TECHNICAL FIELD OF THE INVENTION

The present invention relates to novel prodrugs of metformin, their pharmaceutically acceptable salts, pharmaceutical compositions containing the prodrugs, and methods of using the prodrugs. In particular, the present invention relates to prodrugs wherein sulfur- containing promoieties are attached to metformin to form novel bioreversible sulfenyl guanidine (N-S) prodrugs of metformin with improved oral absorption, and consequently promoted bioavailability.

BACKGROUND OF THE INVENTION

Metformin, iV,./V-dimethyl imidodicarbonimidic diamide, is a potent insulin-sensitizing biguanide used to treat type 2 diabetes (Davidson and Peters 1997; Kirpichnikov, McFarlane, and Sowers 2002) and usually considered to be a first-line treatment, particularly in obese and/or hyperlipidemic non-insulin-dependent diabetes mellitus patients (Effect of intensive blood-glucose control with metformin on complications in overweight patients with type 2 diabetes (UKPDS 34). UK Prospective Diabetes Study (UKPDS) Group 1998). Moreover, metformin is used as adjunct therapy to virtually every other antihyperglycemic medicine available today. The antihyperglycemic effects of metformin are not only the inhibition of intestinal glucose absorption and the improvement of peripheral and hepatic insulin sensitivity but also the reduction of hepatic glucose production and the enhancement of peripheral glucose utilization, although the exact mechanism of action is still uncertain (Jackson et al. 1987; Klip and Leiter 1990). Also a number of other conditions, such as polycystic ovary syndrome (PCOS) and nonalcoholic fatty liver disease (NAFLD), to be treated with metformin have been explored. However, metformin is highly basic and fully protonated under physiological conditions (pH 1.2-7.4), and therefore, is slowly and incompletely absorbed from the upper intestine after oral administration resulting in the poor bioavailability. Previously, various formulation strategies, for example, extended-release formulations, have been developed attempting to ameliorate the malabsorption of metformin with trivial results (Belcher et al. 2005; Cullen et al. 2004).

Metformin (Λ/,Λ/-dimethyl imidodicarbonimidic diamide)

Prodrugs are pharmacologically inactive or impaired, bioreversible derivatives of drug molecules utilised to improve the unfavourable physicochemical, pharmaceutical or biopharmaceutical properties of a parent drug (Prodrugs: Challenges and Rewards. 2007; Rautio et al. 2008; Stella and Himmelstein 1985). Accomplishing good membrane permeability for high passive transcellular absorption after oral administration by masking hydrogen bonding groups of an active compound is probably one of the most commonly introduced prodrug strategy.

Recent publications (Guarino, Karunaratne, and Stella 2007; Hemenway et al. 2007; Guarino, Karunaratne, and Stella 2003; Gilead Sciences Inc. Viread® (Tenofovir Disoproxil Fumarate) full prescribing information. Foster City (CA): Gilead Sciences Inc., 2006 March, WO 03/032908) report prodrugs wherein sulphur-containing promoieties are attached to pharmaceutical compounds which contain one or more N-H bonds to produce prodrugs containing at least one N-S bond. A patent application by Zhaojun (Zhaojun 2005, CN1583717) describes amino acid derivatives of metformin which are more likely amides. Moreover, a US patent (Rapin and Halbitte 2007, US 7,265,156 B2) describes metformin linked with a carrier, in that case an amino acid arginine, via linker group. However, no such "sulfenyl guanidine prodrug(s)", wherein "sulfenyl guanidine prodrug(s)" refers to prodrug(s) having a N-S bond, and wherein the S is bivalent, have been described for guanides including metformin. Moreover, up to date, the study of prodrugs of guanidines is confined (Hernandez-Luis et al. 2001; Saulnier et al. 1994; Zhang et al. 2002; Schuster, Bernhardt, and Buschauer 1997; Guan et al. 2005), despite commonness and importance of guanidines and biguanidines as medical applications (Greenhill and Lue 1993; Arafa et al. 2005). For example, no prodrug derivatives of metformin have been reported. Thus, there is a need for metformin prodrugs, which are readily bioconverted to metformin in vivo and able to improve oral bioavailability of metformin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to novel sulfenyl guanidine prodrugs of metformin and their pharmaceutically acceptable salts, as well as to methods of using such prodrugs. The particular embodiments described herein are intended in all respects to be illustrative rather than restrictive. Alternative embodiments will become apparent to those skilled in the art to which the present invention pertains without departing from its scope.

