FIELD OF THE INVENTION

The invention relates to method of making preparations comprising synthetic thymoquinone. The invention further relates to methods of preparing compositions comprising phosphatidylcholine and/or guggulsterol and/or guggulsterol derivatives and/or sodium cholesteryl sulfate. In some embodiments, the invention relates to methods of preparation containing phosphatidylcholine, and guggulsterol or guggulsterol derivatives or sodium cholesteryl sulfate. In certain embodiments, the invention also relates to method of preparing thymoquinone and excipients in a tablet or capsule or topical gel or an ointment. The invention further relates administering preparation to a human subject in the treatment or prevention of diseases. Methods according to the present invention are suitable for practice on an industrial manufacturing scale, and may be practiced, e.g., as a continuous method.

BACKGROUND OF THE INVENTION

Nigella saliva, often referred to as black cumin seed or black caraway seed, is an annual flowering herb that belongs to the family Ranunculaceae, native to south and southwest Asia.N. sativaseeds have been used as a spice and a condiment. It has also been used to treat diseases, including asthma, hyperglycemia, urine retention, hypertension, inflammation, cough, and pain (see, e.g., Ali B H, and Bluden, G. 2003; Entok E. et al. 2014; Burtis M. and Bucar, F. 2000; Bourgou S. et al. 2010; Mariod A A et al. 2009).

The biological effects ofN. sativaare attributed to the various ingredients including thymoquinone, dithymoquinone, thymohydroquinone, and thymol (Omar, A. et al. 1999).N. sativaseed contain other ingredients as well, such as carbohydrates, fats, vitamins, proteins, and essential amino acids (Bhatia, I S and Bajaj K. 1972; Chun, H. et al. 2002; Correa, A D et al. 1986). Thymoquinone has also been shown to exert anti-inflammatory, anti-oxidant, and anti-neoplastic effects both in vitro and in vivo (Mansour, M A et al. 2002). Thymoquinone showed activity against human pancreatic adenocarcinoma, uterine sarcoma, and leukemia cell lines, while it is minimally toxic to normal cells (Worthen, D R et al., 1998). Oral administration of thymoquinone in streptozotocin (STZ) and nicotinamide (NA) induced diabetic rats showed antihyperglycemic effects unassociated with glycogen phosphorylase inhibition (El-Ameen, N H M et al., 2015).

Most of the work on thymoquinone has been done at non-clinical level and very few attempts have been made to test its therapeutic effect at the clinical level. An efficacy study ofN. sativaseed extract was conducted in patients with mild hypertension and the results suggest that the daily use ofN. sativaseed extract for 2 months may have blood pressure lowering effect in patient with mild hypertension (Dehkordi F R and Kankhah, A. F. 2008). Another report showed thatN. sativapowder administered to the hypercholesteremia patients for two months was found to reduce the total cholesterol and triglycerides (Bhatti, I U et al. 2009).

Thymoquinone as an active constituent of black cumin seed was first extracted in 1963 (El-Dakhakhany, M. 1963). However, isolation of thymoquinone fromN. sativaseeds is a cumbersome process and requires large amounts of organic solvents. The essential oil fraction ofN. sativaconstitutes only one percent of the seed oils. Thus, thymoquinone comprises only 0.3 to 0.6% of the fixed oil fractions, which are the most common commercially available product ofN. sativa(Pacioretty, L M and Babish J G, US 2010/0028468 A1). Considering the low percentage of thymoquinone in theN. sativaseed extracts, it is imperative to utilize synthetic thymoquinone to determining its clinical benefits.

Synthetic thymoquinone (2-Isopropyl-5-methyl-1,4-benzoquinone) is a yellow crystalline solid soluble in organic solvents such as ethanol, chloroform, DMSO. It is sparingly soluble in aqueous buffers (approx. 500 μg/mL), which may be enough to exert pharmacological effects if administered via parenteral route. However, thymoquinone is highly unstable in aqueous solutions and is very sensitive to light (Salmani, J M M et al. 2014), limiting its usage in parenteral administration. Hence, there is a need for methods or processes for preparation of stable compositions comprising thymoquinone for oral administration.

