preferred embodiment, a pH adjustment agent is sodium carbonate and is present in an amount from about 0.05 to 5% of the composition.

In the yet embodiment, the composition further comprises one or more pharmaceutically acceptable excipients selected from a solubilizer, a solvent, an antioxidant, and combinations thereof.

In an embodiment, the antioxidant is selected from the group consisting of a-tocopherol acetate, ascorbic acid, citric acid, erythorbic acid, butylated hydroxytoluene (BHT), d-a-tocopherol natural, monothioglycerol, thioglycolic acid, sodium bisulfite, sodium sulfite, sodium metabisulfite, potassium metabisulfite, acetone sodium bisulfite, ascorbyl palmitate, cysteine, d-a-tocopherol synthetic, nordihydroguaiaretic acid, sodium formaldehyde sulfoxylate, sodium thiosulfate, acetylcysteine, ascorbyl palmitate, butylated hydroxyanisole (BHA), cysteine hydrochloride, dithiothreitol, propyl gallate, and thiourea or a combination thereof.

In another embodiment of the invention, the antioxidant is present in the amount of 0.01 to 5% of the composition.

In another embodiment, wherein a solvent is selected from the group consisting of dichloromethane, water, acetonitrile, ethyl acetate, acetone, propylene carbonate, glycerin, coconut fatty acid diethanolamide, medium and/or long chain fatty acids or glycerides, monoglycerides, diglycerides, triglycerides, structured triglycerides, polyethylene glycol, caprylocaproylmacroglycerides, caproyl 90, propylene glycol, oils, propylene glycol esters, polyoxyethylenesorbitan fatty acid esters, polyoxyethylene castor oil derivatives, castor oil, oleic acid, methanol, ethanol, isopropyl alcohol, butanol, polyethylene alcohol, acetone, methyl isobutyl ketone, methyl ethyl ketone, N-methylpyrrolidone, dimethylacetamide, dimethylsulfoxide, and dimethylisosorbide or a combination thereof.

In an embodiment, the composition is stable when subjected to stability conditions of a temperature of 25°C and 60%RH; 30°C and 75%RH; and 40°C and 75%RH for six months.
In another embodiment, the composition is also stable when subjected to stability conditions of a temperature of 2°C to 8°C for at least six months.
In another embodiment, the composition is also stable when subjected to freeze-thaw stability conditions of a temperature of approximately -15°C to -25°C for 24 hours and then stored it at a higher temperature (approximately 25°C) for 24 hours.

Neutropenia and thrombocytopenia can be caused by several factors, including:
Infections: Infections like hepatitis C, HIV, dengue, sepsis, tuberculosis, and Lyme disease can cause neutropenia.
Medications: Some medications, such as antibiotics, antipsychotics, chemotherapy, clozapine, chlorpromazine, ticlopidine, and sulfasalazine, can cause neutropenia.
Radiation therapy: Radiation therapy can cause neutropenia.
Genetic problems: Some genetic problems can cause neutropenia.
Immune system issues: When the immune system attacks neutrophils, it can cause neutropenia. This is known as autoimmune neutropenia.
Bone marrow problems: Cancer and other blood and/or bone marrow disorders, including leukemia and lymphoma, can prevent the body from making enough healthy white blood cells, causing neutropenia.

Evaluation of the parenteral composition of cinnamic acid (CA):
The following studies were conducted for the parenteral composition:
? % Neutrophils on Day 0, 3 and 10
? % Monocyte on Day 0, 3, 10
? %Lymphocytes Day 0, 3, 10
? Total WBC Count (per mm3 of blood) on Day 0, 3, 10
? No of Platelets (per mm3 of blood) on Day 0, 3, 10
? Histopathological studies on Kidney and Liver

EXAMPLES
The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of present disclosure. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the subject matter.

EXAMPLE 1
Formulation:
0.5 g of sodium carbonate was dissolved in 100 ml of 0.2 M and 0.5M sodium phosphate buffer of pH 6.8. The pH of the solution was measured to be pH 7-7.2. To the resultant solution, 2 g of trans-cinnamic acid was added and dissolved completely.
The above compositions were subjected to stability studies based on ICH guidelines. Table 1 provides one month stability data of trans-cinnamic acid prepared in 0.2M and 0.5M sodium phosphate buffer comprising sodium carbonate. Table 1A shows that the pharmaceutical composition for parenteral administration was stable for one month, when subjected to various stability conditions.

Table 1A: Stability data for one month
Parameters Stability period Stability conditions
2-8°C 25°C 60%RH 30°C 75%RH 40°C 75%RH
Trans-cinnamic acid 20mg/mL in 0.2 M buffer with 0.5% sod. carbonate






1 Month

Clear solution Clear solution Clear solution Clear solution
pH 6.20 6.13 6.13 6.15
Trans-cinnamic acid 20mg/mL in 0.5 M buffer with 0.5% sod. carbonate Clear solution Clear solution Clear solution Clear solution
pH 6.35 6.40 6.36 6.32

The above composition comprising Trans-cinnamic acid prepared in 0.5M sodium phosphate buffer comprising 0.5% sodium carbonate was subjected to stability studies. Table 1B provides one, three and six month stability data of the composition. Table 1B shows that the pharmaceutical composition for parenteral administration was stable for six month when subjected to various stability conditions.

