PREVENTION OF CALCIUM OXALATE KIDNEY STONES BY POTASSIUM HYDROXYCITRATE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 62/546,669, filed August 17, 2017. The contents of which are incorporated into the present application by reference. BACKGROUND OF THE INVENTION

A. Field of the Invention

[0001] The present invention relates generally to the fields of biology, chemistry, and medicine. More particularly, it concerns methods and compositions relating to treating or preventing the formation of calcium oxalate (CaOx) kidney stones by oral administration of potassium hydroxycitratc (KOHCit).

B. Description of the Related Art

[0002] CaOx is the most common constituent of kidney stones. CaOx kidney stones are commonly associated with hypocitraturia (low urinary citrate), hypercalciuria (high urinary calcium) and hyperoxaluria (high urinary oxalate). Potassium citrate (KCit) is often used to prevent CaOx stone formation, since it increases urinary citrate, which is an inhibitor of stones, and reduces urinary saturation of CaOx by complexing calcium. (Pak, 1985).

[0003] When KCit is taken orally, the absorbed citrate is mostly oxidized in the liver to carbon dioxide and water. This reaction consumes H ⁺ and imposes an alkali load on the body. The alkali load is responsible for increasing urinary citrate by affecting renal metabolism and reabsorption of citrate. (Melnick, 1996). Increased urinary citrate inhibits crystal growth (Chung, 2016) and agglomeration of CaOx (Kok, 1986). Moreover, citrate forms soluble complexes with calcium, reducing the fraction of ionized calcium available for CaOx formation. Thus, the rise in urinary citrate reduces the urinary saturation of CaOx. The urinary saturation of CaOx is also reduced by increased urinary pH from alkali load that increases saturation of calcium phosphate and enhances the formation of soluble complexes of calcium and oxalate. (Pak, 2009). When urine is over-alkalinized (pH >6.9) by a high dose [0004] of Cit, this treatment might invite formation of calcium phosphate stones. At urinary pH >6.9, the saturation of calcium phosphate rises steeply, causing precipitation- crystallization of calcium phosphate. (Pak, 1971). Moreover, KCit does not increase urinary hydroxycitrate, another inhibitor of CaOx crystallization. [0005] Hydroxycitric acid (HCA), is commonly found in fruits of Garcinia Cambodia. Commercial preparations of Garcinia extract (touted to contain HCA) are widely used as a food supplement. These preparations allegedly lead to weight loss by inhibiting the ability of citrate to provide acetyl groups to CoA in the cytoplasm (Heymsfield, 1998). While their value in weight control might be disputed, Garcinia HCA preparations are well tolerated with safety of usage at recommended doses. It has been suggested that HCA might be a stone- prevention drug that might be devoid of the complication of calcium phosphate stones (Chung, 2016). When examined by atomic force microscopy, HCA inhibited crystal growth and disrupted CaOx crystals in vitro. This inhibitory effect of HCA was greater than that of citric acid, especially at lower concentrations. In human subjects taking a commercial preparation of HCA, a sufficient amount of HCA appeared in urine to exert inhibitory action. Orally administered HCA should not increase urinary pH to cause calcium phosphate crystallization, since HCA does not confer an alkali load because it doesn't have an accompanying non-metabolizable cation. Although HCA would not increase urinary citrate without alkali load, HCA might enhance urinary citrate by inhibiting renal citrate lyase (Melnick, 1996). Thus, it was speculated that HCA might inhibit CaOx urolithiasis (by enhancing both urinary hydroxycitrate and citrate), without potential complication of calcium phosphate stones (in the absence of urinary alkalization).

[0006] Unfortunately, HCA is not readily available in a pure form. It is not an ideal oral agent, since it is unstable and gradually converts to inactive HCA lactone (HCAL) in water. A recent article by Chung (2017) revealed that HCA might have a promoter (enhanced) activity against calcium oxalate crystallization at either extremely low or extremely high pH. Thus, since pH is not altered by HCA, HCA has the potential for aggravation of stones in patients with very low urinary pH.

SUMMARY OF THE INVENTION

[0007] KOHCit meets the need for an agent that confers a modest alkali load to avoid abnormally high urinary pH or undue urinary acidity, produces an optimum rise in urinary citrate, and yields urinary excretion of another inhibitor - hydroxycitrate. KOHCit is just as effective as KCit in increasing urinary citrate, a key inhibitor of CaOx stone formation. Moreover, unlike KCit, KOHCit does not overly alkalinize the urine and thereby does not pose a risk of calcium phosphate stones. Moreover, unlike KCit, KOHCit increases urinary hydroxycitrate, another inhibitor of CaOx crystallization. In addition, unlike HCA, KOHCit is stable in water.

