Background of the invention Calcineurin inhibitors, such as cyclosporine and tacrolimus, are currently the keystone of most immunosuppressive regimens used in clinical organ transplantation (Denton et al., Lancet 1999; 353: 1083-1091). Calcineurin inhibitors have a selective inhibitory effect on T lymphocytes suppressing the early cellular response to antigenic and regulatory stimuli. Both cyclosporine and tacrolimus bind to their cytoplasmic receptors, called cyclophilin and FKBP, respectively.

Cyclophilin-cyclosporine and FKBP-tacrolimus complexes associate with calcineurin at the endofacial surface of T cells and inhibit calcineurin catalytic activity (Liu et al., Cell 1991; 66: 807-815). As a consequence, the nuclear translocation of nuclear factor of activated T cells (NFAT) and the induction of several cytokine genes are not initiated.

Calcineurin inhibitors have dramatically improved long-term survival after organ transplantations. However, cyclosporine as well as tacrolimus have been shown to markedly increase blood pressure, deteriorate renal function, and induce lipid disorders (Paul, Transplant Proc 2001; 33: 2089-2091). In fact, hypertension, nephrotoxicity, and hyperlipidemia are very common side effects during CsA and

tacrolimus treatments, and these side effects may also contribute to the increased risk of cardiovascular morbidity and mortality after organ transplantation. Clinical and experimental studies have revealed that hypertension and nephrotoxicity induced by calcineurin inhibitors are mediated by several mechanisms, such as sodium retention, renal vasoconstriction, stimulation of the renin-angiotensin system, activation of the sympathetic nervous system, impaired synthesis of nitric oxide, increased synthesis of endothelins, and alterations in renal prostanoid and thromboxane production. Recent studies have demonstrated that calcineurin inhibitors induce magnesium wasting both in experimental animals and in humans, suggesting that magnesium depletion may also be involved in the pathogenesis of calcineurin inhibitor toxicity.

Alpha-lipoic acid (6,8-thioctic acid) is a sulfur-containing substance that is readily converted to and from its reduced form, dihydrolipoic acid. It acts as a coenzyme in mitochondrial reactions that lead to ATP formation. More specifically, alpha-lipoic acid is involved in the decarboxylation of pyruvate and some other alpha-keto acids. Alpha-lipoic acid is considered to be extremely safe in the amounts utilized clinically. During more than three decades of scientific research and clinical usage no serious adverse effects have been reported as a consequence of alpha-lipoic acid supplementation, when used at daily doses from 25 mg to 1800 mg. Clinical applications for alpha-lipoic acid include, for example, the following conditions: diabetic polyneuropathy, cataracts, glaucoma, ischemia-reperfusion injury, and Amanita mushroom poisoning. Because of its unique characteristics alpha-lipoic acid is likely to have therapeutic application in a wide range of additional clinical conditions and it has also shown great promise, for example, in the treatment of HIV and in diabetes mellitus.

Alpha-lipoic acid has recently gained considerable attention as an endogenous and universal antioxidant that is readily absorbed from an oral dose. Alpha-lipoic acid and its reduced form, dihydrolipoate, are potent natural antioxidants both in fat and water-soluble media. Lipoic acid reacts with superoxide, hydroxyl radicals,

hypochlorous acid, peroxyl radicals, and singlet oxygen, and thereby protects against increased oxidative stress. Lipoic acid also effectively strengthens the antioxidant network of the human body by recycling vitamin C, vitamin E, and by increasing intracellular glutathione concentrations.

Summary of the invention It was discovered that, by administering lipoic acid simultaneously or separately in combination with the calcineurin inhibitor cyclosporine or by increasing the level of lipoic acid in the diet, development of the major adverse effects induced by the calcineurin inhibitor cyclosporine, namely arterial hypertension, nephropathy, and hyperlipidemia are prevented unexpectedly effectively. The beneficial effect of lipoic acid on the adverse effects induced by cyclosporine treatment greatly exceeds any beneficial effect which one could expect on the basis of current knowledge.

Within the scope of the present invention the term'lipoic acid'is intended to mean alpha-lipoic acid, which is a chiral molecule known also as thioctic acid; 1,2- dithiolane-3-pentanoic acid; 1, 2-dithiolane-3-valeric acid; 6,8-thioctic acid. The term covers the racemic mixture as well as any other (non 50/50) mixture of the enantiomers including substantially pure forms of either the R- (+) or the S- (-) enantiomer. The term also covers pharmaceutically acceptable salts (e. g. Na and K salts) and amides, esters, and metabolites of the acid, the reduced form of lipoic acid (dihydrolipoic acid) and naturally occuring forms of lipoic acid, such as lipoamide and lipoyllysine.