Table 1B: Assay of Trans-cinnamic acid composition when subjected to various temperature and humidity conditions
Stability period Stability conditions Trans-cinnamic acid content
mg/ml %Assay
Initial NA 20.31 101.6

1 M 2-8°C 20.22 101.1
40°C 75%RH 20.2 101

3 M 2-8°C 19.96 99.8
25°C 60%RH 19.91 99.6
30°C 75%RH 20.08 100.4
40°C 75%RH 20.04 100.2
6 M 2-8°C 20.05 100.30
25°C 60%RH 20.00 100.00
30°C 75%RH 20.11 100.60
40°C 75%RH 20.03 100.20

The above composition were also subjected to freeze-thaw stability studies, wherein the composition was exposed to exposing the product to freezing temperatures (A cycle of -5°C, 20°C, 40°C of 24 hours each which goes to 21 days).

Table 2: Freeze-thaw stability data
Visual observation pH % solubility by HPLC
Trans-cinnamic acid 20mg/mL 0.2M sodium phosphate buffer with 0.5% Sod. carbonate Clear solution 6.22 100%
Trans-cinnamic acid 20mg/mL 0.5M sodium phosphate buffer with 0.5% Sod. carbonate Clear solution 6.37 100%
Trans-cinnamic acid 20mg/mL in Methanol Clear solution - 100%

The above data indicates that Trans-cinnamic acid composition is not affected by temperature variation even in a freeze-thaw study.

It is important for the parenteral composition to remain soluble throughout the shelf life as well as during dilution with suitable diluting media.

Stock solution (20 mg/mL) Visual observation Visual observation after dilution
20mg/mL in Methanol Complete soluble Precipitation
20mg/mL in 0.25% sodium carbonate Partly soluble Insoluble
20mg/mL in 1% sodium carbonate Complete soluble Clear solution
20mg/mL in Water Partly soluble Insoluble

Example 2
The effect of parenteral Trans-cinnamic acid on Cyclophosphamide induced neutropenia and other haematological, and histopathological changes
Methods:
Female Swiss Albino mice were randomized into 4 groups of 6 animals each based on body weight as follows: Group 1-vehicle control group, Group 2-cyclophosphamide control, Group 3-GCSF 2 µg per animal through subcutaneous route and Group 4 trans-cinnamic acid composition of Example 1 with 0.5M Sodium Phosphate Buffer and 0.5% sodium carbonate (0.6 mg/mice of 25 g body weight) treated groups through intravenous (i.v.) route. On day 0, blood was withdrawn from all animals for basal haematological parameters. All the groups of animals except the vehicle control group were injected with 150 mg/kg of cyclophosphamide through the intraperitoneal route (i.p.), while group 1 was injected with water for injection, i.p. On Day 3, blood was withdrawn from all animals for Total and differential count (neutrophils, lymphocytes, and monocytes). The animals of groups 2 to 4 were dosed with one additional dose of 100 mg/kg CPH on Day 4. From day 5, group 4 were administered with trans-cinnamic acid by i.v. and group 3 was administered with GCSF2 µg per animal through S.C. route. The cyclophosphamide control group were injected with water for injection from day 5 to day 9. On day 10, blood will be withdrawn from all animals for total and differential count (neutrophils, Lymphocytes and monocytes) and platelets.

Results:
Intravenous administration of trans-cinnamic acid in mice exhibited significant improvement in total and differential count as evident by the increased neutrophils, monocytes and also platelets on day 10 as shown Table 3. The lesions observed in the histopathology of liver were minimal upon treatment with trans-cinnamic acid when compared to the Cyclophosphamide control, in which there were mild to moderate lesions as shown in Table 4. Further there were no abnormality detected in the histopathology of kidneys upon treatment with trans-cinnamic acid when compared to the Cyclophosphamide control animals. There were mild changes in the vasculature, cellular swelling and degenerative changes in the renal tubules in cyclophosphamide control animals.

Table 3: Effect of Trans-Cinnamic acid on hematological parameters in Cyclophosphamide induced Neutropenia
Parameters Day Vehicle Control CP Control G-CSF (2 µg) Trans-cinnamic acid
% Neutrophils 0 62.33±4.06 62.33 ± 0.67 64.83 ± 2.12 64.33 ±0.76
3 61.33±2.31 11.00 ±0.97 9.83 ±0.75 10.33 ±0.42
10 61.83 ± 1.87 13.00 ±0.97 58.33 ± 3.90 57.83 ±1.28
% Monocytes 0 7.00±0.45 6.00 ±0.63 6.10 ±0.70 5.50 ±0.76
3 7.17 ±0.31 2.00±0.37 2.33 ±0.33 2.00 ±0.37
10 7.00 ±0.37 2.50 ± 0.26 4.67 ±0.21 4.50 ±0.22
% Lymphocytes 0 29.50±4.06 30.50±1.41 27.67±2.33 29.17±1.11
3 30.33±2.47 85.83±0.91 86.50±0.85 86.67±1.09
10 30.00±2.00 83.83±0.60 35.67±3.60 36.67±1.43
Total WBC Count (per mm3 of blood) 0 7355 ±594.40 7806 ±616.6 6581.67± 1279.0 8558.33±136.20
3 7996.67 ± 663.70 1921.67 ±166.60 1983.33 ±171.70 1978.33±110.10
10 7961.67 ±581.00 3686.67 ± 184.90 7395 ±316.70 7291.67 ±224.60
No of Platelets (per mm3 of blood) 0 935833.33 ±
74890.00 1199000 ±
55910.00 920500 ±
37570.00 909333.33 ±54680.00
3 938216.67 ±
75220.00 638166.67 ±
9282.00 615333.33 ± 21560.00 655666.67 ±
28430.00
10 981500.0 ±
82310.00 638833.0 ±
27720.00 985500.0 ±
17170.00 937833.33 ±
18340.00