SUMMARY OF THE INVENTION

Provided herein are methods of preparing a stable composition containing thymoquinone, preferably synthetic thymoquinone. In some embodiments, the composition comprises phosphatidylcholine and/or one or more of guggulsterol, guggulsterol derivatives, and/or sodium cholesteryl sulfate. In some embodiments, the composition further comprises phosphatidylcholine. In some preferred embodiments, the composition further comprises other excipients. Certain embodiments comprise a method of preparing a stable formulation comprising thymoquinone and administering the preparation to a subject. In certain embodiments, the subject is a mammal. In preferred embodiments, the subject is human.

Provided herein in some embodiments are methods of preparing compositions comprising thymoquinone and guggulsterol for oral delivery to a human subject. In some embodiments, the preparation for oral delivery is in the form of tablet or capsule including gel capsule, pill, dragees, and suppositories. In preferred embodiments, the tablets and/or capsules comprises enteric coating.

Provided herein in some embodiments are methods of preparing a composition comprising thymoquinone and a guggulsterol derivative for oral delivery to a human subject. In some embodiments, oral delivery is in the form of tablet or capsule. In more preferred embodiments, the tablets and/or capsules comprises enteric coating.

Provided herein in some embodiments are methods of preparing a composition comprising thymoquinone and sodium cholesteryl sulfate for oral delivery to a human subject. In some embodiments, the oral delivery is in the form of tablet or capsule. In more preferred embodiments, the tablets and/or capsules comprises enteric coating.

Provided herein in some embodiments are methods of preparing a composition comprising thymoquinone and phosphatidylcholine for oral delivery to a human subject. In some embodiments, the oral delivery is in the form of tablet or capsule. In more preferred embodiments, the tablets and/or capsules comprises enteric coating.

Provided herein in some embodiments are thymoquinone preparations comprising thymoquinone, guggulsterol, and phosphatidylcholine for topical administration to a human subject. In some embodiments, the preparation for topical administration is in the form of a gel, ointment, or cream.

Provided herein in some embodiments are thymoquinone preparations comprising thymoquinone, a guggulsterol derivative, and phosphatidylcholine for topical administration to a human subject. In certain preferred embodiments, the preparation for topical administration is in the form of gel, ointment, or cream.

Provided herein in some embodiments are thymoquinone preparations comprising thymoquinone, sodium cholesteryl sulfate and phosphatidylcholine for topical administration to a human subject. In certain preferred embodiments, the preparation for topical administration is in the form of gel, ointment, or cream.

In some embodiments, a thymoquinone preparation of present invention is co-administered with other drugs. Drugs that can be co-administered along with thymoquinone preparations include but are not limited to anti-diabetic drugs such as metformin, glimepiride, glipizide, glyburide, tolazamide, rosiglitazone, alogliptan, linagliptan, saxagliptin, sitagliptin, insulin, etc.; anticancer drugs such as doxorubicin, paclitaxel, docetaxel, methotrexate, tamoxifen, letrazole, fluvestrant, aromasin, cyclophosphamide, daunomycin, bleomycin, irinotecan, bendamustine, mitoxantrone, ifosfamide, BCNU, streptozotocin, cytrabine, capecitabine, carboplatin, cisplatin, etoposide, 5-flurouracil, etc.; lipid lowering agents such as atorvastatin, simvastatin, rosuvastatin, etc.; anti-inflammatory drugs such as for treating rheumatology, arthritis, psoriasis; antihypertensive agents, such as dihydropyridines, antidepressants, antiallergic agents, etc.; drugs for treating skin diseases such as psoriasis, atopic dermatitis, eczema, melanoma, mycosis, acne, cellulitis, ichthyosis, hives, antifungal agents such as polyene antibiotics or poly antimycotics, azole antifungal drugs, etc. Examples of polyene antibiotic or polyene antimycotics include but not limited to amphotericin B, nystatin, hamycin A & B, aureofungin A & B, natamycin, pimaricin etc. Examples of azole antifungal drugs include but not limited to ketoconazole, itraconazole, fluconazole, etc.