[0008] In some aspects, disclosed is a method of preventing or treating calcium oxalate kidney stones in a subject, the method comprising orally administrating a composition comprising an effective amount of potassium hydroxycitrate to treat or prevent calcium oxalate stones.

[0009] The term "effective," as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result. "Effective amount" or "therapeutically effective amount" or "pharmaceutically effective amount" means that amount which, when administered to a subject or patient to accomplish a desired, expected, or intended results, is sufficient to achieve the desired, expected, or intended result. In some embodiments, the effective amount raises urinary citrate, raises urinary hydroxycitrate, increases urinary excretion of both citrate and hydroxycitrate, and/or maintains normal urinary pH (5.5-6.9). A concentration is raised if there is a measurable increase in concentration, or if there is a 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100%, or more, increase. In some embodiments, the subject (for a 70 kg human being), is administered a dose of 5-80 meq per day.

[0010] In some embodiments, the effective amount is the above said amounts given in divided doses. The composition may be administered to (or taken by) the patient 1 , 2, 3, 4, 5, or 6 times, or any range derivable therein, and they may be administered every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 18, or 24 hours, or 1 , 2, 3, 4, 5, 6, or 7 days. It is specifically contemplated that the composition may be administered once daily, twice daily, three times daily, four times daily, five times daily, or six times daily (or any range derivable therein) and/or as needed to the patient. In some embodiments, the composition is administered two, three, or four times per day. Alternatively, the composition may be administered every 2 to 24 hours (or any range derivable therein) to or by the patient. It is specifically contemplated that the composition may be administered daily over the course of multiple months or years, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 months or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 years (or any derivable range therein) or for an indefinite period of time. The compositions may be administered one or more times in such daily administration. In some embodiments, the compositions are administered 1 to 10 times or more.

[0011] As used herein, the term "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dogs, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the subject is a primate. In some embodiments, the subject is a human. Non-limiting examples of human subjects are adults and juveniles. In some embodiments, the subject has calcium oxalate kidney stones. In some embodiments, the subject is at risk for developing calcium oxalate kidney stones. [0012] The composition may be administered in any suitable manner. For example, it may be administered systemically, orally, via infusion, via continuous infusion, via a lavage, in cremes, or by other method or any combination of the foregoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 1990). In some embodiments, administration comprises oral administration. In some embodiments, the composition is in the form of a tablet, a solution, or a powder.

[0013] "Treatment" or "treating" includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.

[0014] "Prevention" or "preventing" includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.

[0015] "Pharmaceutically acceptable" means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use.

[0016] The phrase "and/or" means "and" or "or". To illustrate, A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, "and/or" operates as an inclusive or.

[0017] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0018] The use of the word "a" or "an" when used in conjunction with the term "comprising" may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0019] The words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0020] The compositions and methods for their use can "comprise," "consist essentially of," or "consist of any of the ingredients or steps disclosed throughout the specification. Compositions and methods "consisting essentially of any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.

[0021] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention. [0022] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0024] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims. Embodiments are contemplated wherein a feature or embodiment described herein is specifically excluded from the invention.

[0025] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0027] FIG. 1 illustrates structural formula of tripotassium hydroxycitrate (KOHCit). KOHCit has three potassiums for each hydroxycitrate molecule. [0028] FIG. 2 illustrates examination of analytical grade KOHCit, KCit, and HCAL by capillary electrophoresis. Peak 1 is HCAL, peak 2 citrate, and peak 3 is hydroxycitrate.

[0029] FIG. 3 is a schematic illustration for the calculation of formation product (FP) and crystal growth (CG) of CaOx in human urine in vitro. Precipitation of CaOx occurring from addition of increasing amounts of oxalate was detected by optical density (absorbance). The arrow indicates the FP, or the minimum amount of additional oxalate needed to elicit precipitation. The CG of CaOx is shown by the shaded area.

[0030] FIG. 4 compares FP of CaOx upon addition of KOHCit, KCit or KC1 6 meq/L each to human urine in vitro. Dots represent data obtained from urine samples collected from 12 separate human subjects. ** p < 0.01 and† p < 0.001 from KC1, (**) p < 0.01 between KCit and KOHCit. Since chloride is physicochemically inactive, KC1 served as the control.

[0031] FIG. 5 illustrates CG of CaOx upon addition of KOHCit, KCit or KC1 6 meq/L each to human urine in vitro, p < 0.05 and† p < 0.001 from KC1, (**) p < 0.01 between KCit and KOHCit. [0032] FIG. 6 reveals the FP and CG of CaOx when HCAL is added to human urine in vitro. Studies in 6 human urine samples are depicted.