Table 4: Histopathological observations of Liver in Cyclophosphamide induced neutropenia
Group Histopathological Observations of LIVER Overall Pathological grade
/ lesion score
Vascular changes-
Congestion /Hemorrhages in hepatic parenchyma Cellular changes /degenerative and granular cytoplasmic changes and vacuolar changes Cellular swelling of hepatocytes with karyomegaly Loss of nuclei and necrotic changes of hepatocytes Inflammatory changes in hepatic tissue, MNC infiltration in the hepatic parenchyma
1 VC NAD NAD NAD NAD NAD NAD
2 VC NAD NAD NAD NAD NAD NAD
3 CP-C +1 +2 +2 +1 NAD Mild (+2) to Moderate (+3)
4 CP-C +2 +2 +2 +1 +1 Mild (+2)
5 G-CSF Focal (+1) Focal (+1) Focal (+1) NAD NAD Minimal (+2)
6 G-CSF Focal (+1) Focal (+1) Focal (+1) NAD NAD Minimal (+2)
7 CA Focal (+1) NAD Focal (+1) NAD Focal (+1) Minimal (+1)
8 CA NAD Focal (+1) Focal (+1) NAD Focal (+1) Minimal (+1)
Note: Overall Grade score as- NAD = No Abnormality Detected, Minimal changes (+1), Mild changes (+2), Moderate changes (+3), Severe changes (+4).

Table 5: Histopathological observations of Kidney in Cyclophosphamide induced neutropenia
Group Histopathological Observations of Kidney Overall Pathological grade
/ lesion score
Vascular changes-
Congestion / Hemorrhages in Renal tissue Cellular swelling, degenerative changes and necrotic changes of Renal Tubules Glomerular pathological changes (atrophy, hypertrophy) Presence of tubular cast and degenerative changes of renal tubules Inflammatory changes in Renal tissue (cortex and medulla)
1 VC NAD NAD NAD NAD NAD NAD
2 VC NAD NAD NAD NAD NAD NAD
3 CP-C +1 Focal (+1) NAD Focal (+1) NAD Minimal (+1)
4 CP-C Focal (+1) Focal (+1) NAD Focal (+1) NAD Minimal (+1)
5 G-CSF Focal (+1) NAD NAD NAD NAD NAD
6 G-CSF NAD NAD NAD NAD NAD NAD
7 CA Focal (+1) NAD NAD NAD NAD NAD
8 CA NAD NAD NAD NAD NAD NAD
Note: Overall Grade score as- NAD =No Abnormality Detected, Minimal changes (+1), Mild changes (+2), Moderate changes (+3), Severe changes (+4).

The study led to the conclusion that intravenous administration of trans-cinnamic acid in mice exhibited significant improvement in total and differential count of blood cells as evident by the increased neutrophils, monocytes and also platelets on day 10. There lesions observed in the histopathology of liver were minimal upon treatment with trans-cinnamic acid when compared to the Cyclophosphamide control, in which there were mild to moderate. Further there were no abnormality detected in the histopathology of kidneys upon treatment with trans-cinnamic acid when compared to the Cyclophosphamide control, in which there were minimal changes in the vasculature, cellular swelling and degenerative changes in the renal tubules.

Example 3: Effect of different concentration of trans-cinnamic acid on Cyclophosphamide induced neutropenia
Methods:
Female Swiss Albino mice were randomized into 7 groups of 6 animals each based on body weight as follows: Group 1 - vehicle control group, Group 2 - cyclophosphamide control, Group 3 - GCSF 2 µg per animal through s.c. route, Group 4 - 6 mg/ml of trans-cinnamic acid; each animal given 50 mcl of this solution to give 0.3 mg trans-cinnamic acid/mice), Group 5 - 12 mg/ml of trans-cinnamic acid; each animal given 50 mcl of this solution to give 0.6 mg trans-cinnamic acid /mice) and Group 6 - 20 mg/ml of trans-cinnamic acid; each animal given 50 mcl of this solution to give 1.0 mg trans-cinnamic acid /mice. On day 0, blood was withdrawn from all animals for basal hematological parameters. All the groups of animals except vehicle control group were injected with 150 mg/kg of cyclophosphamide through intraperitoneal route (i.p.), while group 1 was injected with water for injection, i.p. On Day 3, blood was withdrawn from all animals for Total and differential count (neutrophils, lymphocytes, and monocytes). The animals of group 2 to 7 were dosed with one additional dose of 100 mg/kg CPH on Day 4. From day 5, group 4, 5 and group 6 animals were administered with 6 mg/ml, 12 mg/ml and 20 mg/ml of Cinnamic acid, respectively as 50 µl per animal intravenously for 5 consecutive days. The cyclophosphamide control group was injected with water for injection from day 5 to day 9. On day 10, blood was withdrawn from all animals for total and differential count (neutrophils, and monocytes) and platelet count.