The amount of phosphatidylcholine included in a thymoquinone preparation according to present invention is not limited to any particular amount or percentage (by weight) of the final preparation or weight. In some embodiments, the proportion of phosphatidylcholine is between about 1% to about 90% of the total weight, preferably about 2% to about 80% of the total weight, more preferably about 3% to about 50% of the total weight.

The amount of guggulsterol or guggulsterol derivative or sodium cholesteryl sulfate included in a thymoquinone preparation according to present invention is not limited to any particular amount or percentage (by weight) of the final preparation or weight. In some embodiments, the proportion of guggulsterol or guggulsterol derivative or cholesteryl sulfate is about 0.1% to about 90% of the total weight, preferably about 0.2% to about 50% of the total weight, more preferably about 0.2% to about 25% of the total weight.

The amount of thymoquinone included in a thymoquinone preparation according to present invention is not limited to any particular amount or percentage (by weight) of the final preparation or weight. In some embodiments, the proportion of thymoquinone is about 0.1% to about 90% of the total weight, preferably about 0.5% to about 75% of the total weight, more preferably about 1% to about 50% of the total weight.

In some embodiments, the technology provides a method of preparing a composition comprising thymoquinone comprising:i) mixing thymoquinone and vitamin E TPGS and melting it together;ii) preparing lipid granules by sifting lipid and magnesium aluminometasilicate (Neusilin US2) and forming a mixture comprising lipid granules and melted thymoquinone of i) and sifting the resulting mixture, wherein the lipid is selected from the group consisting of phosphatidylcholine, guggulsterol, a guggulsterol derivative, and sodium cholesteryl sulfate;iii) co-sifting magnesium aluminometasilicate, silicified microcrystalline cellulose (SMCC HD90), cross carmellose sodium, hydrophilic fumed silica; sodium lauryl sulfate, and anhydrous citric acid;iv) sifting Poloxamer and blending with thymoquinone-lipid granules of ii) and granules of iii); andv) sifting magnesium stearate and combining with the thymoquinone-lipid granules of iv) and mixing to form a lubricated mixture.

In some embodiments, the lubricated mixture of v) is pressed into tablets, and in certain preferred embodiments, the tablets are seal-coated. In certain embodiments, the seal coating comprises one or more polymers selected from hydroxymethyl propyl cellulose, methyl hydroxyethylcellulose, ethyl cellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, povidone, sodium carboxy methylcellulose, acrylate polymer, Opadri, and polyethylene glycol. In some embodiments, the seal-coated tablets are enteric coated with polymers, and in some embodiments, the enteric coating comprises one or more polymers selected from hydroxypropyl methyl cellulose phthalate, cellulose acetate phthalate, acrylate polymers, and polyvinyl acetate phthalate, Opadri enteric, Eudragit, and Acryl-EZE.

In certain embodiments of any embodiments of the tablets described above, the amount of thymoquinone in a single tablet is at least 50 mg, preferably at least 500 mg, more preferably at least 1000 mg. In certain embodiments of any embodiments of the tablets described above, the guggulsterol or guggulsterol derivative in a single tablet is between 10 mg and 1000 mg, preferably between 25 mg and 50 mg. In certain embodiments of any embodiments of the tablets described above, the phosphatidylcholine in a single tablet is between 10 mg and 500 mg, preferably between 10 and 250 mg, more preferably between 10 and 100 mg

In some embodiments, the technology provides a method of preparing a gel or ointment composition comprising thymoquinone, phosphatidylcholine, guggulsterol or guggulsterol derivative or sodium cholesteryl sulfate, comprising:i) mixing phosphatidylcholine, guggulsterol, or guggulsterol derivative or sodium cholesteryl sulfate and thymoquinone in a first organic solvent;ii) diluting the mixture of i) with a second organic solvent; andiii) mixing the diluted mixture of ii) with a gelling agent to form a uniform viscous gel or ointment.