[0033] FIGS. 7A-7D shows the results of ex vivo experiments in rat liver slices examining hepatic uptake and presumed oxidation of hydroxycitrate (lower panels, FIGS. 7C and 7D) and citrate (upper panels, FIGS. 7A and 7B). Concentration (left panels, FIGS. 7A and 7C) and percent change (right panels, FIGS. 7B and 7D) in hydroxycitrate or citrate in culture media are depicted. Open circles on the left corner of the slide pointed by arrows represent original hydroxycitrate or citrate concentration at time zero. Each line represents separate experiments.

[0034] FIGS. 8A-8F demonstrates the change in urinary pH (FIG. 8A), ammonium (FIG. 8B), net acid excretion (NAE) (FIG. 8C), citrate (FIG. 8D), FP (FIG. 8E), and CG (FIG. 8F) of CaOx in rats from before treatment to treatment with KOHCit or KCit. The vertical line above or below the bars indicates plus or minus SD.

[0035] FIG. 9 demonstrates the relationship between urinary citrate and corresponding NAE during KOHCit and KCit treatments. In three rats, a 24-hour urine was collected during two days before treatment and on days 6 and 7 of treatment. Each dot represents result from separate urine sample.

[0036] FIG. 10 illustrates the effect of four commercial Garcinia products on the FP and CG of calcium oxalate when added to human urine in vitro. Studies in 8 human urine samples are shown.

DETAILED DESCRIPTION OF THE INVENTION

[0037] Disclosed herein is a method of preventing CaOx urolithiasis by oral administration of KOHCit. KOHCit demonstrates: (a) a greater inhibition of CaOx crystallization by KOHCit versus KCit when added to human urine in vitro, (b) incomplete hepatic uptake and presumed oxidation of hydroxycitrate from ex vivo experiments with liver slices, and (c) partial alkali load and urinary alkalization but optimum citraturic response by KOHCit from in vivo studies in rats. Additional in vivo studies in rats are planned to further clarify the action of KOHCit in the prevention of CaOx stones. Thus, an ideal agent is one that increases urinary pH mild-modestly (avoiding over-alkalization or undue urinary acidity) but increases the inhibitor activity (by raising both urinary citrate and HCA). KOHCit is such an agent.

[0038] When KCit is administered orally, the absorbed citrate is nearly completely metabolized in the liver to carbon dioxide and water; this process consumes 3H ⁺ for each trivalent citrate. The H ⁺ consumption is tantamount to an alkali load. Alkali load enhances urinary citrate excretion by affecting the metabolism and reabsorption of citrate in the renal tubule (Melnick, 1996). Citraturic response is directly equivalent to the amount of KCit absorbed and alkali generated.

A. KOHCit

[0039] KOHCit is a tripotassium salt of hydroxycitrate. Its structural formula is shown in FIG. 1. The third dissociation constant (pKa ₃ ) of hydroxycitrate is 5.1 1 (Chung, 2017). Thus, in normal urinary pH, hydroxycitrate is mostly present as a trivalent anion. KOHCit is readily soluble in water. Once KOHCit is dissolved, the released hydroxycitrate is stable.

B. Pharmaceutical Formulations and Routes of Administration

[0040] The compositions described herein may be administered to a subject in need of treatment by a variety of routes of administration, including orally and parenterally, (e.g., intravenously), as a suppository or using a "flash" formulation, i.e., allowing the medication to dissolve in the mouth without the need to use water, topically, and/or administration via mucosal routes in liquid or solid form. The composition can be formulated into a variety of dosage forms, e.g., extract, pills, tablets, microparticles, capsules, powder in sachet or packets, or oral liquid.

[0041] There may also be included as part of the composition pharmaceutically compatible binding agents, and/or adjuvant materials. The compositions can also be mixed with other active materials including antibiotics, antifungals, other virucidals and immunostimulants which do not impair the desired action and/or supplement the desired action.

[0042] In one embodiment, the mode of administration of the pharmaceutical composition described herein is oral. Oral compositions generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the aforesaid compounds or agents may be incorporated with excipients and used in the form of tablets, powder in sachet or packets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. Some variation in dosage will necessarily occur, however, depending on the condition of the subject being treated. However, the concentration of active ingredient in the composition itself depends on bioavailability and other factors known to those of skill in the art. [0043] In another embodiment, the mode of administration of the pharmaceutical compositions described herein is topical or mucosal administration.