Result
The lowest dose of 0.1mg trans-cinnamic acid /mice of 25 g showed only marginal increase in neutrophil counts, while treatment with all the other concentrations (0.3mg/mice, 0.6 mg/mice and 1 mg/mice) when given intravenously and caused statistical significant alleviation in cyclophosphamide induced decrease in % neutrophil, % monocytes, total WBC count, while significantly increased % lymphocyte count, as shown in Table 6.

Table 6: Effect of different concentrations on hematological parameters in Cyclophosphamide induced Neutropenia
Parameters Day Vehicle Control CP Control G-CSF
(2 µg) t-CA 6 mg/ml t-CA 12 mg/ml t-CA 20 mg/ml
% Neutrophils 0 39.50 ±
1.23 43.00 ±
1.57 40.50±
0.96 38.17±
1.72 40.67±
1.09 40.33±
1.09
3 42.00 ±
0.52 7.67 ±
0.49 7.17±
0.95 6.67±
0.49 7.00±
0.68 6.17±
0.79
10 43.00 ±
1.18 9.67 ±
0.42 37.17±
0.48 27.6±
1.03 33.17±
0.83 36.00±
0.45
% Monocytes 0 5.17 ±
0.48 6.67 ±
0.21 6.50±
0.81 6.50±
0.50 6.83±
0.54 6.00±
0.58
3 9.33 ±
0.42 3.17 ±
0.40 2.00±
0.37 2.17±
0.48 2.17±
0.31 2.83±
0.31
10 10.00 ±
0.37 2.33 ±
0.21 5.50±
0.34 4.20±
0.20 5.17±
0.31 5.50±
0.34
Total WBC Count (per mm3 of blood) 0 7327 ±
392.90 7310 ±
574.00 7095±
553.30 7601±
384.80 7493±
546.20 7135±
432.40
3 7308 ±
340.00 2061 ±
181.00 2457±
190.00 2210±
161.20 2318±
265.90 2204±
175.10
10 7158 ±
369.70 2218 ±
199.20 5870±
181.70 3701±
283.8 4489±
506.00 5287±
172.30
No of Platelets (per mm3 of blood) 0 750100±
11160 743900 ±
10140 752100±
8788 759300±
9647 736600±
5727 772400±
23720
3 762500±
27850 625600 ±
39160 645600±
30490 624000±
33590 630500±
25790 636900±
12560
10 763400 ±
28000 607900 ±
27660 672800±
39380 625900±
30430 660800±
22400 683000±
32710
% Lymphocytes 0 46.83 ±
0.98 42.33 ±
1.17 45.67±
0.95 47.17±
1.80 44.83±
1.66 46.67±
1.09
3 41.67 ±
0.61 81.67 ±
1.45 84.67±
0.67 84.17±
0.40 83.33±
1.20 85.17±
0.95
10 40.00 ±
1.03 80.50 ±
0.76 51.17±
1.08 61.20±
1.66 54.17±
0.98 52.67±
0.99
t-CA - trans-cinnamic acid
Example 4: Effect of Cinnamic Acid and its salts as well as derivatives on haematological parameters in Cyclophosphamide induced Neutropenia through subcutaneous route
Methods:
Female Swiss Albino mice were randomized into 8 groups of 5 animals each based on body weight as follows: Group 1-vehicle control group, Group 2-cyclophosphamide control, Group 3-GCSF 2 µg per animal through s.c. route, Group 4 trans-cinnamic acid (0.1 mg/mice of 25 g) treated, Group 5 sodium salt of cinnamic acid- (called sodium cinnamate) treated, Group 6 potassium salt of cinnamic acid (termed potassium cinnamate), Group 7 hydrocinnamate (also called as 3 phenyl-propionic acid; benzyl acetic acid), and Group 8 D-allyl cinnamate (also called Propenyl cinnamate) treated groups. On day 0, blood was withdrawn from all animals for basal hematological parameters. On day 1, all the groups of animals except vehicle control group were injected with 150 mg/kg of cyclophosphamide through intraperitoneal route (i.p.), while group 1 was injected with water for injection, i.p. On Day 3, blood was withdrawn from all animals for Total and differential count (neutrophils, lymphocytes, and monocytes). The animals of group 2 to 8 were dosed with one additional dose of 100 mg/kg CPH on Day 4. From day 5, group 4, 5, 6, 7, 8 animals were administered with trans-cinnamic acid, sodium cinnamate, potassium cinnamate, hydrocinnamate, and D-allyl cinnamate, respectively as 50 µl per animal (equivalent to 0.1 mg cinnamic acid and their derivatives) subcutaneously for 5 consecutive days. The cyclophosphamide control group was injected with water for injection from day 5 to day 9, subcutaneously. On day 10, blood was withdrawn from all animals for total and differential count (neutrophils, and monocytes) and platelet count.
Result:
Treatment with trans-cinnamic acid and its sodium, potassium salts as well as tested derivatives caused alleviation in cyclophosphamide induced decrease in number of neutrophil, number of monocytes and total WBC count, as shown in Table 7.