The invention is hereinbelow described in more detail referring to the accompanied drawings.

Brief description of the drawings Figure 1. Lipoic acid prevents the development of cyclosporine-induced renal damage.

Figure 2. Lipoic acid prevents the development of cyclosporine-induced hypertension.

Figure 3. Lipoic acid prevents the development of cyclosporine-induced cardiac hypertrophy.

Figure 4. Lipoic acid prevents the development of cyclosporine-induced increase in serum total cholesterol concentration.

Figure 5. Lipoic acid prevents the development of cyclosporine-induced increase in serum LDL-cholesterol concentration.

Figure 6. Lipoic acid prevents the development of cyclosporine-induced increase in serum triglyceride concentration.

Detailed description of the invention The invention is directed to products containing lipoic acid and a calcineurin inhibitor as a combined preparation for simultaneous, separate or sequential use in immunosuppressive therapy. A further object of the invention is the use of lipoic acid for the manufacture of a medicament for the prevention and treatment of the adverse effects of calcineurin inhibitors.

Another object of the invention is the use of lipoic acid and a calcineurin inhibitor as a combination for the manufacture of a medicament for therapeutic application as an immunosuppressant.

A still further object of the invention is a pharmaceutical composition comprising lipoic acid and a calcineurin inhibitor, preferably together with pharmaceutically acceptable carriers and adjuvants.

The invention is also directed to a method for the treatment and prevention of the adverse effects of calcineurin inhibitors in a mammal, said method comprising administering to said mammal lipoic acid in an amount ranging preferably from 25 mg to 1800 mg per day, even more preferably in an amount ranging from 600 to 800 mg per day, and preferably in an amount not exceeding the level of 350 mg/kg or 5% of the dry weight of the diet.

In preferred embodiments of the invention the calcineurin inhibitor is cyclosporine or tacrolimus, preferably cyclosporine, such as cyclosporine A, or a biologically active metabolite of cyclosporine or tacrolimus.

Preferred pharmaceutical compositions according to the invention may be for example in the form of pharmaceutical tablets, capsules, injection solutions, eye drops or ointments. The pharmaceutically acceptable carriers and adjuvants and their amounts can easily be selected by a person skilled in the art.

Lipoic acid may be administered together with the calcineurin inhibitor in a single dosage form, for instance in a capsule containing both agents, or lipoic acid and the calcineurin inhibitor may be administered in separate dosage forms. For instance, the calcineurin inhibitor may be administered intravenously, orally, transdermally, or topically, while lipoic acid is administered orally, intravenously, transdermally or topically.

Preferably, lipoic acid is administered simultaneously with the calcineurin inhibitor. However, it is also possible to administer lipoic acid as part of the diet at the level of ranging from 0.005% to 5% of the dry weight of the diet, preferably at

the level of 0. 1% to 0.5% of the dry weight of the diet, while the calcineurin inhibitor is administered in separate usual dosage forms. Lipoic acid may also be administered in the form of a dietary supplement or as a food ingredient. It may also be administered in separate doses several hours (for example up to 6 hours) before or after the administration of the calcineurin inhibitor.

In order to effectively protect a patient treated with calcineurin inhibitors against the adverse effects of said inhibitors, it is sufficient to use lipoic acid in amounts such as 25-1800 mg/d, corresponding in a 70-kg adult a daily dose of ranging from 0.3 to 25 mg/kg. However, in view of the known safety of alpha-lipoic acid, the upper limit of the amount of lipoic acid is of minor importance, and based on data from the experimental studies may be as high as 350 mg/kg per day. Cyclosporine and tacrolimus are typically used at doses ranging from 2 to 15 mg/kg, and 0.05 to 0.3 mg/kg, respectively. The preferred ratio of cyclosporine to lipoic acid is thus from about 2: 350 to 15: 0.3, more preferably from about 2: 25 to about 15: 3. The preferred ratio of tacrolimus to lipoic acid is from about 0.05 : 350 to 0.3 : 0.3, more preferably from about 0.05 : 25 to about 0.3 : 3.

Lipoic acid proposed for use in accordance with the present invention allows to avoid the undesired side effects induced by calcineurin inhibitors, when administered before the administration of the calcineurin inhibitor, simultaneously therewith or thereafter. Even more surprising is that lipoic acid produces this effect upon administration spread over a prolonged period of time before and after administration the calcineurin inhibitor.

