WO2016181214A1

WO2016181214A1 - Compositions and methods for the treatment of ebola virus disease

Info

Publication number: WO2016181214A1
Application number: PCT/IB2016/000655
Authority: WO
Inventors: Erick GBODOSSOU
Original assignee: Gbodossou Erick
Priority date: 2015-05-13
Filing date: 2016-05-13
Publication date: 2016-11-17

Abstract

Methods and compositions for the treatment of Ebola virus disease (EVD). In particular, pharmaceutical compositions prepared from Momordica balsamina, Aframomum melegueta and Cyperus articulates and methods of treating EVD comprising administering one or more of the pharmaceutical compositions to a human subject suffering from EVD.

Description

TITLE OF TH E INVENTION

[0001] Compositions and Methods for the Treatment of Ebola Virus Disease

REFERENCE TO RELATED APPLICATION

[0002] Priority is claimed to U.S. Provisional Patent Application No. 62/160,735 filed on May 1 3, 2015. The content of this provisional application is hereby incorporated by reference in its entirety.

BACKGROUND OF TH E INVENTION

[0003] The invention relates generally to methods and compositions for the treatment of Ebola virus disease (EVD). Compositions in accordance with the instant invention are prepared from Momordica balsamina, Aframomum melegueta and Cyperus articulatus and compositions or pharmaceutical formulations of the compositions are administered to patients suffering from EVD.

[0004] EVD, formerly known as Ebola hemorrhagic fever, is a severe and often fatal illness in humans. The average fatality rate is around 50%, though fatality rates have been as high as 90% in past outbreaks.

[0005] Initial signs and symptoms of EVD infection may include elevated body temperature or subjective fever, chills, myalgias, and malaise. However, because of the nature of these symptoms, EVD often can be confused with other more common infectious diseases such as malaria, typhoid fever, meningococcemia, and other bacterial infections.

[0006] Within a week, those infected with EVD can develop other conditions, such as severe diarrhea, nausea, vomiting, and abdominal pain. Other symptoms, such as chest pain, shortness of breath, headache, or confusion may also be observed. The most severe cases usually result in patient mortality, with death typically occurring between one to two weeks after infection from complications including multi-organ failure and septic shock. In non-fatal cases, patients may have fever for several days and improve. However, long-term complications, such as joint and vision problems, may await those that survive.

[0007] While the first EVD outbreaks occurred in remote villages in Central Africa, the most recent outbreak in West Africa has involved major urban areas with tens of thousands of cases. However, treatment of patients with EVD is limited because there are yet no approved vaccines or other medicines! In view of the severity of this disease and the lack of any treatment, new therapies are desperately needed.

BRIEF SUMMARY OF THE INVENTION

[0008] Disclosed herein are compositions - in particular pharmaceutical compositions - and methods for the treatment of EVD. Pharmaceutical compositions comprise an extract prepared from one or more plants selected from the group consisting of Momordica balsamina, Aframomum meleg et, and Cyperus articulates. Pharmaceutical compositions further comprise one or more substances selected from the group consisting of a pharmaceutical ly-acceptable carrier, an excipient, a stabilizer, a solubilizer, and a preservative. Pharmaceutical compositions may be formulated for oral, parenteral or topical administration. Pharmaceutical compositions may also be formulated for sustained release. Formulations may be in the form of, e.g., a capsule, pill, or liquid suspension.

[0009] Method of treating EVD comprise administering a therapeutically effective amount of a pharmaceutical composition in accordance with the invention to a subject. In some instances, a preferred dosage may be from about 0.075 mg/kg/day to about 0.45 mg/kg/day.

BRIEF SUMMARY OF THE SEVERAL VI EWS OF THE DRAWINGS

[0010] Figure 1 shows the dose response curve of the control solution in HeLa cells.

[0011 ] Figure 2 shows the dose response curve of Extract A in HeLa (top) and HFF- I (bottom) cells. Concentration is shown in units of g/mL, not as molarity as indicated below the X-axis of the figure.

