WO1999044598A2 - Methods for inhibiting βcell apoptosis - Google Patents

Abstract

The present invention is directed to methods and compositions for preventing β-cell destruction and for ameliorating obesity related NIDDM. More particularly the present inventors have found that β-cell destruction occurs in obesity related NIDDM through the action of ceramides. The present invention suggests that ceramides mediate their cytotoxic influence through increased NO production. More particularly, the active agents for preventing β-cell destruction in obesity related NIDDM are troglitazone, leptin, triacsin and fumosinins, and no-production inhibitors, like aminoguanidines.

Description

DESCRIPTION

FATTY ACID-INDUCED β-CELL APOPTOSIS

BACKGROUND OF THE INVENTION

1. Field of the Invention The present invention relates generally to the fields of diabetes and obesity. More particularly, it concerns the modulation of ceramide levels in β cells to overcome diabetic ailments in obesity.

2. Description of Related Art

The mechanism by which obesity, now the most common American disease (National Center for Health Statistics, 1994), leads to non-insulin-dependent diabetes mellitus (NIDDM), probably the second most common American disease, is unknown. It is generally agreed that insulin resistance is an invariable accompaniment of obesity, but that normoglycemia is maintained by compensatory hyperinsulinemia until the pancreatic β cells become unable to meet the increased demand for insulin, at which point NIDDM begins. The mechanism by which β cells become unable to meet rising insulin demand has never been elucidated, primarily because of the unavailability of human pancreatic islets for appropriate study. However, post-mortem studies in patients with NIDDM indicate that the β cell mass is reduced (Rahier et α/., 1983).

Obesity-linked non-insulin-dependent diabetes mellitus (NIDDM) is preceded by years of insulin resistance, during which normal blood glucose levels are maintained through effective compensation by pancreatic β cells (DeFronzo, R.A. 1988). In approximately 20% of obese individuals, the compensation wanes, hyperglycemia appears, and overt NIDDM is diagnosed. The depressed β cell function is thought be due to excess free fatty acids released from adipocytes in obesity (Campbell et al. , 1994) acting to initially stimulate, but ultimately impair, the function of β cells, and thus limit their compensatory capability. Thus, impaired β- cell function is a characteristic of NIDDM.

A phenomenon thought to play a role in β-cell dysfunction is the cytokine-mediated destruction of β-cells. It is thought that cytokine-induced destruction of islet β-cells is due to the generation of toxic oxygen radicals (Mandrup-Poulsen et al, 1987; Malaisse et al 1982). Such a mechanism for cytokine -mediated islet cell injury is based on the observation that islet cells possess very low oxygen free radical scavenging enzyme activities and as such are extremely vulnerable to free radicals (Asayama et al, 1986; Malaisse et al, 1982). Indeed Sumoski et al. , suggested that oxygen free radical scavengers may protect against cytokines IL- 1 , TNF and IFN-γ. However, it also has been demonstrated external application of free radical scavenging enzymes, such as superoxide dismutase or catalase, has no protective effect on cytokine toxicity (Burkart and Kolb, 1993; Yamada et al 1993).

High levels of NO and various free radicals correlate with two levels of damage to β- cells and β-cell lines. In the first instance, the free radical result in reduction in insulin secretion, that is, they induce β-cell dysfunction. In the second instance, these free radicals are cytotoxic and cause β-cell destruction In either case, diabetes results due to aberrations in insulin production or secretion.

Zucker Diabetic Fatty (ZDF) rats provide a useful replica of the human phenotype of adipogenic NIDDM in which to study the islets (Peterson et al . 1990). Such studies implicate fat deposition in islets as the cause of the β cell decompensation, so-called "lipotoxicity" (Lee et al. 1994; Unger, 1995).

β cell decompensation in this form of diabetes may involve exaggerated induction by FFA of inducible nitric oxide synthase (iNOS) and excess nitric oxide (NO) generation (Shimabukuro et al, 1997a). Because intracellular NO is an important mediator of programmed cell death (Moncada et al, 1991; Corbett et al, 1992; Kaneto et al, 1995), it seems possible that the loss of the β cells observed late in the course of adipogenic NIDDM (Ohneda et al, 1995) might be the result of NO-induced apoptosis. Apoptosis has been reported in fat-laden hepatocytes (Yang et al, 1997).

Thus, it is clear from the foregoing discussion that adipogenic NIDDM is a complicated, multifactorial phenomenon that debilitates a large population of individuals. Unfortunately, to date, there has been no unifying characteristic that can be manipulated to provide a cure or ameliorate the symptoms of the deleterious effects of this disease. SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions for preventing β-cell destruction and for ameliorating diabetes. More particularly, the present inventors have found that β-cell destruction occurs in obesity related NIDDM through the action of ceramides. The present invention suggests that ceramides mediate their cytotoxic influence through increased

NO production.

Therefore, in one aspect the present invention provides a method of inhibiting ceramide- mediated apoptosis in a mammalian cell comprising contacting the cell with an agent that reduces levels of ceramides in the cell as compared to an untreated cell. In certain embodiments, the agent inhibits the synthesis of ceramide in the cell; inhibits the uptake of ceramide by the cell or removes ceramide from the cell. In preferred embodiments, the agent is selected from the group consisting of fumonisin B, and fuminosin B₂. In particular aspects the agent that mediates a ceramide decrease in cells, may be one that blocks activation of ceramides thereby preventing the ceramides from further metabolism. In other embodiments, the agent may be one that selectively lowers ceramide content of islets, for example, fumonisin B B₂ or an analogue thereof. In still further embodiments, a generalized ceramide lowering agent such as leptin.

In further embodiments, the cell is further contacted with an agent that inhibits NO production in the cell. In particular aspects of the present invention the apoptosis is cytokine- mediated. The cytokine may be IL-lβ, IL-lα, γlFN, TNF-α, TNF-β, an IL-8, an IL-12, IL-6,

IL-2, IL-3, IL-5, IL-7, IL-9, IL-14, IL-17, granulocyte-macrophage colony stimulating factor or monocyte chemoattractant protein- 1.

Certain aspects of the present invention further comprise introducing into the cell a gene operatively linked to a promoter. In preferred embodiments, the gene may encode insulin, growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone, adrenocorticotropin (ACTH), angiotensin I, angiotensin II, β-endorphin, β-melanocyte stimulating hormone (β-MSH), cholecystokinin, endothelin I, galanin, gastric inhibitory peptide (GIP), glucagon, amylin, lipotropins. neurophysins, GLP-1, leptin, leptin receptor, calcitonin and somatostatin. In defined embodiments, the promoter may be an inducible promoter. In other particular embodiments, the promoter is selected from the group consisting of CMV IE, SV40 IE, RSV LTR, RIP, modified RIP, POMC, βgal, lac operon, ecdysone-inducible expression system, tetracycline operon, glucocorticoid response element, heat shock promoter and GHP promoter.

It is contemplated that the mammalian cell may be independently a human cell or a non- human cell. The mammalian cell may be a secretory cell, a neuroendocrine cell, a pancreatic beta cell, a pituitary cell, a hepatic cell, a skeletal muscle cell, or a myocardial cell. In other embodiments, the cell may be independently defined as secretagogue responsive; glucose responsive or non-glucose responsive.

In particularly preferred embodiments, the cell is derived from a CM-1, TRM, TRM6 or

HAP5 cell.

In certain embodiments, it is contemplated that the gene is introduced into the mammalian cell along with a selectable marker gene. In preferred embodiments, the selectable marker may be selected from a group consisting of hygromycin resistance, neomycin resistance, puromycin resistance, zeocin, gpt, DHFR and histadinol.

Also provided herein is a method of treating a subject for β-cell destruction comprising contacting the subject an agent that reduces levels of ceramides in the cells of the subject as compared to the untreated level of ceramides, wherein reduction in ceramide levels protects cells of the subject against ceramide-mediated lipotoxicity. The agent may independently inhibit the synthesis of ceramide in the subject; inhibit the uptake of ceramide by the cells of said subject or remove ceramide from the cells of said subject.