Table 7: Effect of Trans-Cinnamic Acid and its salts as well as derivatives on hematological parameters in Cyclophosphamide induced Neutropenia through subcutaneous route
Parameters
Day Vehicle Control CP Control G-CSF
(2 µg) Trans-Cinnamic Acid in Sodium Carbonate buffer Sodium Cinnamate in Phosphate buffer Potassium Cinnamate in Phosphate buffer Hydrocinnamate in sodium phosphate buffer Allyl cinnamate in Ethanol
Number of Neutrophils (per mm3 of blood) 0 1612 ±
48.78 1605 ±
97.16 1639 ±
101.40 1668 ±
60.12 1617 ±
57.52 1653 ±
77.08 1626 ±
45.62 1682 ±
26.86
3 1687.00 ±
48.08 498.80 ±
21.80 504.60 ±
48.18 495.20 ±
26.30 490.20 ±
20.30 502.40 ±
25.46 486±
32.86 501.20 ±
18.04
10 1730 ±
37.98 436.2 ±
21.88 1570 ±
53.26 1494 ±
61.00 1053 ±
12.76 721.8 ±
44.18 1157 ±
39.92 1288 ±
40.74
Number of Monocytes (per mm3 of blood) 0 214 ±
6.473 247.8 ±
16.76 231.2 ±
30.18 215.8 ±
22.52 248.6 ±
35.50 267 ±
23.36 221.2 ±
27.10 222 ±
7.94
3 213.6 ±
5.90 109 ±
3.50 104 ±
3.58 104.6 ±
1.08 103 ±
7.86 106 ±
0.88 105.8 ±
1.06 104.4 ±
1.86
10 217.8 ±
7.70 98.60±
2.58 205.8±
2.82 191.6±
9.30 164 ±
5.98 148.8±
7.60 174.2±
3.38 184.8±
0.58
Total WBC Count (per mm3 of blood) 0 7036 ±
581.70 7860 ±
1081.00 7880±
565.00 7778±
352.30 8630 ±
338.80 7780 ±
242.70 7594 ±
742.50 7650±
243.90
3 7121 ±
572.20 2225 ±
103.70 2229 ±
41.98 2241±
128.90 2249±
52.42 2274 ±
156.70 2279 ±
96.50 2300 ±
117.80
10 7205 ±
566.10 1906 ±
129.70 6005±
218.90 5548±
139.50 4403±
41.18 3656 ±
104.30 4624 ±
26.74 4825 ±
63.60
No of Platelets x 103 (per mm3 of blood) 0 943.6 ±
54.32 956±
44.48 900.4 ±
92.24 929.8 ±
68.06 975.4 ±
77.40 953.8 ±
106.40 929.6 ±
73.72 936.2 ±
18.22
3 864 ±
69.60 631.4 ±
47.74 641 ±
7.82 627.9 ±
28.30 634.1 ±
40.84 637.9 ±
30.58 642.4 ±
12.12 634.4 ±
24.78
10 857.8 ±
51.42 648.8 ±
13.68 774 ±
10.80 755.8 ±
7.60 728.9 ±
3.18 718±
3.86 727.3 ±
3.10 746.2 ±
2.94
Number of Lymphocytes (per mm3 of blood) 0 1729 ±
95.22 1723 ±
22.62 1738 ±
63.86 1708 ±
14.36 1766 ±
30.74 1713 ±
57.08 1751 ±
62.10 1717 ±
43.50
3 1753 ±
92.39 992.4±
43.08 972.8±
28.02 947.0±
40.40 960.4±
21.72 966.8±
15.88 957.8±
15.96 968.6 ±
34.52
10 1828 ±
85.08 812.4 ±
18.30 1194 ±
57.82 1097 ±
18.12 1036 ±
45.04 966.6 ±
48.20 1047 ±
39.16 1074 ±
48.80

All the experiments show that the parenteral composition of trans-cinnamic acid, its salts and its derivatives are stable under various stability condition as well effective through various route of administration and even at a concentration of 0.1 mg/25 g of mice, the therapeutic efficacy was evident when the drug was administered subcutaneously. Thereby, the effective dose strength for trans-cinnamic acid is 4 mg/kg for mice. Based on the dose conversion based on the species table provided in Nair et. al. (A simple practice guide for dose conversion between animals and human, J Basic Clin Pharm. 2016 Mar; 7(2):27-31), the corresponding effective dose for human being would be 0.33 mg/kg.

EXAMPLE 5

Effect of route of administration on the neutropenic effect of Trans-Cinnamic Acid
Female Swiss Albino mice were randomized into 5 groups of 5 animals each based on body weight as follows: Group 1-vehicle control group, Group 2-cyclophosphamide control, Group 3-trans-Cinnamic Acid 0.1 mg/mice of 25 g through s.c. route, Group 4 trans-cinnamic acid (0.1 mg/mice of 25 g) through oral route, Group 5 trans-cinnamic acid (0.1 mg/mice of 25 g) through intravenous route treated groups. On day 0, blood was withdrawn from all animals for basal haematological parameters. On day 1, all the groups of animals except the vehicle control group were injected with 150 mg/kg of cyclophosphamide through the intraperitoneal route (i.p.), while group 1 was injected with water for injection, i.p. On Day 3, blood was withdrawn from all animals for Total and differential count (neutrophils, lymphocytes, and monocytes). The animals of groups 2 to 8 were dosed with one additional dose of 100 mg/kg CPH on Day 4. From day 5, groups 4, 5, 6, 7, and 8 animals were administered with trans-cinnamic acid through different routes as mentioned above (0.1 mg cinnamic acid) subcutaneously for 5 consecutive days. The cyclophosphamide control group was injected with water for injection from day 5 to day 9, subcutaneously. On day 10, blood was withdrawn from all animals for neutrophil counts/ml.
Result:
It was found that the subcutaneous route of administration provided the maximum therapeutic effect, followed by the intravenous and then oral route as given in Table 8.

