WO2000066101A2

WO2000066101A2 - Method of inhibiting glycation product formation

Info

Publication number: WO2000066101A2
Application number: PCT/US2000/011354
Authority: WO
Inventors: Jerry L. Nadler; Samuel Rahbar
Original assignee: City Of Hope
Priority date: 1999-04-30
Filing date: 2000-04-28
Publication date: 2000-11-09
Also published as: AU4671100A; WO2000066101A3

Abstract

This invention provides a method of preventing the formation of advanced glycation products through administering a xanthine-based compound, for example, lisofylline. The method is useful in treatments for diseases associated with advanced glycation end-products, such as diabetes, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Down's syndrome, osteoarthritis, cataracts, cardiac hypertrophy, arterial stiffening, atherosclerosis and kidney toxicity.

Description

METHOD OF INHIBITING GLYCATION PRODUCT FORMATION

BACKGROUND OF THE INVENTION

Advanced glycation end-products (AGEs) are protein adducts that result from the non-enzymatic covalent reaction of simple sugars, like glucose, with proteins. They are thought to arise in vivo primarily through the Maillard reaction. In the first step of the Maillard reaction, an amino acid condenses with a simple sugar to form an N-glycosylamine (Schiff-base) . This product undergoes the "Amadori" rearrangement to form a 1- amino-l-deoxy-2-ketose (Amadori product) , which in the presence of reactive oxygen species (e.g, superoxides) forms highly reactive dicarbonyl species. The dicarbonyl further reacts with proteins to form advanced glycation products. The Maillard reaction is bypassed in the presence of certain metals, which can directly catalyze the formation of the dicarbonyl species from the sugar. Either process results in progressive and irreversible intermolecular protein crosslinking. Monier et al., Enσ J. Med. 314:403, 1986.

The pathophysiological consequences of the accumulation of AGEs are great. They have been associated with a wide variety of disorders and conditions, many of which are characterized by oxidative stress and/or elevated levels of carbohydrates. They have been linked, for example, to aging, Parkinson's disease (PD) , Alzheimer's disease (AD), sporadic amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), Down's syndrome (Trisomy 21), osteoarthritis, cataracts, cardiac hypertrophy, arterial stiffening, atherosclerosis, retinopathies and kidney toxicity in a variety of disorders. See, for example, Munch et al., Ann. Neurol. 44(Supp. 1): S85-88 (1998); Niwa et al., Bioche . Biophvs. Res. Comm. 248:93-97 (1998); Chou et al., Mol. Med. 4:324-32 (1998); Odetti et al. Biochem.

Biophys. Res. Comm. 342:849-51 (1998); Bank et al., Biochem. J. 330:345-51 (1998); Corman et al . , Proc. Nat ' 1 Acad. Sci. 95:1301-06 (1998); Jinnouchi et al., J. Biochem (Tokyo) 123:1208-17 (1998); Witko-Sarsat et al., J. Immunol. 161:2524-32 (1998); and Sakata et al., Nephron 78:260-65.

Mononuclear leukocyte binding to the endothelium and their entry into the vessel wall are among the earliest events in atherogenesis . Faggiotto et al. Arteriosclerosis 4:323-40 (1984); Gerrity et al., Am. J. Pathol . 95: 755-85 (1979). Several observations suggest that elevated blood glucose levels exacerbate this process. First, atherosclerosis is accelerated in both type 1 and type 2 diabetes. Ruderman et al . , Prog. Cardiovasc. Res. 26: 373-412 (1984); Ruderman et al., FASEB J. 6: 2905-14 (1992). Moreover, monocytes more readily bind to human aortic endothelial cells (HAEC) when exposed to elevated (25 mM) glucose levels, suggesting that high glucose may be sufficient alone. Kim et al., Diabetes 43:1103-07 (1994).