In other embodiments, the subject is further contacted with an agent that inhibits NO production in the cell. In certain embodiments, the β-cell destruction is cytokine-mediated. In other preferred embodiments, the subject exhibits at least one pathologic condition selected from the group consisting of insulin-dependent diabetes mellitus (IDDM); insulin-independent diabetes mellitus (NIDDM) and obesity.

In certain other embodiments, the method further comprises introducing into the mammalian cell a gene operatively linked to a promoter. The gene may encode insulin, growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone, adrenocorticotropin (ACTH), angiotensin I, angiotensin II, β-endorphin, β-melanocyte stimulating hormone (β-MSH), cholecystokinin, endothelin I, galanin, gastric inhibitory peptide (GIP), glucagon, amylin. lipotropins, neurophysins, GLP-1, leptin. leptin receptor, calcitonin and somatostatin.

Also contemplated is a method of preventing non-insulin dependent diabetes mellitus (NIDDM) in a subject comprising the steps of identifying a subject at risk of diabetes mellitus; and providing to the subject a composition comprising an agent that reduces levels of ceramides in the cells of the subject as compared to the untreated level of ceramides, wherein the reduction in ceramide level protects cells of the subject against ceramide-mediated apoptosis of β-cells.

In particular embodiments, the NIDDM is NO-mediated NIDDM. In certain aspects of the present invention the subject is further contacted with an agent that inhibits NO production in the subject. In other embodiments, the NO-mediated NIDDM is cytokine-mediated NIDDM. In particularly preferred embodiments, the cytokine is IL-lβ, IL-lα, γlFN, TNF-α, TNF-β, an IL-8, an IL-12, interleukin-6, IL-2, IL-3, IL-5, IL-7, IL-9, IL-14, IL-17, granulocyte- macrophage colony stimulating factor or monocyte chemoattractant protein- 1.

Other embodiments contemplate a method for inhibiting ceramide-mediated

of a target cell comprising blocking ceramide production or accumulation in the cell. In preferred embodiments, the blocking results in a decrease in the ceramide content of the cell when compared to the level of ceramide in the cell in the absence of the inhibitor. In more preferred embodiments, the decrease in the ceramide content of the cell is mediated b> contacting the cell with leptin. In particular aspects, the contacting comprises contacting the cell with an expression construct comprising a leptin encoding gene operatively linked to a promoter. The promoter may be selected from the group consisting of CMV IE, SV40 IE, RS V LTR, RIP, modified RIP, POMC, βgal, lac operon, ecdysone-inducible expression system. tetracyline operon, glucocorticoid response element, heat shock promoter and GHP promoter. In particularly preferred embodiments, the cell is a pancreatic beta cell. Also provided herein is a method of preventing non insulin dependent diabetes mellitus (NIDDM) in a subject comprising blocking ceramide production in a pancreatic beta cell in the subject. In particularly preferred embodiments, the blocking ceramide production is accomplished by administering to the cell an amount of an inhibitory agent sufficient to protect the cell from ceramide-mediated lipotoxicity. In other preferred embodiments, the blocking ceramide production is accomplished by administering to the cell an amount of an inhibitory agent sufficient to protect the cell from NO-mediated lipotoxicity. In particularly preferred aspects of the present invention the cell is engineered to secrete insulin.

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 preferred 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.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

FIG. 1A and FIG. IB. FIG. 1A. DNA fragmentation in freshly isolated islets from

5 (preobese)- 7 (obese and prediabetic)- and 14 (diabetic)-wk-old α//α ZDF rats and lean +/+ controls, fragmented DNA percent; Lane 1, 100-bp DNA size marker (Boeringer, Mannheim). FIG. IB. Ceramide content in pancreatic islets of obese fa/fa ZDF (solid bars) and lean +/+ controls (open bars). Values are the mean ± SEM of three or four studies, *P < 005 vs. +/+ V < 0.05 vs. 7 wk.

FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D. FIG. 2A. Effect of FFA on DNA fragmentation in pancreatic islets from lean wild-type (+/+) and obese prediabetic homozygous (fa/ fa) ZDF rats. Pancreatic islets were isolated from 7-wk-old rats and cultured with 1 mM FFA as indicated. Ladder % presents fragmented DNA percent. FIG. 2B. Effect of FFA on ceramide content in pancreatic islets of ZDF rats. Islets from lean wild-type (•) and obese homozygous ( D) ZDF rats were cultured with 0 or 1 mM FFA at the indicated times, and ceramide contents were determined. FIG. 2C. De novo synthesis of [ H] ceramide from [³H]palmitate in islets of wild-type (+/+) and fa/fa ZDF rats in the absence and presence of the ceramide synthase inhibitor fumonisin B] (FB,). Rats were 7 wk of age. Values are the mean ± SEM of triplicate studies. FIG. 2D. Comparison of [ H] ceramide and [ H]H₂O to assess relative rates of de novo ceramide synthesis vs. oxidation of [Ηjpalmitate by isolated islets of +/+ and fa/ fa rats. Values are the mean ± SEM of triplicate studies. *P < 0.05 vs. +/+.

FIG. 3. Effect of exogenous ceramide and of blockage of ceramide synthesis on DNA fragmentation in islets from obese fa/fa ZDF rats. Islets isolated from 7-wk-old obese prediabetic fa/fa ZDF rats were cultured for 24 h in medium containing 15 μM C₂-ceramide (C2-Cer) without FFA or 1 mM FFA plus 50 μM fumonisin B, (FB^, an inhibitor of ceramide synthetase. Lane 1, 100-bp DNA size marker (Boeringer Mannheim); Ladder (%): fragmented DNA percent.

FIG. 4A, FIG. 4B and FIG. 4C. FIG. 4A. Inhibitory effect of triacsin C, troglitazone, and aminoguanidine on FFA-induced DNA fragmentation in islets from obese fa/ fa ZDF rats. Islets isolated from 7-wk-old fa/fa ZDF rats were cultured for 24 h at 0 or 1 mM FFA with lOμM triacsin C, 10 μM troglitazone, or 0.5 mM aminoguanidine, M, 100-bp DNA size marker. FIG. 4B. Inhibitory effect of triacsin C. troglitazone, and aminoguanidine on FFA-induced iNOS mRNA induction. FIG. 4C. FFA-induced NO production in islets of obese fa/ fa ZDF rats. Effect of triacsin C, troglitazone, and aminoguanidine on islets were cultured as described in FIG. 4A except for 48 h.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Obesity-linked non-insulin-dependent diabetes mellitus (NIDDM) is preceded by years of insulin resistance, during which normal blood glucose levels are maintained through effective compensation by pancreatic β cells (DeFronzo. 1988). In approximately 20% of obese individuals, the compensation wanes, hyperglycemia appears, and overt NIDDM is diagnosed. The mechanisms by which obesity initially enhances and subsequently depresses β- cell function have eluded investigators. It has been suggested that depressed β cell function is due to excess lipids, for example, free fatty acids (FFA) released from adipocytes in obesity (Campbell et al, 1994) acting to initially stimulate, but ultimately impair, the function of β cells, thus limiting their compensatory capability. Chronic elevation of lipids has been shown to interfere with glycolysis and glucose oxidation and to contribute to both insulin resistance and to alterations in β-cell function that are characteristic of obesity. The present invention demonstrates the definitive role that ceramides, a class of lipids, play in diabetic abnormalities. This mechanism of action, as well as related methods and compositions for ameliorating the deleterious effects of ceramides in diabetes are presented herein below.

A. The Present Invention Fat deposition in islets has previously been suggested as the cause of the β cell decompensation, so-called "lipotoxicity" (Lee et al, 1994; Unger, 1995). Excess fat in β cells and other nonadipocytes in this form of obesity is ascribed to the high plasma levels of free fatty acids (FFAs) (Lee et al, 1994; Unger, 1995), coupled with a greatly enhanced capacity for lipogenesis (Lee et al, 1997).