Table 8: Effect of Trans-Cinnamic Acid on neutrophils count when administered through different routes of administration
Parameters
Day Vehicle Control CP Control Trans-Cinnamic Acid through s.c. route Trans-Cinnamic Acid through oral route Trans-Cinnamic Acid through i.v. route
Number of Neutrophils (per ml of blood) 10 1745.50
442.00 1312.50 730.16 955.50
PHARMACOKINETIC STUDY IN WISTAR RATS

To compare the pharmacokinetic profile of the trans-cinnamic acid after single dose intravenous administration in Wistar rats. The trans-cinnamic acid was administered as a single dose to 9 male and 9 female rats. For intravenous administration, the test item was dissolved in 0.5% sodium phosphate buffer with 0.5% sodium bicarbonate.
Blood samples were collected on day 1 of dosing at 15 minutes, 30 minutes, 1, 2, 4, 6, 8, 12, 24 and 36 h from rats. Blood samples were collected in pre-labeled microcentrifuge tubes containing K2EDTA. After completion of the last (36 h) blood collection, rats were euthanised by carbon dioxide asphyxiation.
The plasma samples were analysed to determine the concentration of the test item. The pharmacokinetics (PK) analysis of the plasma concentration vs time was performed using the non-compartmental model of the WinNonlin® software (version 8.4.0).
Results:
No mortality, morbidity or abnormal clinical signs were observed throughout the experiment.
The plasma concentration of trans-cinnamic acid was detectable up to 36 hours post-dosing.
However, the concentration was found below the limit of quantification at 8 h post-dose in many rats. Hence, it indicates that the Cinnamic acid was eliminated by 8 h post-dose. Concentration observed at 12, 24, and 36h post-dose was due to the endogenous Cinnamic acid levels in food, which the animals consumed during night-time.

Table 9A: Mean plasma concentration of Trans-Cinnamic Acid after single dose IV administration
Mean Concentration (ng/ml)
0.25h 0.5h 1h 2h 4h 6h 8h 12h 24h 36h
Dose

10 mg/kg 1137.92 296.21 154.42 130.10 39.86 63.40 80.36 49.98 112.64 124.52

Table 9B: Mean plasma concentration of Trans-Cinnamic Acid after single dose IV administration
Mean Concentration (ng/ml)
Dose
(mg/kg body weight) Cmax (ng/mL) Tmax (h) AUC
(ng*h/mL) T1/2
(h) Vd
(L/kg) Cl
(L/h/kg) Bioavailability
Male Rats 10 1137.92 0.25 1501.23 2.79 22.07 5.48 100
Female rats 10 3408.99 0.25 3476.04 3.38 13.27 2.72 100
Cmax - mean maximum plasma levels, AUC - area under the plasma concentration curve; Tmax - time to peak plasma concentration, Vd – volume of distribution

EXAMPLE 6: RECEPTOR BINDING STUDY
Protein name: Granulocyte colony-stimulating factor receptor, Organisms(s): Homo sapiens, Sequence Length= 313, Resolution: 2.80ÅUn iprot ID -Q99062, Gene Names:CSF3R, GCSFR.
Grid data info:
Grid Point Spacing =0.375Angstroms,
Even Number of User-specified Grid Points = 36x-points; 30y-points; 42z-points
Coordinates of Central Grid Point of Maps = (-70.340, 29.359, 28.313)
Minimum coordinates in grid = (-77.090, 23.734, 20.438)
Maximum coordinates in grid = (-63.590, 34.984, 36.188)
Ligands used:
1) Ligand name: Trans-cinnamic acid, Molecular Formula: C9H8O2, Molecular Weight: 148.16, PubChem CID: 444539

Table 10: Ligand interactions observed between G_CSF and trans-cinnamic acid
Proteins Name
Ligand Name Binding Energy (kcal/mol) No. of H Bonds Interacting residue Final Intermolecular Energy (kcal/mol) vdW + Hbond + desolv Energy (kcal/mol) Electrostatic
Energy (kcal/mol) Torsional Free Energy (kcal/mol)
2d9q
Granulocyte colony-stimulating factor receptor Trans-Cinnamic acid -4.01 02
H1:2.08Å
H2:2.61Å TYR:172(H1),
ARG:287(H2) -4.91 -3.83 -1.08 +0.89

G-CSF Receptor and ligand interaction details
Granulocyte colony-stimulating factor (G-CSF or GCSF), also known as colony-stimulating factor 3 (CSF 3), is a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream. Functionally, it is a cytokine and hormone, a type of colony-stimulating factor, and is produced by a number of different tissues. The pharmaceutical analogs of naturally occurring G-CSF are called filgrastim and lenograstim. G-CSF also stimulates the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils.