[0012] Figure 3 shows the dose response curve of Extract B in HeLa (top) and HFF- l (bottom) cells. Concentration is shown in units of g/mL, not as molarity as indicated below the X-axis of the figure.

[0013] Figure 4 shows the dose response curve of Extract C in HeLa (top) and HFF- I (bottom) cells. Concentration is shown in units of g/mL, not as molarity as indicated below the X-axis of the figure.

[0014] Figure 5 shows the dose response curve of Extract D in HeLa (top) and HFF- l (bottom) cel ls. Concentration is shown in units of g/mL, not as molarity as indicated below the X-axis of the figure. [0015] Figure 6 shows the, dose response curve of Extract E in HeLa (top) and HFF- 1 (bottom) cells. Concentration is shown in units of g/mL, not as molarity as indicated below the X-axis of the figure.

[0016] Figure 7 shows the dose response curve of Extract F in HeLa (top) and HFF- 1 (bottom) cells. Concentration is shown in units of g/mL, not as molarity as indicated below the X-axis of the figure.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention encompasses compositions and methods for the treatment of EVD. Compositions in accordance with the instant invention comprise extracts derived from a leaf or leaves of one or more of Momordica balsamina, Aframomum melegueta and Cyperus articulatus.

[0018] Extracts of the foregoing plants may be prepared by a number of means. Maceration, crushing and grinding into a fine powder are particularly contemplated. While extracts may be prepared from one or more parts of these plants, the leaf of Momordica balsamina, the seed of Aframomum melegueta, and the root of Cyperus articulatus are particularly contemplated.

[0019] In certain embodiments, compositions in accordance with the instant invention may comprise an extract prepared from one part of one of the plants described herein. In other embodiments, compositions in accordance with the instant invention may comprise an extract prepared from more than one part of one of the plants described herein. In other embodiments, compositions in accordance with the instant invention may comprise an extract prepared from one or more parts of one or more of the plants described herein.

[0020] In certain embodiments, compositions in accordance with the present invention are formulated as pharmaceutical compositions. The term "pharmaceutical composition" is intended to encompass a composition that is prepared, processed, and/or manufactured for use as a medicinal drug. In particular embodiments, pharmaceutical compositions are formulated as pharmaceutically-acceptable salts. Lists of suitable salts are found in, for example, Remington: The Science and Practice of Pharmacy, 21^s' Edition (Lippincott Williams & Wilkins, 2006).

[0021] In certain embodiments, pharmaceutical compositions in accordance with the instant invention comprise a pharmaceutically-acceptable carrier. Suitable pharmaceuticaly- acceptable carriers are described, for example, in Remington: The Science and Practice of Pharmacy, 21^s' Edition (Lippincott Williams & Wilkins, 2006). In other embodiments, pharmaceutical compositions in accordance with the instant invention comprise an excipient. Excipients encompass substances that facilitate processing of the active compounds into formulations for delivery to the site of action. In other embodiments, pharmaceutical compositions in accordance with the instant invention may comprise a stabilizer and/or a solubilizer. depending upon how the pharmaceutical composition is formulated. In other embodiments, pharmaceutical compositions in accordance with the instant invention may also comprise a preservative. Pharmaceutical compositions in accordance with the instant invention may also comprise other therapeutic agents intended to ameliorate one or more symptoms of EVD.

[0022] Pharmaceutical compositions in accordance with the instant invention may be formulated for administration in a variety of ways. Oral administration is particularly contemplated. Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, elixirs, suspensions, syrups or powders and controlled release forms thereof.

[0023] Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, e.g., water-soluble salts. Oily injection suspensions of the compositions may also be prepared and administered. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil or synthetic fatty acid esters (e.g., ethyl oleate or triglycerides). Aqueous injection suspensions can contain substances that increase the viscosity of the suspension. These include, for example, sodium carboxymethyl cellulose, sorbitol, and dextran.