Other observations suggest that a downstream product of elevated glucose levels, and not elevated glucose per se, is responsible for atherosclerosis, for example, glycosylation of low density lipoprotein (Brownlee et al, Diabetes 34:938-41 (1985)) and advanced glycation end-products (Bucala et al., Proc. Nat'l Acad. Sci. USA 90:6434-38 (1993); Kirstein et al . , Proc. Nat'l Acad. Sci. USA 87:9010-14 (1990)). Irrespective of the mechanism, however, an agent which inhibits monocyte adhesion should have therapeutic value in preventing or treating atherosclerosis, particularly at an early stage. Enhanced formation and accumulation of AGEs also is associated with the high blood glucose levels found in Type 1 and Type 2 diabetes and likely plays a substantial role in the pathogenesis of these diseases. Accumulation of AGEs correlates with the development of a range of diabetic complications, including nephropathy, retinopathy and neuropathy. Nicholls et al., Lab. Invest. 60:486 (1989); Hammes et al., Proc. Nat'l. Acad. Sci. USA 88:11555 (1991); Cameron et al., Diabetoloσia 35:946 (1992). Tissue damage to the kidney by AGEs leads both to a progressive decline in renal function and end-stage renal failure (ESRD) and to accumulation of low molecular weight (LMW) AGE peptides (glycotoxins) in the serum of patients with ESRD. Makita et al., Lancet 343:1514 (1994); Koschinsky et al . , Proc. Nat'l. Acad. Sci. USA 94:6474 (1997); Makita et al., Enσ J. Med. 325:836 (1993). The low molecular weight AGEs readily form new cross-links with plasma or tissue components, e.g., low density lipoprotein (LDL) or collagen. This accelerates the progression of tissue damage and morbidity in diabetes. Bucala et al., Proc. Nat'l. Acad. Sci. USA 91:9441 (1994); Miyata et al., J_^ Clin. Invest. 92:1243 (1993).

Direct evidence indicating the contribution of AGEs in the progression of diabetic nephropathy has been recently reported. Vlassara et al . , Lab. Invest. 70:138 (1994). In addition, infusion of pre-formed AGEs into healthy rats induces glomerular hypertrophy and mesangial sclerosis, gene expression of matrix proteins and production of growth factors. Brownlee et al., Enσl. J. Med. 18:1315 (1988); Vlassara et al., Mol . Med. 1:447 (1995). Further studies have revealed that amino- guanidine, an inhibitor of AGE formation, ameliorates tissue impairment of glomeruli and reduces albuminuria in induced diabetic rats. Soulis-Liparota et al . , Diabetes 40:1328 (1991); Itakura et al . , Life Science 49:889 (1991). In humans, decreased levels of hemoglobin AGE (Hb-AGE) are produced along with concomitant amelioration of kidney function by aminoguanidine therapy in diabetic patients, further substantiating the importance of AGEs in the pathogenesis of diabetic complications. Makita et al . , Science 258:651 (1992); Bucala and Vlassara, Am. J. Kidnev Pis. 26:875 (1995).

Several other AGE inhibitor studies have been reported recently. These studies tested AGE formation and AGE-protein crosslinking both in vi tro and in vivo compared to the known AGE inhibitor, aminoguanidine (AG) . Nakamura et al., Diabetes

46:845 (1997); Kochakian et al . , J. Diabetes 45:1694 (1996); Rice et al., Proc. Nat'l. Acad. Sci. USA 91:3857 (1994). These studies are still quite preliminary. In fact, there is no AGE inhibitor currently available to the clinician. In view of the foregoing, it is apparent that a need exists in the medical arts for new and improved therapeutics, and for methods useful in treating and preventing diseases associated with the formation of glycation products. The need extends both to new methods of treatment and methods which augment known treatments.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide methods of treating disorders associated with glycation products. According to this object, the invention provides a method of preventing the formation of advanced glycation end-products. This method generally involves administering an effective amount a xanthine-based compound, or its pharmaceutically acceptable salt, having the following formula:

wherein: R_x is an R enantiomer of an ω-1 secondary alcohol-substituted alkyl (C₅.₈) , wherein said alcohol moiety is optionally esterified; R₂ is alkyl (C₁_₁₂) , optionally containing one or two nonadjacent oxygen atoms in place of a carbon atom; and R₃ is CH₃ or H. A preferred method uses lisofylline as the xanthine-based compound. It is a further object of the invention to provide methods of treating complications arising from diabetes. In accord with this object, a method is provided for treating the complications of diabetes. This method typically entails administering an effective amount of a xanthine-based compound or its salt, as described above. This method is useful, for example, to treat diabetes-associated nephropathy, retinopathy, neuropathy and end-stage renal disease.

It is still another object to provide compositions useful in treating diseases associated with glycation. Further to this object, the invention provides a composition, comprising, and a xanthine-based compound or its salt, as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows oral glucose tolerance in ZDF rats untreated or treated with lisofylline. Figure 1A shows the curve and Figure IB shows the integration of that curve. Figure 2 shows the ability of lisofylline to inhibit glycation product formation in the BSA-glucose assay.

Figure 3 shows the ability of lisofylline to inhibit glycation product formation in the G.K. peptide-ribose assay. Figure 4 shows the ability of lisofylline to inhibit monocyte adhesion in a model system for atherosclerosis.

Figure 5 shows the ability of lisofylline to inhibit the crosslinking of glycated BSA to collagen.