The present invention concerns sulfenyl guanidine prodrugs of metformin and the pharmaceutically acceptable salts thereof. As used herein, the term "sulfenyl guanidine prodrug(s)" refers to prodrug(s) having a N-S bond, wherein the S is bivalent.

The formula below depicts the general structure of sulfenyl guanidine prodrugs of the present invention wherein -S-R represents the sulphur-containing promoiety.

Preferred groups of R in the sulphur-containing moiety include but are not limited to a saturated or unsaturated, straight or branched chain of 1 to 10 carbon atoms or a saturated or unsaturated cyclic ring of 3 to 10 carbon atoms optionally containing a heteroatom, or an aromatic or heteroaromatic ring and all these backbones can optionally be substituted with 1 to 3 groups selected from hydroxyl, amino, mono- or dialkylamino, acylamino, carboxyl, alkylcarboxyl, acyl, aryl, aroyl, aralkyl, cyano, nitro, alkoxy and alkenyloxy.

Even more preferably R is a saturated or unsaturated straight or branched alkyl chain of 2 to 8 carbon atoms, or a saturated or unsaturated cyclic ring of 4 to 8 carbon atoms optionally containing a heteroatom, said straight or branched alkyl chain or cyclic ring optionally being substituted with 1 to 3 groups selected from amino, mono- or dialkylamino, acylamino, carboxyl or alkylcarboxyl, or a phenyl ring optionally with 1 to 2 substituent groups selected from amino, mono- or dialkylamino, acylamino, carboxyl or alkylcarboxyl.

Examples of most preferred groups of R include cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, tetrahydro-2H-pyranyl, tetrahydrofuranyl, phenyl, cysteine (without double S in the structure, -N-Cys), branched -CHR' ₂ or -CR' ₃ , in which R' is straight or branched chain of 1 to 5 carbon atoms, such as i-propyl, sec-butyl, £-butyl, optionally substituted with 1 to 3 groups selected from amino, mono- or dialkylamino, acylamino, carboxyl or alkylcarboxyl.

A straight or branched alkyl chain preferably contains 1 to 10 carbon atoms, even more preferably 2 to 8 carbon atoms.

Aromatic or heteroaromatic groups include but are not limited to groups such as phenyl, naphthelenyl, benzyl, benzoyl, pyridyl, furanyl, pyrronyl, thiophenyl, thiazol, imidazolyl, indolyl, benzofuranyl.

Heteroatoms, if present, are selected from the group consisting of N, O and S.

A suitable pharmaceutical composition according to the invention comprises a metformin prodrug of the invention and a pharmaceutical acceptable vehicle, with which the compound is administered to a patient. Water is a preferred vehicle when the compound is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions. Suitable pharmaceutical vehicles also include excipients such starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, ethanol, and the like. The present pharmaceutical compositions, if needed, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents may be used.

The compositions according to the invention can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquid, powders, sustained- release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for pharmaceutical use.

The present prodrug and/or pharmaceutical compositions thereof may be administered through a variety of routes of administration. Administration can be systemic or local.

Methods for administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intravaginal, transdermal, rectal, topical (e.g., skin, eyes, nose, ears), or by inhalation. Various delivery systems can be used for administration (e.g., encapsulation in liposomes, microparticles, microcapsules, capsules).

A further object of the invention is the use of the novel sulfenyl guanidine prodrugs of metformin for the preparation of a medicament for the treatment of type 2 diabetes.