The present invention demonstrates that apoptosis is increased in the islets of ZDF rats that are progressing through the prediabetic and diabetic stages of disease. The inventors show that this apoptotic effect on β cells is related to the high fat content of the islets and that this greatly exaggerates the apoptotic effect of fatty acyl-CoA. The present invention for the first time shows that ceramide is a mediator of the FFA-induced apoptosis in β-cells.

FFA, a precursor of ceramide, raises the ceramide levels in fat-laden ZDF islets via a greater rate of de novo biosynthesis as indicated by increased incorporation of [ HJpalmitate into [ Hjceramide in the fa/fa islets. The present inventions shows that, in diabetes, there is a striking increase in de novo ceramide formation mediated by an underlying increase in ceramide synthase.

Thus, the inventors showed that the β-cell apoptosis seen in diabetes is directly correlated to increased FFA and ceramide levels in adipogenic NIDDM. This rise in FFA results in an increase in FFA metabolites, namely, ceramide and triglycerides. These molecules are thought to increase β-cell apoptosis through a mechanism involving NO production and iNOS expression. It is, therefore, suggested by the present inventors that prophylactic interventions that reduce ceramide production in islets will be useful in preventing, ameliorating or otherwise decreasing the deleterious effects of obesity-associated NIDDM. Methods and compositions for achieving such a beneficial outcome are presented herein below.

a) Factors Involved in β-Cell Damage High levels of NO and various free radicals correlate with two levels of damage to β- cells and β-cell lines. In the first instance, the free radicals result in reduction in insulin secretion, that is, they induce β-cell dysfunction. In the second instance, these free radicals are cytotoxic and cause β-cell destruction. In either case, diabetes results due to aberrations in insulin production or secretion. Thus, the compositions of the present invention may be used to alleviate the deleterious effect of β-cell dysfunction and β-cell destruction caused by lipotoxicity.

As used herein, "lipotoxicity" refers to any event that results in cellular damage and can range from mild cellular dysfunction to cell death that has been mediated through the effects of a lipid, in particular a ceramide. Lipotoxicity can be associated with obesity-linked disorders including diabetes, cardiovasucular disease, stroke, muscoskeletal degeneration, and hypertension. Lipotoxicity may occur as a result of, or be associated with, aberrant levels of intracellular and extracellular ceramide, and can exert cellular damage through increases in free radicals and cytokines. Lipotoxicity may also exert effects on cellular metabolism and contribute in this way to cellular damage and dysfunction. Lipotoxicity is associated with severe progressive functional and morphologic alterations of β cells at the onset of NIDDM in Zucker diabetic fatty (ZDF) rats (Lee et al, 1994; Unger, 1995).

NO has been shown to mediate IL-lβ-induced impairment of β cell function (Corbett et al, 1992; Corbett and McDaniel, 1995; Eizirik et al, 1996; McDaniel et al, 1996), and ultimately cause β cell death (Kaneto et al, 1995). This mechanism is likely operative in the deterioration of β cells that occurs in obesity. Additionally, it is suggested that in vitro and in vivo evidence that therapeutic strategies to reduce NO production in islets will be useful in preventing adipogenic NIDDM.

Long-chain fatty acids influence pancreatic β-cells via the NO system. In other tissues. NO is thought to have a dual role, serving as a regulator under physiologic conditions (Moncada et al, 1991) and as a cytotoxin under pathophysiologic circumstances (Radons et al, 1994). As a physiologic regulator, NO mediates diverse functions in many organs (Moncada et al, 1991), including the cardiovascular, neuromuscular, neurological, genitourinary, gastrointestinal, and renal systems; in pancreatic islets, NO regulates islet blood flow (Svensson et al., 1994).

The constitutive forms of nitric oxide synthase, NOS I and III, have been identified in rat islets and in β-cell lines (Moncada et al, 1991), and iNOS (NOS II) has been induced in islets by IL-lβ (Corbett et al, 1992; Corbett and McDaniel, 1995; Eizirik et al, 1996; McDaniel et al, 1996; Kaneto et al, 1995; Akabane, 1995). The induction of iNOS by IL-l β results in cytotoxicity (Corbett et al , 1992, Corbett and McDaniel, 1995; McDaniel et al. 1996; Kaneto et al, 1995). NO donors have been shown to cause both β-cell dysfunction and damage (Eizirik et al, 1996).

The cytotoxic role of NO can be induced by FFA in islets of rats predisposed to NIDDM. FFA caused a reduction in both basal and glucose-stimulated insulin secretion in islets from lean fa/+ and obese prediabetic fa/fa ZDF rats in association with a greater than 20- fold FFA-induced increase in NO.

The addition of NIC, an inhibitor of iNOS expression, prevents the induction of iNOS by FFA, reduces NO production, and prevents the FFA-induced decrease in insulin secretion in islets. AG, both a competitive inhibitor of iNOS and a suppressor of its expression (Corbett and McDaniel, 1996; Joshi et al, 1996), also prevents the decrease in insulin secretion in islets, as does NAME, a purely competitive inhibitor.

Thus, NO causes, or is required for, FFA-induced attenuation of insulin secretion in prediabetic rat islets. The relevance of the in vitro findings to clinical diabetes in vivo is supported by the fact that iNOS mRNA levels are 20 times higher in diabetic rats than in lean nondiabetic controls and that immunostainable iNOS can be detected only in islets of diabetic obese ZDF rats. The cellular source of iNOS and NO may well be the β-cells rather than macrophages. NO production by purified β-cells has been reported previously (Corbett et al . 1992) and iNOS expression in β-cells has been documented (Corbett and McDaniel, 1995). Moreover, there was no evidence of macrophages in pancreatic sections from ZDF rats, using two immunochemical stains specific for macrophages.

In these rats, plasma FFA are elevated and triacylglycerol content of islets is increased. Further, there is evidence that intracellular FFA is high (Lee et al, 1994; Unger, 1995). ZDF rats are leptin-resistant because of a Glu 269- Pro mutation in the leptin receptor (Phillips et al, 1996), and their islets have an increased capacity to esterify and a decreased capacity to oxidize FFA (Lee et al, 1997). This defect, which must be related to the leptin resistance, somehow causes the greater induction of iNOS expression by FFA, perhaps by increasing intracellular levels of FFA. Although the mechanism by which FFA or high triacylglycerol increases iNOS expression and NO production in pancreatic islets is unknown, increased levels of diacylglycerol and/or ceramide are among the possibilities.

Diacylglycerol (DAG) is an intracellular signalling mediator that has been associated with abnormalities induced by elevated glucoses levels and in the development of diabetic vascular complications (Chen et al, 1991 ; Wolf et al , 1991 ; Lee et al. 1989; Inoguchi, et al, 1992). The skilled artisan is referred to U.S. Patent No. 5,674,862 (specifically incorporated herein by reference), which discusses the involvement of DAG in diabetes and provides methods and compositions for treating diabetes and its complications.

NIC decreases plasma FFA levels and inhibits iNOS expression in obese prediabetic ZDF rats, while AG suppresses FFA-induced iNOS mRNA expression without lowering the high plasma FFA. This raises the possibility that clinical adipogenic NIDDM and its associated β-cell abnormalities (loss of glucose-stimulated insulin secretion, loss of GLUT-2, and a reduction in β-cell mass) can be prevented by treatment with agents that reduce the FFA levels and/or decrease FFA-mediated NO generation.