DETAILED DESCRIPTION OF THE INVENTION

The present invention responds to a need for medicaments able to reverse and inhibit pathological AGE formation by providing a method of treatment in involving administration of one or more therapeutic xanthine-based compounds. Due to their ability to prevent the formation of AGEs, the methods provided are useful in treating any disease having a pathological association with AGEs. In view of the chemistry behind the formation of AGEs, the disorders and conditions which would benefit from treatment and/or prevention according to the present method generally are characterized by oxidative stress and/or elevated levels of carbohydrates. These include, but are not limited to, Parkinson's disease (PD) , Alzheimer's disease (AD), sporadic amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), Down's syndrome (Trisomy 21), osteoarthritis, cataracts, cardiac hypertrophy and also arterial stiffening, atherosclerosis, retinopathies and kidney toxicity associated with a variety of disorders. AGE-disease association has been most closely studied concerning diabetes. Accordingly, diabetes (both types 1 and 2) , and especially the associated complications associated with glycation products in diabetics, are contemplated as within the scope of the inventive treatments . Without being bound to a particular theory as regards diabetes, the present inventive methods and compositions are useful in at least two different ways, to alleviate the symptoms. First, the active compounds can be used to lower blood glucose levels, acting to by-pass the insulin receptor. They are believed to act as a pseudoinsulin, stimulating the uptake of glucose by intervention in the insulin signaling pathway. In this way, they may be employed alone or in conjunction with insulin (or insulin-like drugs) to reduce blood glucose levels. This may be particularly useful in Type 2 disease which is characterized by insulin resistance.

Second, the methods can be used to inhibit the formation of AGEs, thereby reducing, for example, the incidence of renal toxicity and vascular toxicity otherwise associated with such products. It is this aspect of the methods that extends their applicability to treating the above-listed disorders. With regard to diabetes, the methods are useful in treating AGE-related complications, like nephropathy, retinopathy and neuropathy. They are useful in treating diabetic end-stage renal failure (ESRD) , and preventing accumulation of low- molecular-weight (LMW) AGE peptides (glycotoxins) in the serum of patients with ESRD.

Unless otherwise clearly indicated by context, used generically, the term "treating" in its various grammatical forms in relation to the present invention includes one or more of preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing and halting the deleterious effects or complications of a disease state, disease progression, disease causative agent or other abnormal condition. Methods of prophylaxis are specifically encompassed by the term "treating." Where the more particular terms within the term "treating" are used, however, they should be read according to standard usage. For instance, treating the complications arising from a disease does not include curing the disease per se; it does not refer to reversing or preventing an etiology, but rather it is addressed to preventing or alleviating symptoms, which are somewhat removed from the underlying etiology.

Compounds Useful in the Invention

Compounds generally useful in the invention are based on a xanthine core, which is typically substituted at the 1, 3 and/or 7 positions. Pharmaceutically acceptable salts of these compounds are envisioned as equally useful. These compounds are referred to herein as "xanthine-based compounds." The prototypical xanthine-based compound is lisofylline (LSF) , and in general, unless otherwise indicated, when lisofylline is referred to herein as useful, that reference may be understood generally to apply to other members of the class of xanthine-based compounds described herein. Particularly useful xanthine-based compounds are disclosed in U.S. Patent 5,648,357, which is hereby incorporated by reference its entirety. Methods of preparing these compounds may be found therein, as well. More specifically, the preferred compounds useful in the invention have the following structure:

wherein R_x is an R enantiomer of an ω-1 secondary alcohol-substituted alkyl (C₅.₈) , wherein R₂ is alkyl (C₁.₁₂) , optionally containing one or two nonadjacent oxygen atoms in place of a carbon atom, and R₃ is CH₃ or H. Among this group of compounds with a xanthine core structure, the particularly preferred compound is lisofylline, 1- (5-R-hydroxyhexyl) -3, 7- dimethylxanthine .

It will be recognized that the alcohol-substituted alkyl portions of the foregoing molecules may be esterified, to yield further xanthine-based compounds that are active per se or as prodrugs. Small C^ carboxylic acid esters are preferred and may be made according to standard synthetic techniques. It is anticipated that such compounds will have improved pharmacodynamic properties, such as improved circulating half-life. In addition, any pharmaceutically acceptable salt of the foregoing (esterified or non-esterified) compounds may also substitute in the methods of this invention.

Pharmaceutical Formulations The pharmaceutical compositions employed in the methods of the invention generally contain a therapeutically effective amount of one or more than one compound with a xanthine core structure as described above. Preferably, one or more than one such compound is mixed with a pharmaceutically acceptable carrier.