[0024] Suitable formulations for topical administration include any common topical formulation such as a solution, suspension, gel, ointment or salve and the like. Preparations of such topical formulations are well described in the art of pharmaceutical formulations as exemplified, for example, by Remington: The Science and Practice of Pharmacy, 21^s' Edition (Lippincott Williams & Wilkins, 2006). For topical applications, a carrier that increases the permeability of the skin (e.g., dimethyl sulfoxide) may be desirable.

[0025] In particular embodiments, the pharmaceutical compositions are formulated for sustained delivery of the compositions for a period of twenty-four hours to a month or more. Such formulations are described, for example, in U.S. Patent Nos. 5,968,895 and 6, 1 80,608. Any pharmaceutically-acceptable, sustained-release formulation known in the art is contemplated.

[0026] As used herein, administering or administration includes dispensing, delivering or applying a compound disclosed herein in a method disclosed herein, e.g., in a pharmaceutical formulation, to a subject by any suitable route for delivery of that compound to the desired location in the subject.

[0027] It is contemplated that the compounds disclosed herein are administered in a therapeutically effective amount, i.e., an amount sufficient to achieve a desired result. An effective amount is also one in which any toxic or detrimental effects associated with administration of the compound are outweighed by the therapeutically beneficial effects. For example, effectiveness may be determined by, e.g., improvement, regression and/or disappearance of clinically-observable symptoms. For example, Ebola viral titers may be measured in blood, urine and other subject fluids.

Effective amounts of the compounds used in the methods of the invention may vary according to factors such as the disease state, age, and weight of the subject, and the abi lity of any particular formulation to elicit a desired response in the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. Accordingly, dosages for the methods disclosed herein range from about 0.5 g to 13.5 g per day and more preferably 3.0 g to 9.0 g per day. The total amount may be administered in a single dose or may be divided into multiple dosages administered over a twenty-four hour period. Three doses of 1 .0 g to 3.0 g administered three times per day are particularly contemplated. In other embodiments, the dosage regimens are formulated in a way that the subject receives from 0.075 mg/kg/day to about 0.45 mg/kg/day of the pharmaceutical composition, and more preferably from 0.15 mg/kg/day to about 0.30 mg/kg/day of the pharmaceutical composition.

[0028] Examples

[0029] The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations which are evident as a result of the teachings provided herein. [0030] Example 1

[0031] Stock solutions of Momordica balsamina, Aframomum melegueta and Cypems articulatus were prepared by suspending finely-ground powdered plant-material in sterile water such that the initial concentration of the stock solution was 100 mg/mL. Stock solutions were subsequently filtered through a 0.2 μηι filter. The resultant filtrate were diluted 100-fold (i.e.. I mg/mL or 1000 μg/mL) and then serially diluted in water nine more times to concentrations of 500, 250, 125, 62.5. 3 1.25, 1 5.625, 7.81 25, 3.90625 and 1 .953 13 g/mL, respectively, to produce ten separate solutions to produce a dose-response curve for each extract.

[0032] Aliquots of each solution were diluted in media ( 1 /10) and dispensed by into assay wells with HeLa and HFF- I cell lines. Doses were repeated four times on a single plate. Sixteen wells were treated with media alone to be used as a neutral control. Additionally, sixteen wells were not infected and were used as a low signal controls.

[0033] Cells were infected with EBOV(Zaire) at MOl=0.5 for Hela and MOl+2.5 for HFF- 1. Infection was stopped after 48h by fixing cells with a formalin solution. To detect infected cell immunostaining was completed with anti-GP antibodies. Images were taken using PERKINEL ER® OPERA® confocal platform using a l Ox objective and were analyzed using PERKINELMER® ACAPELLA® High Content Imaging and Analysis Software. Signal for GP-staining was converted into percentage of infection. The number of nuclei per well was used to determine percent viability of cells (in comparison to infected but untreated controls, n= 16).

[0034] Data was further analyzed using GENEDATA® software. Percentage of infection was converted into percent inhibition (%INH) for each well using plate controls.