Thus, like IL-l β, long-chain fatty acids (FFA) upregulate iNOS expression and enhance NO generation in rat islets, thereby implicating the lipid content of islets in the cytotoxic effects of IL-lβ. It is, therefore, likely that islet tissue lipopenia might protect β-cells against NO- mediated cytotoxicity. Indeed, there is a striking relationship between islet triglyceride (TG) content and IL-l β-mediated NO production and cytotoxicity. Further, leptin and troglitazone, agents that lower islet TG content (Fulgencio et al, 1996; Shimabukuro et al, 1997), reduce IL-1 β-induced NO production and cytotoxicity.

iNOS expression and nitrite production are upregulated in both immunogenic (IDDM) (Mandrup-Poulsen, 1996; McDaniel et al, 1996; Eizirik et al , 1996; Corbett and McDaniel 1992; Kleemann et al, 1993; Corbett et al, 1993; Lindsay et al, 1995) and adipogenic (NIDDM; see section on Examples) diabetes. Agents that inhibit iNOS expression or nitrite production in vitro and in vivo, for example, nicotinamide and aminoguanidine, prevent β-cell abnormalities and hyperglycemia in both forms of diabetes (Corbett et al, 1993; Lindsay et al, 1995).

It is believed that islet TG content might influence the cytotoxic potency of IL-lβ, a cytokine implicated in the pathogenesis of autoimmune diabetes (Mandrup-Poulsen, 1996; Bendtzen et al, 1986; McDaniel et al, 1996; Krδncke et al, 1991 ; Eizirik et al, 1996; Corbett and McDaniel 1992; Kleemann et al, 1993; Corbett et al, 1993; Lindsay et al, 1995; Shimabukuro et al, 1997). This premise is consistent with a recent report that obesity increases sensitivity to endotoxin-induced liver injury (Yang et al, 1997).

The islets of obese ZDF rats are fat laden, while those of leptin-overexpressing rats are fat depleted. Normal rats pair-fed to the hyperleptinemic rats exhibit an islet TG content intermediate between the hyperleptinemic group and normal controls. There is a remarkable relationship between TG content of islets, NO generation, and cell viability was observed. NO production is minimal in islets of the hyperleptinemic rats and highest in those of the obese. Viability is maximal in the islets of hyperleptinemics and minimal in those of the obese rats.

These in vivo effects also are evident in vitro in cultured islets subjected to maneuvers that alter islet TG content. FFA increase islet TG content and NO production rises and viability declined. Troglitazone reduces TG content and NO production declines and viability improved. This was observed even in the islets of the obese ZDF rats, which are resistant to the lipopenic action of leptin because of the mutation in their leptin receptor (Phillips et al, 1996; Iida et al, 1996).

Orally administered agents such as troglitazone should lower islet TG content in humans as it did in the rat islets. Thus, one can assess its co-administration with nicotinamide for the prevention of autoimmune diabetes without the use of immunosuppression. The striking reduction in nitrite production and improvement in cell viability associated with a decrease in islet TG content from normal to subnormal is consistent with the usefulness of this strategy. In addition, the leptin-like effects of troglitazone in lowering TG content provide a new mechanism for the treatment of adipogenic non-insulin-dependent diabetes (Fujiwara et al, 1988).

It has previously been speculated that intracellular FFA are required for the formation of diacyl glycerol and ceramide (Lee et al, 1997), both of which are believed to participate in the IL-lβ signal cascade (Mandrup-Poulsen, 1996; McDaniel et al, 1996; Kleemann et al, 1993; Corbett et al, 1993). Ceramide is a fatty acid-containing messenger in cytokine-induced apoptosis, the inventors found that ceramide was significantly increased in prediabetic and diabetic islets. When the prediabetic islets were cultured in 1 mM FFA, it was found that apoptosis as measured by DNA laddering rose to 19.6% vs. 4.6% in lean control islets. This increase in apoptotic activity was immediately preceded by an 82% increase in ceramide.

The inventors^' also observed apoptosis when ceramide (C₂-Ceramide) is added without the presence of FFA. However, if a ceramide synthetase inhibitor, such as fumonisin B_b is added to the prediabetic islets in culture there was a complete block of FFA-induced DNA laddering in cultured ZDF islets.

Triacsin C, an inhibitor of fatty acyl-CoA synthetase, and troglitazone, an enhancer of FFA oxidation in ZDF islets, also both blocks islet cell apoptosis. These agents also reduced inducible nitric oxide (NO) synthase mRNA and NO production, which are involved in FFA-induced apoptosis. Thus the present invention demonstrates that in obesity, β cell apoptosis is induced by increased FFA via de novo ceramide formation and increased NO production. It is suggested that methods of decreasing the levels of ceramide in the islet cells of diabetic individuals will be effective in the treatment of disease. Methods and compositions for preventing this apoptosis are provided herein below.

B. Compositions for Modulating Lipid Levels and iNOS Activity in Diabetes

The inventors have shown that β-cell lipotoxicity in obesity and adipogenic NIDDM involves ceramide- and/or NO-mediated apoptosis. Also, the inventors' studies have demonstrated that increased ceramides induce an increase in iNOS activity. Thus, it is possible that there is an increase in ceramide as a result of increased presence of free fatty acids in the islets of prediabetics and diabetics and this ceramide induces β-cell apoptosis through NO. Formulations that decrease ceramide or free fatty acid content of cells will result in beneficial effects in that such modulation will decrease iNOS expression and NO production in pancreatic islets. In other embodiments, it may be useful to directly inhibit iNOS activity and/or expression. The present section describes various methods of achieving such a beneficial decrease in the cellular ceramide content.

a) Leptin In certain embodiments, it may be beneficial to contact cells with, or engineer cells to express and overexpress the obesity-associated protein known as leptin or a receptor therefore. Leptin is a peptide hormone that controls body composition and is believed to do so, at least in part, via interaction with hypothalamic receptors that regulate food intake and body weight. The various isoforms of leptin receptor (Ob-R), including the long isoform (OB-Rb), are widely expressed in various tissues, suggesting that leptin may play an important role in actions on extraneural tissues as well.

Additional evidence that leptin has non-neural function comes from a report that extraordinary changes in body fat are seen in rats made chronically hyperleptinemic by treatment with an adenovirus vector expressing the leptin cDNA (Chen el al, 1996). In this report, rats lost all discernible body fat within 7 days of adenovirus infusion, while animals that were "pair-fed" at the same low rate of food intake as the hyperleptinemic animals retain more of their body fat. The magnitude and rapidity of the lipid depletion suggested the possibility of a direct "hormone-to-cell" action by leptin, in addition to effects cause through the sympathetic nervous system.

Chen et al (1996) also examined the effects of leptin overexpression on plasma glucose, insulin, plasma triglycerides and free fatty acid levels. While glucose did not change, both plasma triglycerides and free fatty acids dropped by about 50% in adenoviral-leptin treated animals, when compared to controls (Ad-β-gal or saline). These studies now have been confirmed and extended with respect to phospholipids. No clear cut changes in phospholipid concentration was observed. However, using an in vitro system, it was established that reductions in triglyceride levels could be achieved in the absence of sympathetic nervous system effects. Studies performed to determine what pathways are involved in the triglyceride depletion indicated that leptin-induced triglyceride depletion involves a novel mechanisms by which triglyceride disappears through enhanced intracellular triglyceride metabolism, rather than through more traditional free fatty acid export pathways.

Insulin levels in adenovirus-leptin infected rats dropped even more dramatically than the fatty acids, being only about 1/3 of the amount seen in controls. As stated above, the glucose levels of these animals was normal, however. These findings are consistent with enhanced insulin sensitivity in treated animals. Pancreata were isolated from hyperleptinemic rats and examined for β-cell function and morphology. The most striking finding was the complete absence of insulin secretion in response to either glucose or arginine. The morphology appeared normal, and it was determined that insulin secretion could be reestablished following perfusion of pancreatic tissue in the presence of free fatty acids, thereby establishing an important role for these molecules in β-cell function. These studies also indicate that leptin- mediated reduction of elevated tissue lipid levels will improve β-cell function, reduce insulin resistance and help restore abnormal glucose homeostasis in obese individuals.