[0044] Various polymeric and/or non-polymeric materials can be used as adjuvants for enhancing mucoadhesiveness of the pharmaceutical composition disclosed herein. The polymeric material suitable as adjuvants can be natural or synthetic polymers. Representative natural polymers include, for example, starch, chitosan, collagen, sugar, gelatin, pectin, alginate, karya gum, methylcellulose, carboxymethylcellulose, methylethylcellulose, and hydroxypropylcellulose. Representative synthetic polymers include, for example, poly(acrylic acid), tragacanth, poly(methyl vinylether-co-maleic anhydride), poly( ethylene oxide), carbopol, poly(vinyl pyrrolidine), poly( ethylene glycol), poly(vinyl alcohol), poly(hydroxyethylmethylacrylate), and polycarbophil. Other bioadhesive materials available in the art of drug formulation can also be used (see, for example, Bioadhesion— Possibilities and Future Trends, Gurny and Junginger, eds., 1990).

[0045] It is to be noted that dosage values also vary with the specific severity of the disease condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted to the individual need and the professional judgment of the person administering or supervising the administration of the aforesaid compositions. It is to be further understood that the concentration ranges set forth herein are exemplary only and they do not limit the scope or practice of the disclosure. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.

[0046] The formulation may contain the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, corn starch and the like; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; and a sweetening agent such as sucrose, sucralose, or saccharin or flavoring agent such as peppermint, methyl salicylate, or orange flavoring may be added. When the dosage unit form is a capsule, it may contain, in addition to material of the above type, a liquid carrier such as a fatty oil. Other dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings. Thus tablets or pills may be coated with sugar, shellac, or other enteric coating agents. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.

[0047] The solutions or suspensions may also include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as dextrose. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0048] The compositions can be prepared as formulations with pharmaceutically acceptable carriers. Preferred are those carriers that will protect the composition against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as polyanhydrides, polyglycolic acid, collagen, and polylactic acid. Methods for preparation of such formulations can be readily performed by one skilled in the art.

[0049] Liposomal suspensions may also be used as pharmaceutically acceptable carriers. Methods for encapsulation or incorporation of compounds into liposomes are described by Cozzani, I.; Jori, G.; Bertoloni, G.; Milanesi, C; Sicuro, T. Chem. Biol. Interact. 53, 131 -143 (1985) and by Jori, G.; Tomio, L.; Reddi, E.; Rossi, E. Br. J. Cancer 48, 307-309 (1983), for example. These may also be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,81 1 (which is incorporated herein by reference in its entirety). For example, liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound is then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.

[0050] Other methods for encapsulating compounds within liposomes and targeting areas of the body are described by Sicuro, T.; Scarcelli, V.; Vigna, M. F.; Cozzani, I. Med. Biol. Environ. 15(1), 67-70 (1987) and Jori, G.; Reddi, E.; Cozzani, I.; Tomio, L. Br. J. Cancer, 53(5), 615-21 (1986), for example.

[0051] The composition described herein may be administered in single (e.g., once daily) or multiple doses or via constant infusion. The compounds may also be administered alone or in combination with pharmaceutically acceptable carriers, vehicles or diluents, in either single or multiple doses. Suitable pharmaceutical carriers, vehicles and diluents include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. The pharmaceutical compositions formed by combining the compounds of this disclosure and the pharmaceutically acceptable carriers, vehicles or diluents are then readily administered in a variety of dosage forms such as tablets, powders, lozenges, syrups, injectable solutions and the like. These pharmaceutical compositions can, if desired, contain additional ingredients such as flavorings, binders, excipients and the like according to a specific dosage form. [0052] Thus, for example, for purposes of oral administration, tablets containing various excipients such as calcium carbonate may be employed along with various disintegrants such as starch, alginic acid and/or certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and/or acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tabletting purposes. Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules. Preferred materials for this include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration, the active pharmaceutical agent therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin and/or combinations thereof.

[0053] For parenteral administration, solutions of the compounds of this disclosure in sesame or peanut oil, aqueous propylene glycol, or in sterile aqueous solutions may be employed.

[0054] Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, and intraperitoneal administration. In this connection, the sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art.

[0055] The pharmaceutical composition provided herein can also be used with another pharmaceutically active agent effective for a disease such as a metabolic disturbance as described herein.