In some embodiments, the phosphatidylcholine used in the method described above is selected from phosphatidylcholine includes soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidycholine, egg phosphatidylcholine.

In any of the embodiments described above, the guggulsterol derivative guggulsteryl sulfate, guggulsteryl phosphate, guggulsteryl phosphocholine, guggulsteryl phosphoglycerol, fatty acid esters of guggulsterol, and polyethylene glycol (PEG) derivatives of guggulsterol. In certain embodiments, the fatty acid esters of guggulsterol include guggulsterol include guggulsteryl acetate, guggulsteryl propionate, guggulsteryl butyrate, guggulsteryl valerate, guggulsteryl hexanoate, guggulsteryl caprylate, guggulsteryl caprates, guggulsteryl laurate, guggulsteryl myrstate, guggulsteryl palmitate, guggulsteryl stearate, guggulsteryl oleate, guggulsteryl linoleate, guggulsteryl linolenate, guggulsteryl eicosapentenoate, guggulsteryl arachidonate, guggulsteryl hemisuccinate, guggulsteryl succinate. In certain embodiments, the molecular weight of PEG in PEG derivatives of guggulsterol is between 500 and 2000.

In certain embodiments of any of the embodiments described above, the guggulsterol or guggulsterol derivative may be in the form of Z-isomer, and/or may be in the form of sodium salt.

In some embodiments of the method of preparing a gel or ointment composition, aid gelling agent is selected from hydroxypropylmethylcellulose, sodium carboxymethylcellulose, methyl cellulose, Simulgel INS 100, carbomers, Guar gum, and gelatin.

In any of the embodiments of the method of preparing a gel or ointment composition described above, the first organic solvent and second organic solvent may be the same or different. In certain embodiments, the first organic solvent and second organic solvent is selected from isopropyl alcohol, ethanol, propylene glycol, polyethylene glycols, oleic acid, oleoyl alcohol, mineral oil, and purified water.

In any of the embodiments of the method of preparing a gel or ointment composition described above, the amount of thymoquinone in the gel or ointment may be between 0.1% and 10% by weight, preferably 3% by weight; the quantity of the phosphatidylcholine in the gel or ointment may be between 1% and 20% by weight, preferably 6% by weight; and/or the amount of guggulsterol or guggulsterol derivative in a gel or ointment may be between 0.1% and 10% by weight, preferably 0.5% by weight.

In some embodiments, the technology provides a method of administering thymoquinone to a subject, comprising:i) providing a composition comprising thymoquinone prepared according to any of the embodiments described above; andii) administering the preparation to a subject.

In some embodiments, the composition is a tablet or capsule and the administering comprises oral administration; while in some embodiments, the composition comprises a gel or ointment and the administering comprises topical administration. In preferred embodiments, the subject is a human.

Definitions

To facilitate an understanding of the present invention, a number of terms and phrases are defined below:

As used herein, the term “composition” “preparation” or “formulation” refers to the combination of an active agent (e.g. an active pharmaceutical compound) with a carrier, inert or active, excipients, making the composition especially suitable for diagnostic or therapeutic use in vitro, in vivo, or ex vivo.

As used herein, the terms “synthetic thymoquinone” or “thymoquinone” refers to thymoquinone that was synthesized (i.e., was not isolated from extract of a plant), e.g., in a laboratory starting from commercially available starting materials. The synthesis of said thymoquinone may be by any route of synthesis and involves any number of synthetic steps.

As used herein, the term “active” as used in reference to an agent, composition, or compound, refers to an agent that, upon administration or application, causes a beneficial, desired, or expected result. The administration may be in one or more administrations, applications, dosages and is not intended to be limited to a particular formulation or administration route. The term is not limited to any particular level of activity. For example, a formulation of an active agent need not have the same level of activity as a different formulation of an active agent, so long as the active agent in the formulation is sufficiently active that an effective amount of the active agent can be administered by administration of the formulation of the agent.