A further connection between diabetes and leptin comes from studies with genetically obese ZDF rats, which contain mutant OB-R genes. The islets of these animals become overloaded with fat at the time that hyperglycemia begins. Because maneuvers that reduce islet fat content prevent diabetes in ZDF rats, it has been proposed that the accumulation of triglycerides in islets plays a causal role in β-cell dysfunction. Thus, the predisposition to diabetes in homozygous ZDF rats may reflect the fact that their tissue have been completely "unleptinized" throughout their life and therefore have accumulated high levels of TG. In normal rats, this accumulation is prevented by the action of leptin. It is expected that any therapy that reduces triglycerides in islets and in the target tissues of insulin will improve β-cell function and reduce insulin resistance.

In hyperleptinemic rats, every tissue that was examined was lipopenic. Thus, it is speculated that normal non-adipocytes carry a minute quantity of triglyceride, perhaps to serve as a reserve source of fuel in adipocytes that are depleted of fat by starvation and become unable to meet the fuel needs of certain tissues. It is suspected that this triglyceride storage function is closely regulated by leptin. In the obese ZDF rats, this regulatory control is absent, and these putative intracellular triglycerides reserves soar to levels of over 1000-times that of hyperleptinemic rats.

In light of these observations, the present application therefore encompasses various engineered cells which express leptin in amounts in excess of normal. The methods by which leptin genes may be manipulated and introduced are much the same as for other genes included herein, such as amylin. A preferred embodiment would involve the use of a viral vector to deliver a leptin-encoding gene, for example, an adenoviral vector. This approach may be exploited in at least two ways. First, in the engineering of cells to produce certain polypeptides in vitro, it may be desirable to express high levels of leptin in order to downregulate various cellular functions, including synthesis of certain proteins. Similarly, leptin overexpression may synergize with cellular functions, resulting in the increased expression of an endogenous or exogenous polypeptide of interest.

Second, it may be desirable to use a leptin-overexpressing cell, or a leptin expression construct, such as a leptin-expressing adenovirus, in an in vivo context. This includes various

"combination" approaches to the treatment of disease states such as obesity, hyperlipidemia and diabetes. For example, leptin expressing cell lines may provide for prolonged expression of leptin in vivo and for high level expression. Preliminary results indicate that injection of recombinantly produced leptin is less efficacious at achieving weight loss and reduction of lipids. Induction of hyperleptinemia using cells lines or expression constructs also may find use in reducing fat content in livestock just prior to slaughter. Moreover, because leptin- induced weight loss may act through different mechanisms than those currently employed, it may be possible to avoid related side effects such as diet-induced ketosis, heart attack and other diet-related symptoms. These regimens may involve combinations of other engineered cells, cells engineered with leptin and at least one other gene or genetic construct (knock-out, antisense, ribozyme, etc.), combination gene therapy or combination with a drug. The methods of delivering such pharmaceutical preparations are described elsewhere in this document

As an alternative to increasing the expression of leptin in a cell-based therapy, it may be possible to deliver cells with a high level of leptin receptor expression. The presence of an active leptin receptor will allow a response to applied leptin. Therefore, in therapeutic applications, it will be possible to deliver these receptor-bearing cells to a patient in need thereof, in such a way that these cells will then be able to respond to leptin. The leptin may be endogenously secreted leptin, recombinant leptin that has been contacted with the cell in the form an expression construct or exogenously applied purified leptin.

As shown in the results presented herein leptin reduced IL-l β-induced NO production and cytotoxicity. The inventors therefore suggest that the use of leptin and other agents that decrease islet ceramide content will likely be useful in the prevention of both type 1 and type 2 diabetes.

b) Troglitazone Out of the many drugs available for the treatment of diabetic ailments, the thiazolidinedione derivatives are very prominent and are considered effective constituents working through a different mechanism than the other class of commonly used anti-diabetic agents, the sulfonylureas. Troglitazone (5-[[4-[3(3,4- dihydro-6-hydroxy-2, 5, 7, 8-tetramethyl- 2H-l-benzopyran-2-yl) methoxy] phenyljmethyl] 2, 4-thiazolidinedione), is one such thiazolidinedione, which exhibits euglycemic effect, was reported first in 1983 by Sankyo Co. Ltd., Japan (Japanese Patent No. 60-051189/ Australian Patent No. 570067) and has created interest in the field ever since.

Pharmaceutical troglitazone is known as Rezulin™ and is sold as an oral antihyperglycemic agent which acts primarily by decreasing insulin resistance, and is used in the management of type II diabetes (NIDDM). It improves sensitivity to insulin in muscle and adipose tissue and inhibits hepatic gluconeogenesis. Troglitazone is not chemically or functionally related to either the sulfonylureas, the biguanides, or the α-glucosidase inhibitors.

Troglitazone is a white to yellowish crystalline compound; it may have a faint, characteristic odor. Troglitazone has a molecular formula of C₂ H₂₇NO₅S and a molecular weight of 441.55 daltons. It is soluble in N,N-dimethylformamide or acetone; sparingly soluble in ethyl acetate; slightly soluble in acetonitrile, anhydrous ethanol, or ether; and practically insoluble in water.

Troglitazone is available as 200 and 400 mg tablets for oral administration formulated with the following excipients: croscarmellose sodium, hydroxypropyl mefhylcellulose, magnesium stearate, microcrystalline cellulose, polyethylene glycol 400, polysorbate 80, povidone, purified water, silicon dioxide, titanium dioxide, and synthetic iron oxides.

Troglitazone is a thiazolidinedione antidiabetic agent that lowers blood glucose by improving target cell response to insulin. It has a unique mechanism of action that is dependent on the presence of insulin for activity. Troglitazone decreases hepatic glucose output and increases insulin-dependent glucose disposal in skeletal muscle. Its mechanisms of action is thought to involve binding to nuclear receptors (PPAR) that regulate the transcription of a number of insulin responsive genes critical for the control of glucose and lipid metabolism. Unlike sulfonylureas, troglitazone is not an insulin secretagogue.

In animal models of diabetes, troglitazone reduces the hyperglycemia, hyperinsulinemia. and hypertriglyceridemia characteristic of insulin-resistant states such as type II diabetes. Plasma lactate and ketone body formation are also decreased. The metabolic changes produced by troglitazone result from the increased responsiveness of insulin-dependent tissues and are observed in numerous animal models of insulin resistance. Treatment with troglitazone did not affect pancreatic weight, islet number or glucagon content, but did increase regranulation of the pancreatic beta cells in rodent models of insulin resistance.

Since troglitazone enhances the effects of circulating insulin (by decreasing insulin resistance), it does not lower blood glucose in animal models that lack endogenous insulin.

Maximum plasma concentration (Cmax) and the area under plasma concentration-time curve (AUC) of troglitazone increase proportionally with increasing doses over the dose range of 200 to 600 mg/day. Following daily drug administration, steady-state plasma concentrations of troglitazone are reached within 3 to 5 days. Thus, the present invention contemplates the use of troglitazone doses including but not limited to 100 mg/day, 150 mg/day, 200 mg/day, 300 mg/day, 400 mg/day, 500 mg/day, 600 mg/day, 700 mg/day.

Clinical studies demonstrate that troglitazone improves insulin sensitivity in insulin- resistant patients. 1 roglitazone increases insulin-dependent glucose disposal, reduces hepatic gluconeogenesis. and enhances cellular responsiveness to insulin and thus, improves dysfunctional glucose homeostasis. In patients with type II diabetes, the decreased insulin resistance produced by Troglitazone causes decreases in serum glucose, plasma insulin, and hemoglobin A_1C. These effects are independent of weight loss and persist with troglitazone treatment.

Following troglitazone treatment, LDL, HDL, and total cholesterol (total-C) increase, although total-C/HDL and LDL/HDL ratios do not change. The increase in total cholesterol is due to the increase in HDL and LDL cholesterol. Despite the observed increase in total and LDL cholesterol, ApoB fraction levels are not increased. Patients treated with troglitazone and concomitant insulin exhibit an initial reduction in triglyceride levels. With the reduction in insulin doses that may occur following Troglitazone therapy, some attenuation of the triglyceride reduction may occur.