Because methods using xanthine-based compounds are useful in treating a variety of diseases, it is beneficial to formulate the xanthine-based compounds in conjunction with other therapeutics. For instance, when treating arthritis, the xanthine-based compounds may be formulated with antiinflammatories . In the case of diabetes, due to the complementary action of the foregoing xanthine-based compounds with the action of insulin, it is advantageous to use and to formulate the two medicaments together. These combined formulations would allow lower doses of insulin. Thus, in addition to reducing the incidence of AGEs, such formulations would enhance the other actions of insulin. The relative amounts of such combination medicaments may be ascertained readily by the skilled clinician, informed by the appropriate amounts of the known therapeutic, and empirically, using standard dose-escalation protocols . A suitable formulation will depend on the nature of the disorder to be treated, the nature of the medicament chosen, and the judgment of the attending physician. In general, the xanthine-based compounds are formulated either for oral administration or injection, although other modes of administration such as transmucosal or transdermal routes may be employed. Suitable formulations for these compounds, including pharmaceutically acceptable excipients, can be found, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, chapters 83-92, pages 1519-1714 (Mack Publishing Company 1990) (Remington's), which is hereby incorporated by reference.

If a solid carrier is used, the preparation of xanthine- based compound can be tableted. The composition can be placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge. The amount of solid carrier will vary widely but preferably will be from about 25 mg to about 1 gram.

Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example, using the aforementioned carriers in a hard gelatin capsule shell. Where the composition is in the form of a soft gelatin shell capsule, any pharmaceutical carrier routinely used for preparing dispersions or suspensions may be considered, for example, aqueous gums, cellulose, silicates or oils and are incorporated in a soft gelatin capsule shell.

When a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous liquid suspension.

A syrup formulation will generally consist of a suspension or solution of the compound or salt thereof in a liquid carrier with a flavor or coloring agent. Examples of liquid carriers include ethanol, polyethylene glycol, coconut oil, glycerine and water.

Although other routes of administration are contemplated, the pharmaceutical compositions of the invention preferably are suitable for oral and parenteral administration. Parenteral administration can include intravenous ("i.v."), intramuscular ("i.m."), subcutaneous ("s.c"), intranasal, intrarectal, intravaginal, intraperitoneal ("i.p.") ex vivo culture, or topical delivery. Preferred administration is accomplished intravenously, especially when administered with insulin. Appropriate dosage forms for each specific route of administration may be prepared by conventional techniques. A typical dosage form for parenteral administration is a solution or suspension of at least one xanthine-based compound, or its pharmaceutically acceptable salt. The parenteral dosage form typically contains a parenterally acceptable sterile aqueous or non-aqueous carrier. The parenteral dosage form optionally contains a parenterally acceptable oil. Examples of such oils include polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil, and sesame oil. Parenteral acceptability is known to the skilled clinician.

Formulation as a standard unit dose is also contemplated. Thus, the pharmaceutical compositions of the invention can be formulated, for example, for oral use in dosage unit form as a pill, a tablet, a caplet, or a capsule. These dosage units may each contain a therapeutically effective amount of one or more xanthine-based compounds. These dosage units also may contain sub-therapeutically effective amounts, where multiple units may be combined to achieve a therapeutically effective amount.

The amount of xanthine-based compound in a unit dose will depend on many factors considered by the skilled clinician. Generally, however, dosage units prepared for use will contain from about 500 mg to about 1500 mg of at least one xanthine-based compound. A typical parenteral dose can be from about 500 mg to about 5 g and may be administered (i.v., i.p., i.m., or s.c.) over a course of 24 hours. A typical topical formulation contains from about 1% to about 4% by weight. An ex vivo culture concentration, for use in the transplantation methods described below, can be maintained from about 10 μM to about 500 μM.

Methods of the Invention The methods of the present invention generally comprise administering a therapeutically effective amount of at least one xanthine-based compound to a patient suffering a disorder associated with the presence of AGEs. These methods result in a decrease in the amount of AGEs, which relieves the patient from the symptoms caused by these detrimental molecules, thereby increasing patient health and well-being. The patient may be a human or a non-human animal .

Some preferred methods further comprise coadministration of a second therapeutic, such as insulin, and at least one xanthine-based compound. It will be recognized that the optimal timing for administering the xanthine-based compound will be determined by the clinician. Thus, in this context, coadministration means concurrent administration, not necessarily simultaneous administration. Preferably, however, the xanthine-based compound (s) will be administered prior to or simultaneously with the second therapeutic. The xanthine-based compound (s), either alone or in combination with second therapeutic are preferably administered by intravenous injection or by continuous subcutaneous infusion pump.