[0056] The compositions described herein can be formulated alone or together with the other agent in a single dosage form or in a separate dosage form. Methods of preparing various pharmaceutical fonnulations with a certain amount of active ingredient are known, or will be apparent in light of this disclosure, to those skilled in this art. For examples of methods of preparing pharmaceutical formulations, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19th Edition (1995). [0057] In some embodiments, the compositions described herein further comprise a carrier. In one embodiment, the carrier may be comprised of sequestering agents such as, but not limited to, collagen, gelatin, hyaluronic acid, alginate, poly(ethylenc glycol), alkylcellulose (including hydroxyalkylcellulose), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl- methylcellulose, and carboxymethylcellulose, blood, fibrin, polyoxyethylene oxide, calcium sulfate hemihydrate, apatites, carboxyvinyl polymer, and poly(vinyl alcohol). See for example, U.S. Pat. No. 6,620,406, herein incorporated by reference.

[0058] In one embodiment, the carrier may include buffering agents such as, but not limited to glycine, glutamic acid hydrochloride, guanidine, heparin, glutamic acid hydrochloride, acetic acid, succinic acid, polysorbate, dextran sulfate, sucrose, and amino acids. See for example, U.S. Pat. No. 5,385,887, herein incorporated by reference. In one embodiment, the carrier may include a combination of materials such as those listed above. By way of example, the carrier may be a PLGA/collagen carrier membrane.

[0059] In one embodiment, the composition according to this disclosure may be contained within a time release tablet. A bioactive agent described herein can be formulated with an acceptable carrier to form a pharmacological composition. Acceptable carriers can contain a physiologically acceptable compound that acts, for example, to stabilize the composition or to increase or decrease the absorption of the agent. Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, further antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the anti-mitotic agents, or excipients or other stabilizers and/or buffers. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the art would appreciate that the choice of a carrier, including a physiologically acceptable compound depends, for example, on the route of administration.

[0060] The composition can have a dosage of about 1 g to about 10 kg, for example, the dose may be at least, at most, or exactly 1 , 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8500, 9000, 9500, 10000, 1 1000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, or 20000 g (or any derivable range therein).

[0061] Embodiments of the composition can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable may include powder, tablets, pills, capsules. In some embodiments, the composition is in the form of a tablet, a solution, or a powder.

C. Examples

[0062] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0063] In the following examples, it is shown that: (a) when added to human urine in vitro, KOHCit is more effective than KCit in retarding crystallization of CaOx; (b) in ex vivo experiments with liver slices, the hepatic uptake (and presumed oxidation) of hydroxycitrate is less than that of citrate, thus, when given as potassium salts, less alkali load is provided by KOHCit than KCit; (c) in an experimental rat model, KOHCit increases urinary pH to a lesser extent than KCit, but produces a nearly equal rise in urinary citrate; and (d) KOHCit but not KCit increases urinary hydroxycitrate.

EXAMPLE 1

GREATER INHIBITION OF CALCIUM OXALATE CRYSTALLIZATION BY KOHCIT VERSUS KCIT WHEN ADDED TO HUMAN URINE IN VITRO

[0064] This experiment was conducted to elucidate the direct effect of hydroxycitrate, citrate and HCAL on crystallization of CaOx in vitro. FP and CG of CaOx were determined after adding analytical grade KOHCit, KCit, KC1 and HCAL to human urine samples.

[0065] Procurement of analytical-grade preparations. The materials tested were: a. Potassium chloride (KC1, sACS Grade, >99% purity, RPI Corp)

b. Potassium hydroxycitrate tribasic monohydrate (KOHCit, Sigma-Aldrich,

Cat#59847-5G, lot#BCBB2015V) (FIG. 1)

c. Potassium citrate tribasic monohydrate (KCit, Sigma-Aldrich, Lot#SLBP7939V) d (+)- Garcinia Acid (HCAL, Garcinia HCA lactone, Sigma-Aldrich, Cat #44292, Lot# BCBR3837V)

[0066] Analytical grade KOHCit, KCit and HCAL were purchased from Sigma-Aldrich.

To show purity of these compounds, they were analyzed by capillary electrophoresis (FIG.

2). Three distinct separate peaks were identified, where HCAL is indicated by #1 , citrate by #2, and hydroxycitrate by #3. Conditions for capillary electrophoresis were: polyacrylamide- coated capillary, 50 μιτι ID, 50 cm total length (39.8 cm effective length); 200 mM phosphoric acid, pH 6.0 (titrated with NaOH), 10% acetonitrile as background electrolyte; 0.5 psi pressure injection for 7 s; -23 kV applied voltage for 8 min; UV detection at 190 nm.

They were used in physicochemical experiments in human urine in vitro. The same sources of KOHCit and KCit were used in ex vivo experiments with rat liver slices and in in vivo rat experiments.