A still further object of the invention is a method for treating type 2 diabetes in a mammal, preferably in a human, which comprises administering to said mammal an effective amount of a compound according to the invention to provide therapeutically effective amount of metformin suitable for the desired treatment and management of type 2 diabetes. An effective amount of metformin refers to an amount which is effective in achieving the desired antidiabetic and antihyperglycemic effects. A typical dose of metformin for an adult human is 500 mg 2 to 3 times a day, total daily doses of for example 0.5 to 3 g per day being possible. However, the improved bioavailability of metformin ascribed to the novel prodrugs of the present invention may result in improved efficacy, reduced gastro-intestinal adverse effects and/or lower required daily orally administered therapeutic doses.

The invention is also directed to a method for increasing oral bioavailability of metformin, comprising the step of preparing a metformin prodrug of the formula

wherein R is as defined above, or a pharmaceutically acceptable salt thereof. The novel sulfenyl guanidine prodrugs of the invention are readily absorbed from the gastrointestinal tract either by passive and/or carrier-mediated absorption and/or any other permeation mechanism and bioconverted to metformin in vivo. The bioavailability of the novel prodrugs of metformin is improved compared to the bioavailability of metformin. Consequently, lower doses of orally administered metformin are necessary to achieve and maintain the required therapeutical effects. This means less side effects and better patient compliance.

The following examples illustrate the invention without limiting the same in any way.

EXAMPLES

The general synthetic pathway to the sulfenyl guanidine prodrugs of metformin is shown below.

I b R = Ph* 2b R = Ph

1c R = CH ₂ CH(CO ₂ Me)NHBoC 2c R = CH ₂ CH(CO ₂ Me)NHBoC

Id R = CMe ₃ 2d R = CMe ₃

* Commercially available

Reaction conditions: a) Acetonitrine (ACN), microwave irradiation (MW) 100 ⁰ C, 10 min, b) metformin (1,1- dimethyl imidodicarbonimidic diamide), dimethylformamide (DMF), room temperature (RT), 24 h.

Example 1: Synthesis of 2-(Cyclohexylthio)isoindoline-l,3-dione.

The following compound was used as an intermediate product in the synthesis of sulfenyl guanidine prodrugs of metformin.

Dicyclohexyl disulphide (0.51 g, 2.21 mmol) and iV-bromophthalimide (0.50 g, 2.21 mmol) in anhydrous ACN (20 ml) were irradiated at 100 ⁰ C (1 bar) in a microwave reactor for 10 min. The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography eluting with hexane/ethyl acetate (2:1) solution to obtain the compound 1 as a light yellow solid (0.29 g, 52%).

¹ H NMR (CDCl ₃ ): δ 7.95-7.91 (2H, dd, ³ J _HH = 5.46 Hz, ⁴ J _HH = 3.07 Hz), 7.81-7.76 (2H, dd, ³ JHH = 5.46 Hz, ⁴ J _HH = 3.07 Hz), 3.22-3.15 (IH, tt, ³ J _HH = 3.63, 10.88 Hz), 1.95-1.88 (2H, m), 1.84-1.75 (2H, m), 1.65-1.57 (IH, m), 1.43-1.33 (2H, m), 1.32-1.18 (3H, m).

Example 2: Synthesis of ^^-Dimethyl-S-cyclohexyl-Λ^-thiohydroxylbiguanidine (R=C ₆ H ₁₁ ).

Metformin (/VyV-dimethyl imidodicarbonimidic diamide hydrochloride) (1.0 g, 6.0 mmol) in 10 ml of 1 M NaOH was stirred at room temperature for 30 min. Water was evaporated in vacuo and the residue was dissolved in 30 ml of MeOH. The solvent was evaporated and the residue was redissolved in 20 ml of MeOH. NaCl was filtered out of the solution and the filtrate was evaporated to yield basic metformin as white solid (0.77 g, 99%). The basic metformin (0.15 g, 1.15 mmol) and 2-(cyclohexylthio)isoindoline-l,3-dione (0.29 g, 1.15 mmol) (the example 1) in anhydrous DMF (10 ml) was stirred overnight. The solvent was evaporated in vacuo and the residue was purified by flash chromatography eluting with MeOH/dichloromethane (DCM) (0.5:10) solution to obtain the white solid compound 2a (88 mg, 33%).

