Theobromine or its derivatives for the treatment or prevention of renal lithiasis

Abstract

The present invention refers to the use of theobromine or its derivatives as an inhibitor of the crystallization of uric acid to avoid the formation of uric acid crystals in urine and as a consequence renal lithiasis or specifically uric acid renal lithiasis.

Claims

1. A method of treating renal lithiasis in a subject, comprising administering to the subject a composition comprising a compound with general formula (I) ##STR00002## where: R.sub.1 and R.sub.2 are methyl, or any of the compound's pharmaceutically acceptable salts, wherein the renal lithiasis is uric acid renal lithiasis.

2. The method of claim 1, for reducing risks and improving health status of the subject with diseases related to the crystallisation of uric acid.

3. The method of claim 1, where the composition is a pharmaceutical composition, functional food, nutraceutical product or food supplement.

4. The method of claim 1, where the compound of general formula (I) wherein R1 and R2 are methyl, or any of the compounds pharmaceutically acceptable salts, is administered in a dose of between 100 mg/day and 380 mg/day.

5. The method of claim 1, where the composition further comprises lactose, sucrose, talc, magnesium stearate, cellulose, calcium salts, gelatine and/or fatty acids.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Kinetics curves for the crystallisation of uric acid (400 mg/l) in synthetic urine at pH=4.67, at different theobromine concentrations. The absorbance (at 550 nm) is plotted against time (in minutes).

(2) FIG. 2. Graphical representation of the induction periods (in minutes) for a solution of 400 mg/l uric acid in synthetic urine, for different pH values and concentrations of theobromine.

(3) FIG. 3. Uric acid crystals observed using scanning electron microscopy, obtained in synthetic urine at pH=4.67 in the absence of theobromine (A) and the presence of 20 mg/l (B) and 40 mg/l (C) theobromine.

(4) FIG. 4. Flow diagram of the system used, consisting of a synthetic urine solution and a uric acid solution, each driven by a peristaltic pump with a different flow rate, a T-shaped connection and a chamber containing the uric acid calculus.

(5) FIG. 5. Percentage increase in mass (with standard deviation bar) in vitro of renal uric acid calculi fragments from a single patient, obtained after extracorporeal shock wave lithotripsy, when subjected to a constant flow of synthetic urine for 48 hours in the presence of different concentrations of theobromine.

(6) FIG. 6. Scanning electron microscope image of fragments of renal uric acid calculi that have grown in contact with synthetic urine without theobromine (A) and with 20 mg/l theobromine (B).

EXAMPLES

(7) A turbidimeter was used to obtain the kinetic curves for uric acid crystallisation corresponding to a solution of 400 mg/l uric acid in synthetic urine (average composition similar to urine) at different pH and theobromine concentrations. FIG. 1 shows the kinetic curves for uric acid (without theobromine) at pH=4.67 and for the same uric acid concentration with 10 mg/l, 20 mg/l and 40 mg/l theobromine added. As can be seen, the induction time (time when crystals begin to appear) increases considerably with increased theobromine concentration, indicating that this substance acts as a uric acid nucleation inhibitor.

(8) The induction times were therefore calculated for 400 mg/l of uric acid in synthetic urine for different pHs and different theobromine concentrations (Table 2). As can be seen in the graphs in FIG. 2, the induction times increase with increased theobromine concentrations. In addition, the inhibitory effect is much more pronounced with increased urinary pH.

(9) TABLE-US-00001 TABLE 1 Induction times (and standard deviation (SD)) for a solution of 400 mg/l uric acid in synthetic urine at different pH and theobromine concentrations. Theobromine conc. Induction time (mg/l) (min) SD pH = 4.39 0 2.3 0.07 10 4.3 0.4 20 6.8 0 40 15 2.8 pH = 4.50 0 3 0 10 3.9 0.5 20 6.9 0.14 40 14.8 1 pH = 4.67 0 6 0.6 10 13 3 20 23 3 40 38 4

(10) The precipitate generated during the turbidimetry experiment was vacuum filtered, the crystals were allowed to dry and they were then examined under a scanning electron microscope to see if there were any morphological differences between the crystals formed under differing theobromine concentrations. FIG. 3 shows the crystals generated in a 400 mg/l solution in synthetic urine at pH=4.67, without theobromine and with 20 mg/l and 40 mg/l theobromine. As can be seen, the uric acid crystals' morphology becomes much narrower and more elongated with increasing theobromine concentrations. This indicates that the inhibitor acts on the crystals' lateral faces, preventing growth in that area. Therefore, in addition to being an inhibitor of uric acid nucleation, it also acts as an inhibitor of crystal growth.

(11) The last experiment performed was a study on the effect of theobromine as an inhibitor of uric acid crystal growth. In order to do this, fragments of uric acid calculi from the same patient (obtained by extracorporeal shock wave lithotripsy) were placed in a flow of synthetic urine with 400 mg/l uric acid and different concentrations of theobromine using a flow system as shown in FIG. 4. This system had two peristaltic pumps. One of them pumped the synthetic urine without uric acid, at pH=3.00 (4-A). The other pumped a 2 g/l solution of uric acid at pH=10.70 (4-B). The temperature of both solutions was regulated at 37? C. (5). For the phases studying the effects of theobromine, it was dissolved in the synthetic urine solution. These solutions (synthetic urine and uric acid) were mixed in the T-shaped connection (3) to produce synthetic urine with 400 mg/l uric acid at a pH or around 5.40. The peristaltic pumps had different flow rates as the final synthetic urine had to have 400 mg/l uric acid and the total daily volume had to be 750 ml, which is the mean volume of urine that passes through a kidney.

(12) The solution resulting from the mixing of the uric acid and synthetic urine solutions is passed through a chamber containing a uric acid calculus of known weight (2); the chamber was located within an oven at 37? C. (1). The flow system is maintained for 48 hours. At the end of this time the calculi are dried in an oven and weighed again. The % increase in mass is then calculated.

(13) FIG. 5. shows the percentage increase in mass in relation to the concentration of theobromine. A total of 5 calculi were used for each theobromine concentration. As can be seen, as the theobromine concentration increases, the % in mass of the calculus fragments decreases, with the increase in mass practically zero at 20 mg/l theobromine.

(14) A total of two fragments were examined using scanning electron microscopy in order to see if there were any morphological differences between the calculi in relation to theobromine concentration after the calculi were exposed to the flow process (FIG. 6). It can be seen that when there is no inhibitor the fragments that have grown on the surface of the calculi are larger than when theobromine is present.

(15) All the previous experiments demonstrate theobromine's high capacity to inhibit uric acid crystal nucleation and growth, with a potential application in the treatment and prevention of renal lithiasis.