[0067] The method for the determination of FP and CG of CaOx was a miniaturized version of a technique developed by the main inventor in 1976 (Pak & Holt). In each experiment, urine was aliquoted into 96 well plate. A sodium oxalate standard solution of increasing concentration was added to each well. The plate was incubated at 37° C for 3 hours. The optical density of each well was read at 630 nm using a BioTek ELx808 Elisa Plate Reader with Gen5 software. In the plot of the increasing added oxalate concentration (increment in Ox) and the optical density (absorbance), the inflection point (coincident with the abrupt rise in optical density indicated by arrow) was calculated using SigmaPlot software (FIG. 3). The increment in oxalate at the inflection point estimated the FP or limit of metastability. CG of CaOx reflected the growth of crystals formed following nucleation. In the plot of the absorbance vs. increment in Ox, the area under the curve (AUC) reflected CG (shaded area, FIG. 3). Thus, a rise in FP indicated greater inhibition of CaOx nucleation, and reduced CG meant inhibition of further growth. [0068] Twelve 24-hour urine samples collected from different human subjects were tested. To each urine sample, KC1, KOHCit, or KCit was added to yield three test urine specimens, containing 6 meq KC1, 6 meq K and 6 meq citrate, and 6 meq K and 6 meq hydroxycitrate, respectively. Once these salts are dissolved in water, active anions are chloride, hydroxycitrate and citrate. Since the amount of K added was the same and chloride is inactive, these experiments compared the effect of hydroxycitrate versus citrate. In each specimen, FP and CG of CaOx was determined. FIG. 4 shows FP of CaOx in 12 human urine samples. FP was significantly higher on KCit and KOHCit than on KC1. FP was higher with KOHCit than with KCit. FIG. 5 shows CG of CaOx in 12 human urine samples. Compared to KC1 control, CG was significantly lower upon adding KCit or KOHCit. CG was lower with KOHCit than with KCit.

[0069] This demonstrates that hydroxycitrate was more potent than citrate in increasing FP of CaOx, indicative of greater inhibition of nucleation. Moreover, hydroxycitrate reduced CG of CaOx more prominently than citrate. These results, showing superior direct inhibitory action of hydroxycitrate, were unexpected.

[0070] Six 24-hour urine samples collected from different human subjects were tested. To each urine sample, HCAL 2 mM was added. Corresponding samples without HCAL represented the control.

[0071] FP of CaOx decreased slightly or did not change after addition of HCAL (FIG. 6). CG of CaOx increased slightly. Thus, HCAL is devoid of direct inhibitor activity against CaOx crystallization. Instead, it seems to have a slight promoter activity. The opposing action of HCAL over KOHCit emphasizes the importance of KOHCit in the prevention of CaOx stones.

EXAMPLE 2

SLOW AND INCOMPLETE HEPATIC UPTAKE AND PRESUMED OXIDATION OF HYDROXYCITRATE FROM EX VIVO EXPERIMENTS WITH RAT LIVER

SLICES

[0072] Ex vivo studies on the hepatic metabolism of hydroxycitrate were performed by incubating KOHCit and KCit with rat liver slices.

[0073] Materials used were: (a) a solution containing KOHCit 2.5 g/dL in sterile distilled water; (2) a solution containing 5 g/dL of KCit in sterile distilled water.

[0074] Under sterile condition, the liver was harvested from normal Sprague-Dawley rats. While kept in ice, ~ 40 mg liver mass was weighed, cut into ~1 mm thick slices in high glucose DMEM culture media. A solution of KOHCit was added to a high glucose DMEM culture medium to reach a concentration of 500 mg/dL. A solution of KCit was added to a high glucose DMEM culture medium to reach a concentration of 250 mg/dL.

[0075] Liver slices were incubated in culture media containing KOHCit or KCit at 37°C in a standard C0 ₂ incubator (95% air and 5% carbon dioxide). At 15 and 120 minutes, 10 μΐ _^ of cultured medium was taken for the measurement of hydroxycitrate and citrate by capillary electrophoresis. After 120 minutes, the hydroxycitrate concentration in the culture media was reduced by 18% from the value at 15 minutes (FIG. 7B). The citrate concentration in the culture media was reduced by 37% (FIG. 7D). This reduction of hydroxycitrate and citrate indicates uptake and presumed oxidation of hydroxycitrate and citrate by liver slices.

[0076] In summary, the above results are compatible with the contention that hepatic uptake (and presumed oxidation) of hydroxycitrate is less efficient than that of citrate. The results of ex vivo studies with liver slices suggest that a larger amount of accompanying anion might be left non-metabolized following KOHCit ingestion than after KCit. Thus, when delivered in equimolar amounts, KOHCit should confer a lower alkali load than KCit.