¹ H NMR (DMSO): δ 8.33 (IH, s), 7.50 (2H, s), 7.18 (IH, s), 2.95 (6H, s), 2.93-2.87 (IH, m), 1.91-1.84 (2H, m), 1.76-1.69 (2H, m), 1.61-1.55 (IH, m), 1.33-1.15 (5H, m). ESI-

MS: m/z = 244.2 (M+H) ⁺ . Example 3: Synthesis of Λ^Λ^-Dimethyl-S-phenyl-Λ^-thiohydroxylbiguanidine

The compound 2b was synthesized as example 2 above.

¹ H NMR (DMSO): δ 8.97 (IH, s), 7.65 (2H, s), 7.40-7.34 (2H, m), 7.30-7.25 (3H, m), 7.23-7.18 (IH, m), 2.90 (6H, s). ESI-MS: m/z = 238.1 (M+H) ⁺ .

Example 4: Synthesis of N ¹ ^V ¹ -Dimethyl-5-[N ⁶ ((ter/-butyloxy)carbonyl)-cysteine methyl ester]-N ⁴ -thiohydroxylbiguanidine

The compound 2c was synthesized as example 2 above.

¹ H NMR (DMSO): δ 8.52 (IH, s), 7.6 (2H, bs), 7.19 (IH, s), 4.83-4.77 (IH, m), 3.59 (3H, s), 3.30-3.15 (2H, m, partially under residual H ₂ O signal), 2.92 (6H, s), 1.48 (9H, s). ESI-MS: m/z = 363.2 (M+H) ⁺ .

Example 5: Synthesis of Λ^jΛ^-Dimethyl-S-tert-butyl-Λ^-thiohydroxyl-biguanidine (R=C ₄ H ₉ ). The compound 2d was synthesized as example 2 above.

¹ H NMR (CD ₃ OD): δ 3.20 (6H, s), 1.39 (9H, s). ¹³ C NMR (CD ₃ OD): δ 159.2 s, 155.9 s, 49.9 s, 38.0 q, 27.2 q. ESI-MS: m/z = 218.1 (M+H) ⁺ .

Evaluation of sulfenyl guanidine prodrugs of metformin The novel sulfenyl guanidine prodrugs of metformin (2a-b) have been evaluated in in vitro and in vivo studies. Aqueous solubility and conversion of the prodrug is described below.

1) Stability of sulfenyl guanidine prodrugs of metformin in buffer solutions. Method

The rates of chemical stability of the metformin sulfenyl guanidine prodrugs were determined at 37 ⁰ C in 50 mM (ionic strength 0.15) HCl buffer at pH 1.0, acetate buffer at pH 4.0, phosphate buffer at pH 7.4 and borate buffer at pH 9.0. The incubation mixtures were prepared by dissolving 5-10 mM stock solutions of prodrugs in H ₂ O in preheated buffer solutions. The prodrug mixtures of about 200 μM were incubated in a water bath at 37 ⁰ C and the samples were withdrawn at appropriate intervals. ACN was added to the samples (1: 1, v/v) to hinder further degradation during the high-performance liquid chromatography (HPLC) analyses. The concentration of metformin and metformin sulfenyl guanidine prodrugs were analyzed by HPLC.

Results

The half-lives (ti _/2 ) for the chemical degradation of prodrugs in aqueous buffer solutions, determined from the linear slopes of the plots by taking the logarithm of remaining prodrug over time, are shown in Table 1. When degraded, the hydrolysis of the prodrugs followed pseudo-first-order kinetics and released metformin. The prodrugs were reasonable stable in aqueous buffer solutions (> 80 h) at pH 4.0 and 7.4. In the acidic conditions prodrugs showed lower stability (ti _/2 = 12 — 478 min).