The need for this treatment ultimately will be based on the evaluation by a skilled clinician. Generally, however, a patient will be in need of such treatment to mitigate the effects of protein glycation. Hence, the methods are useful in treating, and at least in alleviating the complications or symptoms of, disorders like diabetes, Parkinson's disease (PD) , Alzheimer's disease (AD), sporadic amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), Down's syndrome (Trisomy 21), osteoarthritis, cataracts, cardiac hypertrophy, arterial stiffening, atherosclerosis and kidney toxicity.

With regard to diabetes, these method are also useful in treating AGE-related complications, like nephropathy, retinopathy and neuropathy. They are useful in treating diabetic end-stage renal failure (ESRD) , and preventing accumulation of low molecular weight AGE peptides (glycotoxins) in the serum of patients with ESRD.

In particular, the invention contemplates a method of treating a patient that entails administering to the patient an effective amount of one or more xanthine-based compound. An effective amount may be an amount sufficient to reduce the formation of glycation products in the patient, compared to control levels. In general, the magnitude of any effect should be on the order of at least about 10%, but more typically at least about 20%. More preferably, the effect should be at least about 30%. Thus, for example, an effective amount is one sufficient to reduce elevated blood glucose level of the patient by at least about 10% or sufficient to reduce the formation of glycation products in said patient by at least about 10%, and so on.

Other preferred methods comprise administering to a patient an effective amount of a second therapeutic, in conjunction with an effective amount of at least one xanthine-based compound.

Although the foregoing detailed description and the following examples set forth representative preferred embodiments of the invention, they are not intended as exclusive embodiments. In view of the material presented, one of ordinary skill in the art readily would appreciate further embodiments that fall within the scope of the invention.

EXAMPLE 1 This example demonstrates that the methods of the invention are useful in preventing AGE formation by reducing blood glucose (a reactant in AGE formation) , either alone or in combination with insulin. Sixteen ZDF male rats and eight ZDF female rats (approximately 8 weeks of age) were divided into two groups. The rats were administered LSF at 25 mg per kg of body weight or vehicle solution only via intraperitoneal injection twice a day. Animals were weighed prior to each injection to precisely determine appropriate dose of agent. After seven days of treatment all rats underwent an oral glucose tolerance test (2g glucose per kg of body weight) after which they remained on their respective treatment for another 48 hrs . Balon et al., Am. J. Physiol. 269:E745-52, 1995. Rats were then euthanized and blood and tissues were obtained. 2-deoxyglucose uptake in vi tro in epitrochlearis muscle was performed as previously described. Balon et al., J. Appl. Physiol. 77 ( 6) : 2519-22, 1994. Blood glucose was analyzed with a YSI glucose analyzer. Animal body weight was not different in the two groups at the time of sacrifice (345 ± 5 vs . 344 ± 7 grams for males and 312 ± 5 vs . 314 ± 10 grams for females) .

Fed state glucose values were measured 3 days after the treatment. Glucose concentration after LSF treatment in the rats was 267 ± 24 mg/dl SEM (male, LSF), 262 ± 18 (male, vehicle control), 119 ± 10 mg/dl (female, LSF), and 105 ± 12 (female, vehicle control). The p value was 0.8773 for males and 0.3926 for females.

Oral glucose tolerance was monitored by analyzing the integrated area under the glucose tolerance curve (AUC) at 0, 15, 30, 60 and 120 minutes. In the female rats, a statistical reduction in the AUC was observed with LSF treatment (24,095 ± 1,191 (LSF) verus 28,958 ± 857 (vehicle control), p <0.02 (Figure 1) . Significant reduction in glucose concentration also was observed at the 15, 30, 45 and 60 minute time points in female rats treated with LSF. However, in the male rats, no significant difference in AUC was observed after LSF treatment (27,301 ± 2,405 (LSF) versus 26,932 ± 1,536 (vehicle control). Several male rats responded to LSF, but the large standard error of measurement (SEM) resulted in a lack of significance. However, there was an indication that fasting glucose was reduced by LSF treatment (95 ± 9 mg/dl (LSF), 106 ± 4 (vehicle control) , p= 0.1304) .

Blood glucose and other blood parameters were obtained at sacrifice. LSF was found to reduce blood glucose levels in female rats. The statistical insignificance (p = 0.423) is due to a small n and variability. Thus, LSF alone increases tissue glucose uptake.

Glucose (mσ/dl) LSF Vehicle . Males 270 ± 27 270 ± 27 0.287

Females 103 ± 11 116 ± 6 0.36

To demonstrate that xanthine-based compounds can act in combination with insulin, glucose uptake in response to insulin exposure (200 μU/ml) was studied in isolated epitrochlearis muscle from both male and female rats. LSF effect on 2-deoxyglucose uptake in response to insulin exposure was analyzed. Treatment with LSF did not produce significant difference in insulin response in the muscle of male rats. Consistently, however, in the presence of insulin, glucose uptake in female rats was improved with LSF treatment. The female rats were more insulin-resistant because their response to insulin involved lowered glucose uptake.