Troglitazone has only been shown to exert its antihyperglycemic effect in the presence of insulin. Because troglitazone does not stimulate insulin secretion, hypoglycemia in patients treated with troglitazone alone is not to be expected.

Thus, troglitazone is considered one of the most effective antidiabetic drugs available today. It has a multipurpose activity not only acting on diabetes itself but also on the reduction of the triglycerides and also on the accompanying complications, such as, cataract, neuropathy, retinopathy etc. (which are usually the chronic ailments accompanied by diabetes). Indeed, the troglitazone is emerging as the first drug candidate of englycemic class of antidiabetic agents. The present invention contemplates the use of troglitazone in order to reduce the triglyceride content of cells and to ameliorate diabetic ailments. Other troglitazone derivatives, for example those disclosed in U.S. Patent No. 5,700,820, have been shown to have enhanced anti-diabetic effects as compared to the native troglitazone structure. In as much as these compounds will also be useful in the present invention, U.S. Patent No. 5,700,820, is specifically incorporated herein by reference.

c) Inhibition of ceramide-mediated response Ceramide is believed to act as a second messenger through the activation of various kinases. In some reports it has been suggested that ceramide and other members of sphingomyelin pathway may mediate senescence and apoptosis (Jayadev et al, 1994; Dbaibo et al, 1993; Haimovitz-Friedman et al, 1994). It has been suggested that in this respect, ceramide mediates the effects of TNFα on intracellular processes (WO 96/20710, specifically incorporated herein by reference).

U.S. Patent No. 5 ,518,879 described the discovery of a variety of fumonisins, and their analogs, that affect the biosynthetic pathway of sphingolipids. In as much as these fumonisin compounds inhibit ceramide biosynthesis as exemplified by Fumonisin B_j described in the Examples herein, U.S. Patent No. 5,518,879 is incorporated herein by reference to provide disclosure of other fumonisin analogues that may be used to inhibit ceramide synthesis in order to prevent, or decrease β-cell apoptosis associated with obesity and NIDDM.

Thus, the present invention uses fumonisins in altering the metabolism of sphingolipids in a cell comprising contacting the cell with a metabolism altering amount of a fumonisin, or an analog thereof. Fumonisins act by binding to ceramide synthase in the conversion of sphinganine to dihydroceramide. Additionally, the invention provides that the alteration in metabolism can be effected by fumonisin, or an analog thereof, binding to ceramide synthase in the conversion of sphingosine back to ceramide. Such compositions for use in these methods are provided in U.S. Patent No. 5 ,518,879.

As used herein, "ceramide synthase" means an enzyme or enzymes which add in N-acyl linkage to sphingosine, sphinganine and other long-chain bases. Thus, both sphinganine N- acyltransferase, which catalyzes the conversion of sphinganine to dihydroceramide. and shingosine N-acyltransferase, which catalyzes the conversion of sphingosine to ceramide. are referred to as ceramide synthase herein.

Ceramide synthesis is inhibited by contacting a cell with fumonisins in an amount effective to inhibit ceramide synthase activity. "Contacting the cell" as used herein means the fumonisin is administered in such a way that the fumonisin can enter the cell and affect the ceramide biosynthetic pathway within the cell.

"Amount effective to inhibit ceramide synthase activity" as used herein means a quantity of a fumonisin capable of effecting a change in the sphingolipid metabolism in cells such that the level of ceramide is decreased or lowered. Naturally, this amount can vary depending on the environment in which the cells are contained. For in vivo administration, the precise quantity of the fumonisin to elicit the desired effect can be determined by standard administration and monitoring until optimal dosage is determined. Such amounts can vary from subject to subject depending on size and condition of the subject. The amount of fumonisin compound which is administered can vary. Preferably the amount is between about 5 and 500 mg, especially between about 25 and 75 mg when fumonisin B, or B₂ is utilized or an analog with a similar activity.

PCT publication WO 96/20710 (specifically incorporated herein by reference) provides details of inhibiting the signal transduction action of ceramides. Such methods and compositions are known to inhibit apoptosis. Thus, in certain embodiments, it may be that the compounds described therein would be useful in inhibiting ceramide action in the present invention.

Cycloserine is a competitive inhibitor of the enzyme serine palmitoyl CoA transferase and therefore blocks ceramide biosynthesis. It has already been used in humans for the treatment of Alzheimer's disease (U.S. Patent Nos. 4,904,681; 5,061,721 ; 5,260,324). In theory, it should block ceramide formation, thus, the skilled artisan is referred to U.S. Patent Nos. 4,904,681 ; 5,061,721; 5,260,324 and 3,932,439 each specifically incorporated herein by reference, in order to provide details on compositions that will be useful in the therapeutic applications contemplated herein.

d) Nitric Oxide Synthase Inhibitors

Aminoguanidine is a known inhibitor of NO formation. The skilled artisan is referred to U.S. Patent No. 5,108,930 (specifically incorporated herein by reference) for discussions regarding the nitric oxide formation inhibiting capacity of aminoguanidine and its use in a warm blooded mammals. Aminoguanidine analogues as described in U.S. Patent No. 4,889,935 (specifically incorporated herein by reference) also may prove useful as NO production inhibitory agents.

U.S. Patent No. 5,585,402 (specifically incorporated herein by reference) discusses a method for inhibiting tissue damage in mammals caused by pathological NO production, by administering a NO synthase inhibitor, preferabl L-NMMA. U.S. Patent No. 5,585,402 describes doses and routes of administration for L-NMMA. C. Host Cells and Modifications thereof

The present invention is directed to decreasing lipotoxicity in cells involved in obesity and NIDDM. In particular aspects, it is contemplated that secretory cells such as neuroendocrine cell will be the targets for the therapies described herein. The lipid components of these cells or organs containing these cells, will be decreased using the strategies described above.

a) Cells

Secretory cells, especially neuroendocrine cells, have several endogenous functions that make them uniquely suited for production of a wide range of proteins, including secreted peptide hormones.

The present invention is designed to take advantage of cell based production of proteins for the purpose of producing leptin to combat the ceramide-induced apoptosis of β-cells in NIDDM and obesity. As such, these cells can be engineered to express leptin as well as other genes necessary to combat the NIDDM phenotype. A variety of different modifications may be made to increase the efficiency of the cell, one example is the blocking of production of an endogenous protein in the host cell. This will, in essence, "make room" for the heterologous protein and, hence, avoid competition between the endogenous and heterologous proteins during synthesis. The components for such a system, and methods of producing proteins therewith, are set forth in detail below.

Engineering of cells to synthesize proteins for the purposes of the present invention, will advantageously make use of many attributes of these cells. Regulated secretory cells present a natural bioreactor containing specialized enzymes involved in the processing and maturation of secreted proteins. These processing enzymes include endoproteases (Steiner et al, 1992) and carboxypeptidases (Fricker, 1988) for the cleavage of prohormones to hormones and PAM, an enzyme catalyzing the amidation of a number of peptide hormones (Eipper et al, 1992a). Similarly, maturation and folding of peptide hormones is performed in a controlled, stepwise manner with defined parameters including pH, calcium and redox states. (i) Glucose Responsive Cells.

For delivery of leptin, and other ceramide-lowering peptides, it may be desirable to cause the polypeptide to be released from cells in response to changes in the circulating glucose concentration. The most obvious example of a secretory cell type that is regulated in this fashion is the β-cell of the pancreatic islets of Langerhans, which releases insulin in response to changes in the blood glucose concentration. Primary β-cells may be used alternatively, cell lines may also be employed and then transplanted to the target organ in order to provide the appropriate level of leptin signal for the surrounding cells to decrease their ceramide production as described above.

Cell lines derived from various source are well known to those of skill in the art.

(Gazdar et al. 1980, Santerre et al, 1981; Efrat et al, 1988, Miyazaki et al, 1990). While insulinoma lines provide an advantage in that they can be grown in essentially unlimited quantity at relatively low cost, most exhibit differences in their glucose-stimulated insulin secretory response relative to normal islets. These differences can be quite profound, such as in the case of RINm5F cells, which were derived from a radiation-induced insulinoma and which in their current form are completely lacking in any acute glucose-stimulated insulin secretion response (Halban et al, 1983).