2) Biocon version of metformin sulfenyl guanidine prodrugs in vitro. Method

The bioconversion of the metformin sulfenyl guanidine prodrugs were determined in 80% human serum, rat serum and 20% rat liver homogenate diluting with phosphate buffer (pH 7.4) at 37 ⁰ C. Prodrugs were dissolved in 50 mM phosphate buffer pH 7.4 (ionic strength 0.15) and the stock solutions as well as the biological material were preheated in a water bath. The reactions were initiated by adding the stock solutions to serum/liver homogenate. The mixtures were placed in a water bath at 37 ⁰ C and the samples were withdrawn at appropriate intervals. Ice-cold ACN was added to the samples (4:1, v/v) to precipitate proteins and hinder further degradation and the samples were centrifuged for 10 min at 12 000 rpm and kept on ice until injected into the HPLC system. The concentration of the prodrugs and metformin were analyzed from the supernatants by the HPLC.

Results

The prodrug 2b released metformin extremely fast with the half-lives ranging from 4 seconds to 40 minutes, whereas the prodrug 2a released hardly any metformin in vitro during 24 hours (Table 1). Due to possible involvement of free radicals in the bioconversion in vitro, the prodrug 2b was activated more readily compared to the prodrug 2a.

Table 1. Half-lives of the Metformin Prodrugs 2a and 2b in 50 mM Buffer Solutions, 80% Human Serum, 80% Rat Serum, 20% Rat Liver Homogenate at 37 ⁰ C (mean + SD, n

= 3).

Solution t _V2 of 2a t _1/2 of 2b pH l.O 477.74 ± 41.37 min 12.13 ± 2.82 min pH 4.0 78.60 ± 3.10 h pH 7.4 78.56 ± 2.41 h pH 9.0 18.14 ± 1.11 min

80% Human Serum 3.74 ± 0.90 s 80% Rat Serum 36.81 ± 2.45 min

20% Rat liver 14.98 ± 3.18 s Homogenate u 10-20% of the prodrug was degraded to metformin during the 4 weeks incubation.

* 40-50% of the prodrug was degraded to metformin during the 4 weeks incubation. c 10-20% of the prodrug was degraded to metformin during the 24 hours incubation. 3) Distribution coefficients of metformin and metformin sulfenyl guanidine prodrugs in octanol/water.

Method

The distribution coefficients (log D) of metformin and metformin sulfenyl guanidine prodrugs were determined at room temperature from the distribution of the compounds between a mixture of 1-octanol and 50 mM acetate/phosphate buffer (pH 4.0 or 7.4) system. 1 mL of metformin or prodrug solution (150 μg/mL) in 50 mM buffer was added to 4 ml of 1-octanol saturated with desired buffer. The mixtures were shaken for 1 h and the phases were separated. The concentration of metformin or the metformin sulfenyl guanidine prodrugs in the buffers before and after the shaking were analyzed by HPLC.

Results

The log D values of the prodrugs 2a and 2b as well as metformin are shown in Table 2. The log D values of the prodrugs were significantly higher than those of metformin. The calculated logP of 2c is 1.74 (ChemBioDraw Ultra 11.0).

Table 2. Octanol/Water Distribution Coefficients (log D) of Metfomin and Metfomin Prodrugs 2a and 2b at pH Values of 4.0 and 7.4 (mean ± SD, n = 3 unless otherwise mentioned).

log D pH 4.0 pH 7.4

Metformin -3.41 ± 0.71 ⁽ⁿ⁼²⁾ -3.37 + 0.39

Prodrug 2a -1.10 ± 0.02 0.49 + 0.01

Prodrug 2b -0.70 + 0.01 -0.76 + 0.02

4) In vivo studies of metformin sulfenyl guanidine prodrugs Methods

Adult male Wistar rats weighing 250 ± 5 g were treated with metformin hydrochloride or metformin prodrugs both intravenously and orally.