Selection of Ligands:
A few of the ligands were got from the PubChem drug databases and docked against Granulocyte colony-stimulating factor receptor (2D9Q) with high AutoDock V4.2 software. The ligand names are Cinnamic acid (-4.01kcal/mol). For the selection of ligands in the current examination, the existing ligands for the mentioned proteins were chosen from a series of literature review. The existing quantities of ligands are 01 which are chosen to promote perceptions.

Ligand Preparation-
The 3-D structure of inhibitors with their respective PubChem CID were redeemed and saved in SDF format. Furthermore, ligand preparations were continued by taking the 3-D structure of all the ligands and were introduced in Pymol software for conversion of 3-D structure from SDF to PDB format. Using Pymol software metals were also removed from the ligands structure for an appropriate docking study. The prepared ligands were saved in PDB format for further docking studies.

Protein preparation-
The crystal structure of Target proteins was retrieved from Protein Data Bank (PDB) with PDB ID2D9Q with chain Band was carried further for more studies of docking process.

Molecular docking
Molecular Docking is an important component of computer-assisted drug discovery. It helps in predicting the intermolecular framework formed between a protein and ligand and outputs the appropriate binding between the molecules. Docking was performed by the AutoDock 4.2.6 program, using the implemented empirical free energy function and the Lamarckian Genetic Algorithm (LGA). The grid maps were calculated using AutoGrid. In all dockings, a grid map with 36 x 30 x 42points and a grid-point spacing of 0.375 Å was applied. The best conformation with the lowest docked energy was chosen from the docking search. The interactions of complex protein-ligand conformations including hydrogen bonds and bond lengths were analyzed using Pymol software, UCSF Chimera and Accelrys Discovery Studio Visualizer software.

Discussion:
The best conformation with the lowest docked energy was chosen from the docking search. On performing the docking of the Granulocyte colony-stimulating factor receptor (2D9Q) with Cinnamic acid (-4.01kcal/mol) it was observed that the binding energy shown by the protein and ligand is good at Cinnamic acid (-4.01kcal/mol). For this ligand, three number of torsions were observed. Hydrogen bond interactions are also calculated and mentioned, presence of H-bonds depicts stable interaction between ligand and protein. Discovery studio 2020 Client and Chimera softwares are used to depict Hydrogen bonds, 2-D images and protein-ligand interactions images for a good visualization of the docking.
Table 11: % inhibition caused by trans-cinnamic acid
S. No. Receptor Test Molecule Concentration % inhibition
1 GCSFR
(Granulocyte colony stimulating factor receptor) Trans-Cinnamic Acid 32 nM 22
65 nM 50
130 nM 90
2. CXCR4
(C-X-C chemokine receptor type 4) Trans-Cinnamic Acid 6.5 mM 28
13 mM 69

Figure 1 provides a 3D model of interaction between Granulocyte colony-stimulating factor receptor (2D9Q) binding domain complexed with Cinnamic acid.

Both GCSF and Trans-cinnamic acid bind to the GCSF receptor showing the mechanism of action for enhancing neutrophils in neutropenia-induced animals is similar between GCSF and trans-cinnamic acid.

Cinnamic acid provides several advantages over G-CSF, which is the standard of care for neutropenia. G-CSF is a recombinant protein, hence can be administered through a parenteral route and is known to have concerns associated with protein such as heat sensitivity, protein aggregation, endotoxin contamination and oxidative degradation. These factors lead to an increased manufacturing cost making this an expensive treatment therapy. On the other hand, Cinnamic acid is a chemical entity, hence not susceptible to oxidative degradation, has no concerns related to endotoxins, heat stable. All these factors lead to a significantly reduced manufacturing cost, thereby providing a reasonable therapy.