RIN 1046-38 cells are also derived from a radiation-induced insulinoma but can be shown to be glucose responsive when studied at low passage numbers (Clark et al, 1990). This response is maximal at subphysiological glucose concentrations and is lost entirely when these cells are cultured for more than 40 passages (Clark et al, 1990). GLUT-2 and glucokinase are expressed in low passage RIN 1046-38 cells but are gradually diminished with time in culture in synchrony with the loss of glucose-stimulated insulin release (Ferber et al , 1994). Ferber et al continued to describe various methods to restored glucose-stimulated insulin secretion (Ferber et al . 1994; (Newgard, U.S. Patent 5,427,940, incorporated herein by reference. By way of example, WO publication numbers WO 97/26334 (published July 24, 1997) and WO 97/26321 (published July 24, 1997) describe various glucose responsive cell lines that will be useful in combination with the present invention. (ii) Non-glucose responsive

An alternative to insulinoma cell lines are non-islet cell lines of neuroendocrine origin that are engineered for insulin expression. The foremost example of this is the AtT-20 cell, which is derived from ACTH secreting cells of the anterior pituitary (Moore et al, 1983). These cells contain a regulated secretory pathway that is similar to that operative in the islet β- cell (Moore et al, 1983, Gross et al, 1989). These and other non-glucose responsive cells have been described in greater detail in WO publication numbers WO 97/26334 (published July 24, 1997) and WO 97/26321 (published July 24, 1997) each incorporated herein by reference

b) Methods for Increasing Production of Peptides from Cells The present invention contemplates augmenting or increasing the capabilities of cells to produce biologically active leptin and other proteins involved in ceramide lowering effects. Engineering the overexpression of a cell type-specific transcription factor such as the Insulin Promoter Factor 1 (IPF1) found in pancreatic β-cells (Ohlsson et al, 1993) could increase or stabilize the capabilities of engineered neuroendocrine cells. Insulin promoter factor 1 (IPF-1 ; also referred to as STF-1 , IDX-1, PDX-1 and bTF-1) is a homeodomain-containing transcription factor proposed to play an important role in both pancreatic development and insulin gene expression in mature β-cells (Ohlsson et al, 1993, Leonard et al, 1993, Miller et al, 1994, Kruse et al, 1993). In embryos, IPF-1 is expressed prior to islet cell hormone gene expression and is restricted to positions within the primitive foregut where pancreas will later form. Indeed, mice in which the IPF-1 gene is disrupted by targeted knockout do not form a pancreas (Jonsson et al , 1994). Later in pancreatic development, as the different cell types of the pancreas start to emerge, IPF-1 expression becomes restricted predominantly to β-cells. IPF-1 binds to TAAT consensus motifs contained within the FLAT E and PI elements of the insulin enhancer/promoter, whereupon, it interacts with other transcription factors to activate insulin gene transcription (Peers et al, 1994).

Stable overexpression of IPF-1 in neuroendocrine β-cell lines will serve two purposes.

First, it will increase transgene expression under the control of the insulin enhancer/promoter.

Second, because IPF-1 appears to be critically involved in β-cell maturation, stable overexpression of IPF-1 in β-cell lines should cause these mostly dedifferentiated β-cells to regain the more differentiated function of a normal animal β-cell. If so, then these redifferentiated β-cell lines could potentially function as a more effective neuroendocrine cell type for cell-based delivery of fully processed, bioactive peptide hormones.

Also, further engineering of cells to generate a more physiologically-relevant regulated secretory response is contemplated. Examples would include engineering the ratios of glucokinase to hexokinase in rat insulinoma cells that also overexpress the Type II glucose transporter (GLUT-2) such that a physiologically-relevant glucose-stimulated secretion of peptide hormones is achieved. Other examples include engineering overexpression of other signaling proteins known to play a role in the regulated secretory response of neuroendocrine cells. These include cell surface proteins such as the β-cell-specific inwardly rectifying potassium channel (BIR; Inagaki et al, 1995), involved in release of the secretory granule contents upon glucose stimulation, the sulfonylurea receptor (SUR), and ATP sensitive channel.

Other cell surface signaling receptors which help potentiate the glucose-stimulated degranulation of β-cells including the glucagon-like peptide I receptor (Thorens, 1992) and the glucose-dependent insulinotropic polypeptide receptor (also known as gastric inhibitory peptide receptor) (Usdin, 1993) can be engineered into neuroendocrine cells. These β-cell-specific signaling receptors, as well as GLUT-2 and glucokinase, are involved in secretory granule release in response to glucose. In this way, glucose stimulated release of superoxide dismutase can be engineered.

c) Methods for Blocking Endogenous Protein Production Blocking expression of an endogenous gene product may be an important modification of cells according to the present invention. For example, in certain aspects of the present invention it may prove useful to inhibit endogenous iNOS activity of cells. Blocking expression of this endogenous gene product, while engineering high level expression of genes of anti-oxidizing capabilities will be useful in the context of the present invention.

Cells generated by this two-step process express heterologous proteins, including a variety of natural or engineered proteins (fusions, chimeras, protein fragments, etc.). Cell lines developed in this way are uniquely suited for in vivo cell-based delivery.

A number of basic approaches are contemplated for blocking of expression of an endogenous gene in host cells. First, constructs are designed to homologously recombine into particular endogenous gene loci, rendering the endogenous gene nonfunctional. Second, constructs are designed to randomly integrate throughout the genome, resulting in loss of expression of the endogenous gene. Third, constructs are designed to introduce nucleic acids complementary to a target endogenous gene. Expression of RNAs corresponding to these complementary nucleic acids will interfere with the transcription and/or translation of the target sequences. Fourth, constructs are designed to introduce nucleic acids encoding ribozymes - RNA-cleaving enzymes - that will specifically cleave a target mRNA corresponding to the endogenous gene. Fifth, endogenous gene can be rendered dysfunctional by genomic site directed mutagenesis. Each of these methods for blocking protein production are well known to those of skill in the art. By way of example, WO publication numbers WO 97/26334 (published July 24, 1997) and WO 97/26321 (published July 24, 1997) describe these methodologies and are specifically incorporated herein by reference.

D. Proteins

In addition, to the ceramide lowering agents described above, the present invention also contemplates genetic based therapies in which the target cells of the present invention are engineered to express a variety of proteins, including but not limited to leptin, insulin, growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone, adrenocorticotropin (ACTH), angiotensin I, angiotensin II, β-endorphin, β-melanocyte stimulating hormone (β-MSH), cholecystokinin, endothelin I, galanin, gastric inhibitory peptide (GIP), glucagon, amylin, lipotropins, neurophysins, GLP-1, calcitonin and somatostatin. The present section discusses the methods and compositions involved in such expression.

In a particular embodiment, the present invention contemplates providing leptin protein in order to abrogate lipotoxic β-cell destruction in a diabetic state. The leptin may be a purified form of leptin, that is commercially available, alternatively, the leptin may be provided to the cell in the form of a genetic construct that will express leptin at the target site it is delivered to. Furthermore, a variety of other proteins can also be expressed in combination with leptin according to the present invention. Proteins can be grouped generally into two categories - non-secreted and secreted - discussions of each are detailed below. The proteins may not necessarily have a single sequence but, rather, will exists in many forms. These forms may represent allelic variation or, rather, mutant forms of a given protein. It is contemplated that various proteins may be expressed advantageously as "fusion" proteins. Fusions are generated by linking together the coding regions for two proteins, or parts of two proteins. This generates a new, single coding region that gives rise to the fusion protein. A third variation contemplated by the present invention involves the expression of protein fragments. It may not be necessary to express an entire protein and, in some cases, it may be desirable to express a particular functional domain, for example, where the protein fragment remains functional but is more stable, or less antigenic, or both.