In case of intravenous administration, compounds were dissolved either in 0.9% NaCl solution or 10% HP-β-CD in 0.6% NaCl solution, and infused via the jugular vein over 0.5 min (total injection volume of 0.3 ml) to the rats. An approximately 200 μl aliquot of blood sample was collected via the jugular vein at regular time intervals after the start of the intravenous administration to micro test tubes containing 3% ethylenediaminetetraacetic acid (EDTA) in 0.7% NaCl solution. The drawn blood volume was substituted by 0.9 % NaCl immediately after each blood sampling.

In case of oral administration, the compounds were dissolved either in 0.9% NaCl solution or 10% HP-β-CD in 0.6% NaCl solution and orally administered (total injection volume of 1.0 ml) to the rats using a feeding tube. An approximately 200 μl aliquot of blood sample was collected via the jugular vein at regular time intervals after the start of the oral administration to micro test tubes containing 3% EDTA in 0.7% NaCl solution. The drawn blood volume was substituted by 0.9 % NaCl immediately after each blood sampling.

Blood samples were centrifuged 5 min at 14 000 rpm immediately after each blood sampling and 100 μl aliquot of each plasma sample and 20 μl of 5.2 mg/ml 1,1,3,3- tetramethylguanidine as an internal standard were vortexed with ice-cold ACN (1:4, v/v) to precipitate proteins and hinder further degradation. The samples were centrifuged 5 min at 14 000 rpm and kept on ice until injected into the HPLC system. The pharmacokinetic analysis was performed by GraphPad Prism software, version 4.03 (GraphPad Software, Inc., San Diego, CA, USA). The area under the plasma concentration - time curve (AUC) was calculated using the linear trapezoidal method. The maximum plasma concentration (C _max ) and the time to reach the maximum plasma concentration (t _max ) were read directly from the plasma concentration - time data.

Results

The bioconversion of sulfenyl guanidine prodrugs of metformin is expected to occur from a nucleophilic substitution reaction by endogenous nucleophiles (glutathione, cysteine, etc.) (Guarino, Karunaratne, and Stella 2007). The prodrugs 2a and 2b were readily and quantitatively converted to metformin in rats after intravenous administration in vivo and only metformin was detected in rat blood. Tables 3 and 4 show the AUC and C _max values of both metformin and prodrug 2a after i.v. and oral administration, respectively. The AUC and C _max values after oral administration of the prodrug 2a (274.97 μg* min/ml, 1.34 μg/ml, respectively) was almost equal to the AUC and C _max after oral administration of metformin (289.37 μg* min/ml, 1.65 μg/ml, respectively) although 25 mg (0,1 mmol) of prodrug 2a was given compared to 35 mg (0,2 mmol) of metformin. The bioavailability (F%) of the prodrug 2a (63.1%) was improved compared to the bioavailability of metformin (42.7%).

Table 3. Pharmacokinetic Parameters of Metformin and the Prodrug 2a after Intravenous Administration into Rats (mean ± SD, n = 3).

Metformin (i.v.) Prodrug 2a (i.v.)

Dose (mg/kg) 35.0 (53 μmol) 67.0 (69 μmol)

AUCo-oo (μg*min/ml) 170 + 20 291 + 47 t _max (min) approx. 1 approx. 10

C _max (μg/ml) 20.9 + 3.7 3.0 + 0.7

Table 4. Pharmacokinetic Parameters of Metformin and the Prodrug 2a after Oral Administration into Rats (mean + SD, n = 3).

Metformin (p.o.) Prodrug 2a (p.o.)

Dose (mg/kg) 140 (0.2 mmol) 100 (0.1 mmol) AUC (μg*min/ml) 289 + 75 275 + 29 t _max (min) 49.78 + 8.35 59.20 + 0.35

C _max (μg/ml) 1.7 + 0.1 1.3 + 0.1

F (%) 43.0 64.5

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