Stimulation of 2-deoxyσlucose Uptake by Insulin Exposure n LSF Vehicle β

Males 8 0.79 ± 0.38 1.08 ± 0.38 0.6136

Females 4 1.51 ± 0.69 -0.29 ± 0.84 0.1483

The female ZDF model is a diabetes model exhibiting glucose-intolerance, insulin resistance, maintained insulin secretion and LSF-improved glucose uptake, both alone and in response to insulin. This suggests that the xanthine-based compounds act to promote tissue glucose uptake both directly, and indirectly by improving/ restoring insulin activity. Accordingly, irrespective of mechanism, LSF may be used to reduce blood glucose, thereby preventing AGE formation. Importantly, these observations may be extended to non-diabetic conditions associated with AGEs, since reducing blood glucose should inhibit glycation by removing a reactant.

EXAMPLE 2

This example illustrates the effect of LSF on glucose control in female ZDF rats maintained on a high fat diet, which has been reported to induce further insulin resistance and hyperglycemia in female ZDF model. The method used here was identical to that described in EXAMPLE 1 except that an in si tu approach, the Hindquarter perfusion technique was used to measure insulin sensitivity. See Balon et al., Hypertension 23(6) [part 2]: 1036-39 (1994). The study was carried out for 30 days to evaluate prolonged effects of LSF.

Although there was variability in baseline glucose values in this particular batch of ZDF female animals, the animals were matched prior to drug group assignment (117 ± 0.17 mg/dl and 114 ± 15 mg/dl) . The glucose concentration showed a lowering trend during the course of LSF treatment. On day 18 of treatment, morning glucose concentration was 208 ± 18 mg/dl (LSF) and 264 ± 20 mg/dl (vehicle control), p < 0.06. Fasting blood glucose concentration (109 ± 9 mg/dl (LSF) and 98 ± 7 mg/dl (vehicle control)) was measured on the day of the glucose tolerance test. While not statistically significant, the data showed a trend of reduced glucose concentration with LSF treatment.

Interestingly, LSF reduced significantly the level of hemoglobin A1C (a glycated hemoglobin) from 10.5 ± 0.5 percent to 8.8 ± 0.48 percent in female ZDF rats. Such results indicate that the xanthine-based compounds reduce blood glucose and protein glycation. Hindquarter-perfusion results demonstrate that LSF significantly affects glucose in the presence of high insulin concentrations. Thus, at 31-33 days, the rats were prepared for hindquarter perfusion as described. Balon et al . , Hypertension 23(6) [part 2]: 1036-39, 1994. The observed glucose uptake by perfused rat (weight-matched animals) hindquarter (μmol/g) is shown as follows.

Insulin (μU/ml) Vehicle LSF 0 0.92 ± 0.27 0.99 ± 0.25

200 2.44 ± 0.21 2.82 ± 0.30

20,000 4.35 ± 0.24 5.88 ± 0.66*

Values are means ± SEM of 7-8 observations . * denotes significance between groups at P = 0. 369.

In conclusion, LSF can improve insulin response in ZDF female rats fed a high fat diet which induces insulin resistance after long-term ( 30 day) treatment with LSF . Interestingly, a quite significant reduction of hemoglobin A1C glycation product was found in LSF-treated animals , indicating that LSF reduces protein glycation in vivo .

EXAMPLE 3

This example confirms the effect of xanthine-based compounds on glucose tolerance as demonstrated in EXAMPLE 1, using a larger population of animals. The same protocol described in EXAMPLE 1 was adopted. First, the effects of LSF on glucose level and tolerance were measured in eleven rats for both LSF and vehicle control groups. On the day of the glucose tolerance curve was obtained, the fasting glucose concentration was 99.5 ± 5 mg/dl (vehicle control) and 97.5 ± 4 (LSF), p = 0.759. The data below show the trend of glucose uptake in LSF- treated animals at 15, 30, and 120 minutes, and statistically significant changes in glucose uptake at 60 min with LSF treatment (p < 0.03). The glucose concentration (mg/dl) is summarized as follows.

Time (min) Vehicle LSF p_

15 189 ± 7 175 ± 6 0.143

30 251 ± 11 230 ± 5 0.107 60 290 ± 12 252 ± 9 <0.03

120 209 ± 13 175 ± 13 0.085

In addition, the integrated area under the glucose tolerance curve was significantly smaller for LSF-treated animals (25,137 ± 809) than for control animals (28,526 ± 976), p <0.02.