Experimental results demonstrating the effectiveness of the invention The spontaneously hypertensive rat (SHR) provides a suitable model for examining the effects of various dietary factors or drugs on, among other things, on blood pressure and renal function and/or morphology. To investigate the adverse effects of calcineurin inhibitors, we developed recently a novel animal model, where

cyclosporine is given to SHR concurrently with a"Western-type"high-salt diet (Mervaala et al., Hypertension 1997; 29: 822-827). We were able to demonstrate that in this animal model, cyclosporine, when given at dosages producing clinically relevant blood drug concentrations, induces both arterial hypertension, nephropathy, and hyperlipidemia. The suitability of this animal model for investigating the adverse effects of calcineurin inhibitors have been confirmed recently by a series of experiments (Pere et al., Nephrol Dial Transpl 1998 ; 13: 904- 910 ; Mervaala et al., Blood Press 1999; 8: 49-56; Pere et al., Surgery 2000; 128: 67- 75; Pere et al., Kidney Int 2000; 58: 2462-2472; Finckenberg et al., Transplantation 2001; 71: 951-8; Lassila et al., J Physiol Pharmacol 2001; 52: 21-38; Lassila et al., Br J Pharmacol 2000; 130: 1339-47; Lassila et al., Eur J Pharmacol 2000; 398: 99- 106).

The effectiveness of the present innovation was examined in cyclosporine-treated SHR on high-salt diet. The protocols were approved by the Animal Experimentation Committee of the Institute of Biomedicine, University of Helsinki, Finland, whose standards correspond to those of the American Physiological Society. The rats had free access to chow and drinking water. In the beginning of the study the 6-7-week-old male SHR were normotensive and without signs of any marked renal damage. The systolic blood pressure was measured indirectly from the pre-trained and unanesthetized rats by plethysmography (Apollo-2AB Blood Pressure Analyzer, Model 179-2AB, IITC Life Science, Woodland Hills. CA). The analog signals obtained were converted to digital values by an on-line microprocessor. Before the measurements the rats were warmed for 10 to 15 minutes at 28°C to make the pulsations of the tail artery detectable. Values for systolic blood pressure and heart rate were obtained by averaging results from 3 to 5 measurements. To minimize stress induced fluctuations in blood pressure, all measurements were taken randomly by the same person at the same time of day (9 to 12 AM). The systolic blood pressure was 122 2 mm Hg and body weight 179 ut 3 g in the beginning of the experimental period. The blood pressure-and body

weight-matched SHR were then divided into three groups to receive the following diets and drug regimens for 6 weeks (n=8-10 in each group).

Group 1 (SHR control group). During the 6-week experimental period these rats received a commercially available rodent diet (R36, Finnewos, Finland) containing all the essential nutrients, also including adequate levels of the mineral elements and vitamins to maintain normal body function. Systolic blood pressure increased in this group to the level of 180 6 mm Hg during the 6-week experimental period.

The degree of cardiac hypertrophy, i. e. the pathological increase of the weight of the heart, was estimated by the heart weight-to-body weight ratio (mg/g). In the SHR control group the average cardiac hypertrophy index was 3.795 0.232 mg/g.

The degree of renal damage was assessed by analyzing the 24-hour urinary excretion of albumin by a commercially available rat albumin ELISA kit (Celltrend, Germany). This technique has generally considered as extremely reliable and sensitive method for detection of renal dysfunction. For albumin measurements, urinary samples were collected in rats kept for 24 hours in metabolic cages. Urine volumes were measured gravimetrically, and the urine samples were stored at-80°C before assayed. The 24-hour albuminuria in SHR control group was 557 i 170 llg/d.

Serum lipids from the samples taken at the end of the experiment were analyzed by an accredited laboratory, United Laboratories Ltd., Helsinki, Finland (Hitachi 912 Automatic Analyzer, Hitachi Ltd. , Tokyo, Japan). Serum total cholesterol was determined with an enzymatic method (Boehringer Mannheim GPO-PAP-method), serum LDL-cholesterol with an enzymatic direct method (Boehringer Mannheim LDL-Chol Plus), and serum triglycerides with an enzymatic method (Boehringer Mannheim GPO-PAP-method). The average serum total cholesterol, LDL- cholesterol, and triglyceride concentrations in SHR controls were 1. 54+0. 075 mm/1, 0. 16+0. 04 mm/1, and 0. 92+0. 097 mm/1, respectively.