,CLAIMS:1. A stable pharmaceutical composition for parenteral administration, comprising cinnamic acid or its isomer, pharmaceutically acceptable salt, or derivative thereof and a pH adjustment agent in an amount more than 0.4% by weight of the composition.
2. The stable pharmaceutical composition according to claim 1 wherein the pH of the composition is in the range from about 6.0 to about 7.5.
3. The stable pharmaceutical composition according to claim 1 wherein the pH adjustment agent is selected from the group consisting of potassium bicarbonate, potassium chloride, potassium citrate, sodium citrate, sodium bicarbonate, sodium carbonate, sodium borate, sodium carbonate, sodium hydroxide, sodium propionate, tromethamine and triethylamine or a combination thereof.
4. The stable pharmaceutical composition according to claim 3 wherein the pH adjustment agent is sodium carbonate.
5. The stable pharmaceutical composition according to claim 1 wherein the pH adjustment agent is present in an amount from about 0.4% to about 10% by weight of the composition.
6. The stable pharmaceutical composition according to claim 5 wherein the pH adjustment agent is present in an amount from about 0.5% to about 5% by weight of the composition.
7. The stable pharmaceutical composition according to claim 1 wherein the concentration of cinnamic acid in the composition is from about 0.5 mg/ml to about 30 mg/ml.
8. The stable pharmaceutical composition according to claim 1 wherein the dose of cinnamic acid is from about 0.2 mg/kg to about 30 mg/kg.
9. The stable pharmaceutical composition according to claim 8 wherein the dose of cinnamic acid is from about 0.2 mg/kg to about 15 mg/kg.
10. The stable pharmaceutical composition according to claim 1 wherein the composition is ready to use injection, infusion, depot injection, concentrated solution for injection or lyophilized powder composition for reconstitution.
11. The stable pharmaceutical composition according to claim 1 wherein the composition is a solution, suspension, emulsion or in a powder form.
12. The stable pharmaceutical composition according to claim 11 wherein the solution is an aqueous solution.
13. The stable pharmaceutical composition according to claim 1 wherein the composition is administered through intravenous, intramuscular, intraperitoneal, or subcutaneous.
14. The stable pharmaceutical composition according to claim 1 wherein isomer of cinnamic acid is trans-cinnamic acid.
15. The stable pharmaceutical composition according to claim 1 wherein the salts are selected from the group consisting of sodium, potassium, magnesium, calcium, zinc, triethylamine.
16. The stable pharmaceutical composition according to claim 1 wherein the composition is buffered.
17. The stable pharmaceutical composition according to claim 16 wherein the composition comprises a buffer.
18. The stable pharmaceutical composition according to claim 17 wherein the buffer is selected from the group consisting of acetate, phosphate, citrate, histidine, TRIS, borate, carbonate, or a combination thereof.
19. The stable pharmaceutical composition according to claim 18 wherein the buffer is a phosphate buffer.
20. The stable pharmaceutical composition according to claim 1 wherein the composition comprises one or more pharmaceutically acceptable excipients selected from a solubilizer, a solvent, an antioxidant, and combinations thereof.
21. A stable aqueous parenteral composition of cinnamic acid, comprising
a) cinnamic acid or isomer, pharmaceutically acceptable salt, or derivative thereof;
b) a buffer; and
c) a pH adjustment agent
wherein the pH adjustment agent is present in an amount from about 0.5% to about 5% by weight of the composition and the pH of the composition is in the range from about 6.0 to about 7.5.
22. The stable composition according to claim 21 wherein the buffer is a phosphate buffer.
23. The stable composition according to claim 21 wherein the pH adjustment agent is sodium carbonate.
24. The stable composition according to claim 21 wherein the composition comprises
a) Trans-cinnamic acid or a pharmaceutically acceptable salt or a derivative thereof;
b) about 0.2M to about 1.0M phosphate buffer; and
c) about 0.5 to about 5% of sodium carbonate.
25. A method for the prophylaxis, treatment, and management of neutropenia and/or thrombocytopenia, the method comprising administering to the patient a stable pharmaceutical composition for parenteral administration, comprising cinnamic acid or isomer, pharmaceutically acceptable salt, or derivative thereof and pH adjustment agent in an amount more than 0.4% by weight of the composition, wherein post-administration patient does not show significant histopathological changes in kidney.
26. The method according to claim 25 wherein post-administration, composition increases the neutrophil counts.
27. The method according to claim 25 wherein post-administration, composition increases the monocytes counts.
28. The method according to claim 25 wherein post-administration, composition increases the white blood cell counts.
29. The method according to claim 25 wherein post-administration, composition increases the platelet counts.
30. The method according to claim 25 wherein post-administration, composition decreases the lymphocyte counts.
31. The method according to claim 25 wherein the concentration of cinnamic acid in the composition is from about 1 mg/ml to about 30 mg/ml.
32. The method according to claim 25 wherein the dose of cinnamic acid in the composition is from about 0.2 mg/kg to about 15 mg/kg.
33. The method according to claim 25 wherein the neutropenia or thrombocytopenia is associated with diseases selected from the group consisting of autoimmune disease, cancer and cancer treatments, and infections.
34. The method according to claim 25 wherein the neutropenia or thrombocytopenia is due to certain medication.
35. The method according to claim 33 wherein the neutropenia or thrombocytopenia is due to cancer and cancer treatment.
36. A method of treating neutropenia and/or thrombocytopenia in a human subject comprising the step of administering to a human subject exhibiting neutropenia and/or thrombocytopenia or at risk of developing neutropenia and/or thrombocytopenia, the method comprising administering to the patient a stable pharmaceutical composition for parenteral administration, comprising cinnamic acid or isomer, pharmaceutically acceptable salt, or derivative thereof and pH adjustment agent in an amount more than 0.4% by weight of the composition, wherein post-administration patient does not show significant histopathological changes in kidney.
37. A method of treating neutropenia and/or thrombocytopenia in a human subject comprising the step of administering to a human subject exhibiting neutropenia and/or thrombocytopenia or at risk of developing neutropenia and/or thrombocytopenia, the method comprising administering to the patient a stable pharmaceutical composition for parenteral administration, comprising cinnamic acid or isomer, pharmaceutically acceptable salt, or derivative thereof and pH adjustment agent in an amount more than 0.4% by weight of the composition, wherein the composition shows improved safety profile with respect to histopathological changes in liver when compared with G-CSF (Granulocyte Colony Stimulating Factor).
38. A stable pharmaceutical composition for intravenous administration, comprising cinnamic acid or isomer or a pharmaceutically acceptable salt or derivative thereof and a pH adjustment agent in an amount more than 0.4% by weight of the composition
wherein the composition exhibits Cmax of around 1000 to around 4000 ng/mL when administered in a dose of 10 mg/kg to a Wistar rat.
39. The stable pharmaceutical composition for intravenous administration according to claim 38 wherein the composition exhibits AUC of around 1200 to around 3000 ng*h/mL to a Wistar rat.