EXAMPLE 3

PARTIAL ALKALI LOAD AND URINARY ALKALINIZATION BUT OPTIMUM CITRATURIC RESPONSE BY KOHCIT FROM IN VIVO STUDIES IN RATS

[0077] To prove that a presumed impairment of hepatic oxidation of hydroxycitrate results in a lower alkali load by KOHCit than KCit, the following metabolic and physicochemical studies were conducted in rats. Metabolic parameters measured included urinary potassium, pH, ammonium, NAE, citrate and hydroxycitrate. Alkali load was indicated by increased pH, and reduced ammonium and NAE. Physicochemical parameters included FP and CG of CaOx. Inhibitors measured included hydroxycitrate and citrate. [0078] Six adult Sprague-Dawley rats were kept in individual metabolic cages while eating the same rat chow. After stabilization, 3 rats received KOHCit and 3 rats KCit for 7 days. KOHCit and KCit were added to drinking water, to target ingestion of -10 meq/kg/day. While rats were kept in individual metabolic cages, a 24-hour urine was collected for 2 days before, and on days 6 and 7 of treatment. Urine samples were analyzed for potassium, pH, ammonium, and NAE. NAE was obtained by calculating titratable acidity from urinary constituents (Kok, 1993), where NAE = titratable acidity + ammonium - citrate. [0079] Table 1 illustrates the effect of KOHCit compared to KCit on acid base balance and urinary citrate in rats evaluated in vivo. The mean data for two days prior to treatment (pre) and on days 6 and 7 of treatment (on) are shown. Urinary potassium rose following both treatments (KCit and KOHCit) to an equivalent degree. Thus, both salts were absorbed to a similar degree. Urinary pH rose modestly with KOHCit; it increased markedly with KCit. Urinary ammonium fell less markedly with KOHCit than with KCit. NAE decreased with both treatments; this decline was less marked with KOHCit. These results provide metabolic evidence that KOHCit confers less alkali load than KCit.

Table 1. Effect of KOHCit and KCit on Urinary Parameters

KCit KOHCit

Pre On Pre On

K, meq/day 1.44 ± 0.30 3.66 ± 0.29 1.54 ± 0.11 3.70 ± 0.51 pH 6.10 ± 0.04 7.17 ± 0.30 6.23 ± 0.34 6.63 ± 0.10

NH ₄ , meq/day 0.46 ± 0.09 0.22 ± 0.06 0.45 ± 0.06 0.38 ± 0.05

NAE, meq/day 0.10 ± 0.06 -1.34 ± 0.13 -0.01 ± 0.14 -0.77 ± 0.26

Citrate, mcq/day 0.48 ± 0.07 1.26 ± 0.19 0.56 ± 0.04 1.20 ± 0.31

FP 96.4 ± 34.9 176.0 ± 0.0 81.8 ± 10.0 160.4 ± 27.0

CG 8.38 ± 1.32 0.00 ± 0.00 6.79 ± 3.90 0.02 ± 0.04 Mean ± SD are presented. FP = formation product; CG = crystal growth.

[0080] FIG. 8 plots the above data in another way by displaying the changes in various urinary parameters from baseline to treatment with KOHCit or KCit. The line above or below the bars indicate plus or minus SD. The same directional changes were shown. Compared with KCit, KOHCit produced a less prominent increment in urinary pH, and less decrement in urinary ammonium and NAE.