Hindquarter perfusion was performed in these rats, which were fed a normal diet, had normal blood glucose, and were treated for 7 days rather than 30 days. In contrast to EXAMPLE 2, no significant effects were seen with LSF treatment. Hence, it is likely that the longer treatments are needed to show an effect in these rats.

Insulin (μU/ml) Vehicle LSF

0 0.53 ± 0.21 0.76 ± 0.34 200 1.92 ± 0.25 1.75 ± 0.10

20,000 4.35 ± 0.24 4.27 ± 0.22

Values are means ± SEM of 7-8 observations. EXAMPLE 4

This example provides assays that confirm the ability of the present xanthine-based compounds to inhibit glycation end-products. The in vitro pharmacological activity demonstrated by these assays is directly relevant to the in vivo formation of glycation products, which negatively correlate with patient health in the various disorders that may be treated according to the invention. These assays thus demonstrate the usefulness of the present compounds in methods of treating Type 1 diabetes.

The following in vi tro assays demonstrate inhibitory effects on the formation of AGEs and AGE-derived crosslinking of proteins and peptides by LSF. Some results show that LSF is 3 to 4 orders of magnitude more potent an inhibitor of AGE-formation than the aminoguanidine control.

The BSA-Glucose assay demonstrated the ability of LSF to inhibit glucose-mediated protein-AGE formation. See Mitsuhashi et al., J. Immunol. 207:79 (1977). Briefly, 50 mg/ml BSA (fraction V, Sigma) was incubated at 37°C for 7 days in 1.5 M phosphate buffer (pH 7.4) containing 400 mM glucose (144 mg/ml) and NaN₃ (0.2 g/1) in the presence or absence of various concentrations of LSF or aminoguanidine (added every 24 hours) . Specific fluorescence (excitation, 370 nm; emission, 440 nm) then was measured. Figure 2 shows the inhibitory effects of LSF (1, 3, and 5 μM) compared to 50 mM aminoguanidine, the positive control. The studies revealed that LSF is able to inhibit glucose-mediated development of BSA glycation at a concentration ranging from 1 to 10 μM.

To measure the ability of LSF to inhibit δ-Glu-mediated formation of glycated hemoglobin (HbA_lc) in a red cell sample, an assay was performed according to known methods. See Rahbar et al., Clin. Che . Acta 287 ( 1-2) : 123-130 (1999); Lindsay et al . , Clin. Acta 263:239 (1997). Briefly, 200 μl blood (drawn within 30 minutes of LSF treatment in potassium-EDTA) was mixed with 40 μl phosphate buffered saline, pH 7.4 alone (control) or containing 50 mM δ-Glu (Sigma) . After incubation for 16 hours at 37°C, the percentage of hemoglobin which was glycated was measured in triplicate versus a control using ion-exchange HPLC (BIORAD DIAMAT) . The assay was repeated using an eight-day incubation period and varying LSF concentration with 800 mg/ml glucose. HbA_lc was monitored as in Smith et al . , J. Clin.

Invest. 69:1164 (1982), using a vitamin E positive control. See Rajewswari et al., J. Cell Physiol. 149:100 (1997). These particular data were inconclusive, however the foregoing in vivo data show a substantial effect on HbA_lc formation. A GK peptide-ribose assay also demonstrated the inhibitory effect of LSF on protein-AGE formation. Figure 3 provides data demonstrating LSF inhibition of GK peptide (N- acetyl-glycyl-lysine methyl ester) crosslinking in the presence of ribose. Equal volumes (0.1 ml) of 0.2 g/1 NaN₃, 80 mg/ml GK peptide (Sigma) and 800 mM (120 mg/ml) ribose, all in 0.5M sodium phosphate (pH 7.4), were mixed and filtered through a 0.2-micron filter (Corning). LSF was added at different concentrations and the solutions incubated under aseptic conditions at 37°C for 24 hours. Samples then were analyzed for their specific fluorescence (Ex 340 nm; Em 420 nm) .

AGE-formation was determined using ion exchange HPLC (BIORAD, DIAMAT) as described above. Aminoguanidine (50 mM) was used as positive control. EXAMPLE 5

This example demonstrates that xanthine-based compounds prevent glucose-enhanced monocyte adhesion. The data indicate that xanthine-based compounds are useful in treating or preventing monocyte adhesion, which is a precipitating event in atherogenesis .