Group 2 (Cyclosporine group). This group of 10 SHR received a diet in which the caloric and other content of diet was otherwise exactly the same as in Group 1, but to mimic the current Western-type human diets, sodium chloride (common salt, Sigma) was added to control diet at the level of 6% of the dry weight of the diet.

The rats received daily subcutaneous cyclosporine injections (Sandimmun, Novartis, Switzerland) for 6 weeks. The dosage of cyclosporine (5 mg/kg) was selected from our previous experiment (Mervaala et al., Hypertension 1997; 29: 822- 827; Mervaala et al. Hypertension 2000; 35: 360-366) to produce blood cyclosporine concentration similar to that found in the cyclosporine-treated patients (approximately 500-1000 ng/ml when measured 24 hours after the cyclosporine injection). Systolic blood pressure increased markedly in this group to the level of 243 i 6 mm Hg (p<0.05 compared to SHR control group). There was also a pronounced 25% increase in the degree of cardiac hypertrophy (p<0.05 compared to SHR control group). 24-hour albuminuria was increased by approximately 220- fold (p<0.05 compared to SHR control group). There was a 48% increase in serum total cholesterol concentration, 69% increase in LDL-cholesterol concentration, and a 35% increase in the level of serum triglycerides (all p<0.05 compared to SHR control group).

Group 3 (Cyclosporine + Alpha-lipoic acid group). This group of 8 SHR received a diet in which the caloric and other content of diet was otherwise exactly the same as in Group 2, but alpha-lipoic acid (DL-6,8-thioctic acid, Sigma T-5625) was added to the diet at the level of 0.5% of the dry weight of the diet to produce an approximate daily dose of 350 mg/kg. Cyclosporine was given exactly as in the group 2. Alpha-lipoic acid decreased systolic blood pressure by 32 mmHg compared to cyclosporine group (p<0.05). Systolic blood pressure in the alpha- lipoic acid-treated rats did not differ from that of SHR controls (p=0. 13). Alpha- lipoic acid also effectively decreased the cardiac hypertrophy index by 15% (p<0.05 compared to cyclosporine group). Cardiac hypertrophy in the alpha-lipoic acid-treated rats did not differ from that of SHR controls (p=0. 58). Alpha-lipoic acid completely prevented the development of cyclosporine-induced renal damage.

The actual decrease in albuminuria by the diet prepared according to the present innovation was remarkable (>99%). Albuminuria in the alpha-lipoic acid-treated rats did not differ from that of SHR controls (p=1.00). Alpha-lipoic acid completely prevented the development of cyclosporine-induced increases in the concentrations of serum total cholesterol, LDL-cholesterol, and triglycerides (all p<0.05). Serum lipid profile in the alpha-lipoic acid-treated rats did not differ from that of SHR controls.

The results are shown in Table 1 and Figures 1 to 6.

Table 1. Effects of alpha-lipoic acid on blood pressure, cardiac hypertrophy, albuminuria, and concentrations of serum total cholesterol, LDL-cholesterol and triglycerides in cyclosporine-treated SHR on high sodium for 6 weeks. Data are presented as means SEM. Statistically significant differences in mean values were tested by ANOVA and the Tukey's test. The differences were considered significant when p<0.05. The data were analyzed using SYSTAT statistical software (SYSTAT Inc, Evanston, IL, USA). * denotes p<0.05 between CsA group and Control group; n denotes p<0.05 between CsA group and CsA+LA group.

Control group CsA group CsA + LA ANOVA (n=8) (n=10) group P-value (n=8) Systolic blood pressure 180 ~ 6 243 ~ 6*# 202 ~ 7 <0.001 (mm Hg) Cardiac hypertrophy 3.795 ~ 0.232 4.736 ~ 0.152*# 4.056 ~ 0.126 0.002 (mg/g)<BR> 24-hour albuminuria 557 ~ 177 124011 ~ 24320*# 963 ~ 524 <0.001 (µg/d) Serum total cholesterol 1.54 ~ 0. 075 2. 28 0. 057 * a 1.38 ~ 0. 086 <0. 001 (mmol/1) Serum LDL-cholesterol 0.16 ~ 0. 04 0.27 0. 042* a 0.1 ~ 0. 001 0.005 (mmol/1) Serum triglycerides 0.92 : E 0. 097 1.24 ~ 0.18*# 0.59 ~ 0. 072 0.01 (mmol/1)