[0081] Despite delivery of lower alkali load, KOHCit elicited a nearly equivalent rise in urinary citrate as KCit (Table 1, FIG. 8). From individual data derived from six rats during all four days (two days before and last two days on treatment), urinary citrate was plotted against corresponding urinary NAE. The regression line for KOHCit (dashed line) is compared with that for KCit (solid line) in FIG. 9. The slope of the line was steeper for KOHCit than KCit (p = 0.01). The shift in NAE to the left of x-axis indicates a delivery of increasing alkali load. Thus, the peak urinary citrate excretion occurred at a lower alkali load from KOHCit, compared with KCit. [0082] A similar discordance was shown for the relationship between urinary citrate and urinary pH (Table 1 , FIG. 8). KOHCit increased urinary pH from 6.23 (within normal range) to 6.63 (still within normal range), an increment of 0.4. KCit raised from pH from 6.10 (within normal range) to 7.17 (high), a much greater increment of 1.07. However, urinary citrate rose similarly to 1.20 meq/day (high) on KOHCit and 1.26 meq/day (high) on KCit, with a similar increment of 0.64 and 0.78 meq/day, respectively. Thus, KOHCit produced an optimum rise in urinary citrate, without over-alkalinizing the urine. In contrast, while it raised urinary citrate optimally, KCit produced alkaline urine conducive to calcium phosphate stone formation. [0083] Urinary hydroxycitrate was measured in urine by capillary electrophoresis. A distinct peak of hydroxycitrate was found in urine of rats treated with KOHCit but not with KCit. Thus, some hydroxycitrate was absorbed and excreted in urine following KOHCit treatment. Hydroxycitrate can potentially increase urinary citrate by inhibiting renal citrate lyase (Melnick, 1996). A nearly equivalent rise in urinary citrate from KOHCit despite a lower alkali load might be explained by inhibition of citrate lyase by hydroxycitrate. From individual data derived from six rats during all four days (two days before and two days on treatment), urinary citrate was plotted against corresponding urinary NAE. The regression line for KCit (solid line) is compared with that for KOHCit (dashed line) in FIG. 9. The slope of the line was steeper for KOHCit than KCit (p = 0.01 ). [0084] Both KOHCit and KCit increased FP, and to an equivalent degree (Table 1 , FIG. 8). CG was reduced by both drugs to an equivalent degree. The marked and equivalent inhibition of CaOx oxalate crystallization during KOHCit and KCit treatments might be ascribed to a similar dramatic rise in urinary citrate by the two treatments. The renal excretion of hydroxycitrate following KOHCit treatment might further inhibit calcium oxalate crystallization. However, the marked hypercitraturia in both groups from the use of a high dose of the drugs might have obscured revelation of a separate inhibitory action of hydroxycitrate. EXAMPLE 4

ADDITIONAL IN VIVO RODENT STUDIES

[0085] Additional rodent studies in vivo, employing lower amount of targeted dose of KOHCit and Cit, and a chow with a neutral or slightly acid in ash content, will be performed.

EXAMPLE 5

EFFECT OF COMMERCIAL GARCINIA PRODUCTS ON CALCIUM OXALATE CRYSTRALLIZATION IN HUMAN URINE IN VITRO

[0086] Four commercial preparations of Garcinia Cambogia were tested.

- Garcinia HCA (Life Extension, lot# Ml 6340C03)

Super HCA (SuperCitrimax, Douglas Laboratories, lot# 50054510)

- HCActive Garcinia Cambogia Extract (Jarrow, lot#57388A17)

- Pure Garcinia Cambogia (Nutrition Forest, lot# 3501 17)

[0087] From the estimated content of hydroxycitrate, sufficient amount of each powdered Garcinia preparation was added to each sample of human urine. After mixing for at least 30 minutes to ensure solubility of all hydroxycitrate and derivatives, the suspension was filtered through 0.22 micron filter, and then through a filter with molecular weight cutoff of 50,000. The urine filtrate containing presumed hydroxycitrate and derivatives was used to obtain FP and CG of calcium oxalate by the method described previously. Urine sample without added Garcinia preparation served as the control.

[0088] As shown in Fig. 10 (top), FP of calcium oxalate was not different or slightly lower in urine samples containing Garcinia products compared with control. Vertical bars indicate mean ± SD. P values from control are shown. Conversely, CG of calcium oxalate was not different or slightly higher in urine samples with added Garcinia products compared with control (Fig. 10, bottom).

[0089] Thus, commercial Garcinia products promoted or did not influence nucleation and growth of calcium oxalate. This distinctly different action of commercial Garcinia products emphasizes the unique inhibitory effect of hydroxycitric acid or KOHCit described above.

* * * [0084] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. All references cited in this application are specifically incorporated by reference for all purposes

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Heymsfield SB, Allison DB, Vasselli JR, et al. Garcinia cambogia (hydroxycitric acid) as a potential antiobesity agent: a randomized controlled trial. JAMA, 1998, 280: 1596-1600

Kok DJ, Bijvoet OLM, Papapoulos SE. Excessive crystal agglomeration with low citrate excretion in recurrent stone formers. Lancet, 1986; i: 1056-1058. Melnick JZ, Srere PA, Elshourbagy NA, Moe OW, et al. Adenosine triphosphate citrate lyase mediates hypocitraturia in rats. J Clin Invest, 1996, 98:2381 -2387.

Pak CYC, Eanes ED, Ruskin B. Spontaneous precipitation of brushite: evidence that brushite is the nidus of renal stones originating as calcium phosphate. Proc Natl Aca Sci, 1971 , 68: 1456-1460. Pak CYC, Fuller CJ, Sakhaee K, et al. Long-term treatment of calcium nephrolithiasis with potassium citrate. J Urol, 134: 1 1-19, 1985.

Pak CYC, Holt K. Nucleation and growth of brushite and calcium oxalate in urine of stone- formers. Metabolism, 1976, 25: 665-673.

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