The methods are essentially as described in Kim et al . , Diabetes 43:1103-07 (1994). Briefly, human aortic endothelial cells were cultured in the presence of 25 mM (elevated) glucose and treated for 4 hours with 20 or 40 μM LSF. Following treatment, cells were washed and normal human monocytes were added. Lipopolysaccharide (LPS) served as a positive adhesion control. Results are depicted in Figure 4. Both concentrations of LSF significantly inhibited monocyte adhesion, as compared to the elevated glucose control (HG) . Moreover, the higher concentration of LSF reduced adhesion levels to those seen with normal glucose levels (NG) .

EXAMPLE 6

This example, measuring the extent of cross-linking of glycated BSA (AGE-BSA) to rat-tail collagen, demonstrates that xanthine-based compounds prevent the formation of advanced glycation products, which result from the cross-linking of glycated proteins. AGE-BSA was prepared by incubating BSA at 100 mg/ml with 200 mM glucose in 0.5 M sodium phosphate, pH 7.4 at 37°C for 12 weeks. The resulting glycated BSA was dialyzed against phosphate buffered saline for 48 hours, with 5 buffer changes.

Collagen-coated 96-well plates (Biocoat Cell Environment, Beckton Dickenson) were first blocked (200 μl/well) with Superbloc blocking buffer (Pierce #37515X) for one hour. The blocking solution was removed and the wells were washed three times with PBS containing 0.5% Tween 20 (PBS-Tween). Fifty microliters of AGE-BSA solution was added to each well, to final amounts of 0, 0.25, 0.5, 0.75 or 1.0 μg/well AGE-BSA. LSF in PBS, pH 7.4, was added at 5 μM, 10 μM or 20 μM and aminoguanidine was used as a positive control. Samples were incubated at 37°C for 4 hours.

Non-crosslinked material was removed by washing three times with PBS-Tween. Sample were then treated for one hour with polyclonal antibodies raised to AGE-RNase, which specifically bind the cross-linked AGE-BSA product followed by four washes with PBS-Tween. Bound antibody was detected by incubation with horseradish peroxidase- conjugated goat anti-rabbit immunoglobulin for 30 minutes, followed by washing and addition of substrate (2, 2-azino-di (3-ethylbenzthiazoline sulfonic acid; ABTS chromogen; Zymed #00-2011) . After 15 minutes, absorbance at 410 nm was measured using a Dynatech plate reader. Results are depicted in Figure 5. AGE represents the AGE-BSA control, with no LSF. LSF inhibited crosslinking of AGE-BSA at all concentrations used, particularly at 10 and 20 μM.

Claims

25

We claim:

l 1. A method of treating a patient to prevent the

2 formation of advanced glycation end-products, comprising 3 administering to patient in need thereof an effective amount of 4 at least one xanthine-based compound, or a pharmaceutically 5 acceptable salt thereof, having the following formula:

7 wherein :

8

9 R_x is an R enantiomer of an ω-1 secondary alcohol- 0 substituted alkyl (C₅_₈) , wherein said alcohol moiety is 1 optionally esterified; 2 R₂ is alkyl (C₁_₁₂) , optionally containing one or two 3 nonadjacent oxygen atoms in place of a carbon atom; and

R₃ is CH₃ or H.

i 2. A method according to claim 1, wherein the patient

2 suffers from a disorder selected from the group consisting of

3 diabetes, Parkinson's disease, Alzheimer's disease, amyotrophic

4 lateral sclerosis, Down's syndrome, osteoarthritis, cataracts, cardiac hypertrophy, arterial stiffening, atherosclerosis and kidney toxicity.

3. A method according to claim 2, wherein said compound is lisofylline (1- (5-R-hydroxyhexyl) -3, 7-dimethylxanthine) .

4. A method according to claim 1, wherein said effective amount is sufficient to reduce the blood glucose level of the said patient.

5. A method according to claim 4, wherein said effective amount is sufficient to reduce the blood glucose level of the said patient by at least about 10%.

6. A method according to claim 1, wherein said effective amount is sufficient to reduce the formation of glycation products in said patient.

7. A method according to claim 6, wherein said effective amount is sufficient to reduce the formation of glycation products in said patient by at least about 10%.

8. A method of treating the complications of diabetes, comprising administering to a patient in need of such treatment, and effective amount of at least one xanthine-based compound, or a pharmaceutically acceptable salt thereof, having the following formula:

wherein : R-_L is an R enantio er of an ω-1 secondary alcohol- substituted alkyl (C₅_₈) , wherein said alcohol moiety is optionally esterified; R₂ is alkyl (C₁_₁₂) , optionally containing one or two nonadjacent oxygen atoms in place of a carbon atom; and R₃ is CH₃ or H.

9. A method according to claim 8, wherein said complication is selected from the group consisting of nephropathy, retinopathy and neuropathy.

10. A method according to claim 8, wherein said complication is end-stage renal failure (ESRD) .