The terms “agent” and “compound” are used herein interchangeably to refer to any atom, molecule mixture or more complex composition having an attributed feature. For example, an ‘active agent” or “active compound” refers to any atom, molecule, preparation mixture, etc. that, upon administration or application, causes beneficial, desired or expected result.

As used herein, the term “administration” or “administering” refers to the act of giving a drug, or active agent, or therapeutic treatment (e.g. composition of the present invention) to a physiological system (e.g., a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs). Exemplary routes of administration to the human body can be through mouth (oral), skin (transdermal), eyes (ophthalmic), nose (nasal), injection (parenteral) and the like. Administration may be in one or more administrations, applications, or dosages, and is not intended to be limited to a particular administration route.

As used herein, the term “co-administration” refers to the administration of at least two agents(s) (e.g. two separate compositions, containing different active agents) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies are concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art.

As used herein, the term “excipient” refers to an inactive ingredient (i.e. not pharmaceutically active) added to the preparation of active ingredient The disintegrates, antiadherants, binders, plasticizers, fillers, coatings, lubricants, preservatives, glidants, enhancers, wetting agents, emulsifying agent, solubilizing agent, dispersing agent, flavors, colors, sorbents, sweetener, antioxidants, permeation enhancer, humectant, emulsifying agent, ointment base, acidifying and/or alkalizing and/or buffering agent, gelling and protective agents described herein may be referred to generally as “excipients”.

As used herein, the term “disease” refers to a state, signs, and/or symptoms that are associated with any impairment of the normal state of a living animal or any of its organs or tissues that interrupts or modifies the performance of normal functions and may be a response to environmental factors.

As used herein, the term “treatment” or grammatical equivalents encompasses the improvement and/or reversal of the symptoms of disease (e.g. diabetes), or reduction of risk of occurrence of disease. A compound which causes an improvement in any parameter associated with disease when used in the screening methods of the instant invention may thereby be identified as a therapeutic compound. The term “treatment” refers to both therapeutic treatment and prophylactic or preventive measures. For example, those who may benefit from treatment with compositions and methods of the present invention include those already with a disease and/or disorder (e.g. diabetes, or symptoms or pathologies consistent with diabetes) as well as those in which a disease and/or disorder is to be prevented (e.g., using a prophylactic treatment of the present invention).

As used herein, the term “oral” refers to application of the compositions of the present invention through mouth. The term oral also includes application of composition through buccal sublabial, sublingual administration.

As used herein, the term “topically” refers to application of the compositions of the present invention to the surface of the skin and mucosal cells and tissues (e.g., alveolar, buccal, lingual, masticatory, or nasal mucosa, and other tissues and cells which line hollow organs or body cavities).

As used herein, the term “transdermal” refers to application of the compositions of the present invention in a form for absorption through the skin into the bloodstream. For example, composition of present invention can be applied in the form of patch (e.g. transdermal patch) to deliver the active agent at a constant rate for a specific period of time, allowing the bioavailability and effectiveness of the drug to remain constant.

The term “polyethylene glycol (PEG) includes polymers of lower alkylene oxide, in particular ethylene oxide (polyethylene glycols) having an esterifiable hydroxyl group at least one end of the polymer molecule, as well as derivatives of such polymers having esterifiable carboxy groups. Polyethylene glycols of an average molecular weight ranging from 200-20,000 are preferred; those having an average molecular weight ranging from 300-2000 are particularly preferred.

The use of terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising”, “including”, “having”, and “containing” are to be construed as open-ended terms (i.e. meaning “including but limited to”) unless otherwise noted. The use of any and all examples, or exemplary language (e.g. ‘such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specifications should be constructed as indicating any non-claimed element as essential to the practice of the invention.