[0035] Example 2

[0036] Dose Response

[0037] The EC₅o for the first test of the control solution was 81 ± 5 nM. The ECso for the second test of the control solution was 104 ± 5 nM. [0038) TABLE 1

[0039] The dose response curve for Extract A is shown in Figure 2. Additional data pertaining to Extract A is shown in Table 2.

[0040] TABLE 2

[0041] The dose response curve for Extract B is shown in Figure 3. Additional data pertaining to Extract B is shown in Table 3.

[0042] TABLE 3

[0043] The dose response curve for Extract C is shown in Figure 4. Additional data pertaining to Extract C is shown in Table 4. [0044] TABLE 4

[0045] The dose response curve for Extract D is shown in Figure 5. Additional data pertaining to Extract D is shown in Table 5.

[0046] TA BLE 5

[0047] The dose response curve for Extract E is shown in both Figure 6. Additional data pertaining to Extract E is shown in Table 6.

[0048] TABLE 6

[0049] The dose response curve for the combined extracts are shown in Figure 7.

Additional data pertaining to the combined extracts is shown in Table 7.

[0051] As demonstrated throughout Figures 1 -7 and Tables 1 -7, all extracts show anti- EBOV activity in both HeLa and HFF cell lines.

[0052] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0053] While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention can be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:

1 . A pharmaceutical composition comprising an extract prepared from one or more plants selected from the group consisting of Momordica balsamina, Aframomiim meieguet, and Cyperus articulates, wherein the pharmaceutical composition further comprises one or more substances selected from the group consisting of a pharmaceutically- acceptable carrier, an excipient, a stabilizer, a solubilizer, and a preservative.

2. A pharmaceutical composition comprising an extract prepared from Momordica balsamina, wherein the pharmaceutical composition further comprises one or more substances selected from the group consisting of a pharmaceutically-acceptable carrier, an excipient, a stabilizer, a solubi lizer. and a preservative.

3. The pharmaceutical composition of claim 2, wherein the extract is prepared from a leaf of Momordica balsamina.

4. A pharmaceutical composition comprising an extract prepared from Aframomiim meieguet, wherein the pharmaceutical composition further comprises one or more substances selected from the group consisting of a pharmaceutically-acceptable carrier, an excipient, a stabilizer, a solubilizer, and a preservative.

5. The pharmaceutical composition of claim 4, wherein the extract is prepared from a seed of Aframomiim meieguet.

6. A pharmaceutical composition comprising an extract prepared from Cyperus articulates, wherein the pharmaceutical composition further comprises one or more substances selected from the group consisting of a pharmaceutically-acceptable carrier, an excipient. a stabilizer, a solubilizer, and a preservative.

7. The pharmaceutical composition of claim 4, wherein the extract is prepared from a root of Cyperus articulates.

8. The pharmaceutical composition of any one of claims 1 -7, wherein the pharmaceutical composition is formulated for oral administration.

9. The pharmaceutical composition of any one of claims 1 -7, wherein the pharmaceutical composition is formulated for parenteral administration.

10. The pharmaceutical composition of any one of claims 1 -7, wherein the pharmaceutical composition is formulated for topical administration.

1 1 . The pharmaceutical composition of any one of claims 1 - 10, wherein the pharmaceutical composition is formulated for sustained release.

12. The pharmaceutical composition of any one of claims 1 -1 1 , wherein the pharmaceutical composition is formulated as a capsule or pill.

13. The pharmaceutical composition of any one of claims 1 - 1 1 , wherein the pharmaceutical composition is formulated as a liquid suspension.

14. A method of treating Ebola virus disease in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition of any one of claims 1 - 1 3 to the subject.

1 5. The method of claim 14, wherein the therapeutically effective amount of the pharmaceutical composition is from about 0.075 mg/kg/day to about 0.45 mg/kg/day.

16. The method of claim 1 , wherein the therapeutically effective amount of the pharmaceutical composition is from about 0.15 mg/kg/day to about 0.30 mg/kg/day.