(i) Non-Secreted Proteins

Expression of non-secreted proteins can be engineered into neuroendocrine cells. Two general classes of such proteins can be defined. The first are proteins that, once expressed in cells, stay associated with the cells in a variety of destinations. These destinations include the cytoplasm, nucleus, mitochondria, endoplasmic reticulum, Golgi, membrane of secretory granules and plasma membrane. Non-secreted proteins are both soluble and membrane associated. The second class of proteins are ones that are normally associated with the cell, but have been modified such that they are now secreted by the cell. Modifications would include site-directed mutagenesis or expression of truncations of engineered proteins resulting in their secretion as well as creating novel fusion proteins that result in secretion of a normally non- secreted protein.

Cells engineered to produce such proteins could be used for either in vitro production of the protein or for in vivo, cell-based therapies. In vitro production would entail purification of the expressed protein from either the cell pellet for proteins remaining associated with the cell or from the conditioned media from cells secreting the engineered protein. In vivo, cell-based therapies would either be based on secretion of the engineered protein or beneficial effects of the cells expressing a non-secreted protein.

(ii) Secreted Proteins

The present invention allows for the production of mammalian cell-produced recombinant leptin and leptin-related species to be used in the treatment of lipotoxicity. The lipotoxicity may give rise to a syndrome such as insulin-dependent diabetes mellitus (IDDM); insulin-independent diabetes mellitus (NIDDM); obesity and various diseases of peptide or hormone deficiency or any other disorder involving free radical generation. For this purpose, it is contemplated that neuroendocrine and other cells can be engineered for the co-expression of leptin with several proteins that are normally secreted. The cDNA's encoding a number of useful human proteins are available. Examples would include growth hormone, prolactin, parathyroid hormone, insulin, glucagon, amylin, glucagon-like peptide I and calcitonin.

F. Genetic Constructs And Their Delivery To Cells

In order for the proteins presented above to be expressed in a given cell they need to be delivered as part of an expression construct. Within certain embodiments expression vectors can be employed to express various genes to produce large amounts of the polypeptide product associated therewith, which can then be purified and used as described to lower ceramide content in cells or to otherwise treat NIDDM. This section provides a description of the production of genetic constructs and their delivery into cells for such scale protein expression.

a) Genetic Constructs Within certain embodiments expression vectors can be employed to express various genes (e.g., leptin or leptin receptor) to produce large amounts of the polypeptide product. Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the leptin products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.

(i) Regulatory Elements. Throughout this application, the term "expression construct" is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest. In preferred embodiments, the nucleic acid encoding a gene product is under transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

The term promoter refers to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a

TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the cell. Thus, where a human cell is used, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde-3 -phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.

By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product. Table 1 lists several inducible elements/promoters which may be employed, in the context of the present invention, to regulate the expression of the gene of interest. This list is not intended to be exhaustive of all the possible elements involved in the promotion of gene expression but, merely, to be exemplary thereof.

Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.

The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization. Below is a list of viral promoters, cellular promoters/enhancers and inducible promoters/enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct. Additionally, any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct. Enhancer/promoter elements contemplated for use with the present invention include but are not limited to Immunoglobulin Heavy Chain, Immunoglobulin Light, Chain T-Cell Receptor, HLA DQ α and DQ β, β-Interferon, Interleukin- 2, Interleukin-2 Receptor, MHC Class II 5, MHC Class II HLA-DRα, β-Actin. Muscle Creatine Kinase, Prealbumin (Transthyretin), Elastase I, Metallothionein, Collagenase. Albumin Gene, α- Fetoprotein, τ-Globin, β-Globin, e-fos, c-HA-ras, Insulin, Neural Cell Adhesion Molecule (NCAM), αl-Antitrypsin, H2B (TH2B) Histone, Mouse or Type I Collagen, Glucose-Regulated Proteins (GRP94 and GRP78), Rat Growth Hormone, Human Serum Amyloid A (SAA), Troponin I (TN I), Platelet-Derived Growth Factor, Duchenne Muscular Dystrophy, SV40, Polyoma, Retroviruses, Papilloma Virus, Hepatitis B Virus, Human Immunodeficiency Virus, Cytomegalovirus, Gibbon Ape Leukemia Virus. Inducible promoter elements and their associated inducers are listed in Table 1 below.

TABLE 1

Element Inducer

MT II Phorbol Ester (TP A), Heavy metal s

MMTV (mouse mammary tumor virus) Glucocorticoids β-Interferon poly(rI)X, poly(rc)

Adenovirus 5 E2 Ela c-jun Phorbol Ester (TP A), H₂O₂

Collagenase Phorbol Ester (TPA)

Stromelysin Phorbol Ester (TPA), IL- 1

SV40 Phorbol Ester (TPA)

Murine MX Gene Interferon, Newcastle Disease Virus

GRP78 Gene A23187 TABLE 1 - Continued Element Inducer α-2-Macroglobulin IL-6

Vimentin Serum

MHC Class I Gene H-2kB Interferon

HSP70 Ela, SV40 Large T Antigen

Proliferin Phorbol Ester-TPA

Tumor Necrosis Factor FMA

Thyroid Stimulating Hormone α Gene Thyroid Hormone

Insulin E Box Glucose

In certain embodiments of the invention, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis. to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden. 1986; Temin, 1986). The first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40. bovine papilloma virus, and polyoma) (Ridgeway. 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). Adeno-associated viruses are also useful in this context (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984). These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals (Nicolas and Rubenstein, 1988; Temin. 1986).

Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a -JJ-

terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

(ii) Selectable Markers.

In certain embodiments of the invention, the cells contain nucleic acid constructs for the introduction of genes into a cell, such a cell may be identified by including a marker in the expression construct. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct. Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed. Immunologic markers also can be employed. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.

(Hi) Multigene constructs and IRES.

In certain embodiments of the invention, the use of internal ribosome binding sites (IRES) elements are used to create multigene. or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picanovirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.

Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker.

b) Delivery of Genetic Constructs

In order to express the proteins from the expression constructs, the nucleic acids need to be delivered into a cell. There are a number of ways in which nucleic acids may introduced into cells. Several methods, including viral and non-viral transduction methods, are outlined below.

(i) Adenovirus.

One of the preferred methods for in vivo delivery involves the use of an adenovirus expression vector. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized.

The expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus and Horwitz, 1992). In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.

Generation and propagation of adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al, 1977). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the E3 or both regions (Graham and Prevec, 1991). Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g. , Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.

Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.

As stated above, the typical adenoviral vector is replication defective and will not have an adenovirus El region. Thus, it will be most convenient to introduce the polynucleotide encoding the gene of interest at the position from which the El -coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention. The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al, (1986) or in the E4 region where a helper cell line or helper vims complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10 - 10 plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al, 1963; Top et al. 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors. Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991 ; Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992).

(ii) Retroviruses. The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse- transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al, 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al, 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).

(Hi) Adeno-Associated Virus (AAV).

AAV utilizes a linear, single-stranded DNA of about 4700 base pairs. Inverted terminal repeats flank the genome. Two genes are present within the genome, giving rise to a number of distinct gene products. The first, the cap gene, produces three different virion proteins (VP), designated VP-1, VP-2 and VP-3. The second, the rep gene, encodes four non-structural proteins (NS). One or more of these rep gene products is responsible for transactivating AAV transcription.

The three promoters in AAV are designated by their location, in map units, in the genome. These are, from left to right, p5, pi 9 and p40. Transcription gives rise to six transcripts, two initiated at each of three promoters, with one of each pair being spliced. The splice site, derived from map units 42-46, is the same for each transcript. The four non- structural proteins apparently are derived from the longer of the transcripts, and three virion proteins all arise from the smallest transcript.

The terminal repeats of the AAV vector can be obtained by restriction endonuclease digestion of AAV or a plasmid such as p201, which contains a modified AAV genome (Samulski et al. 1987), or by other methods known to the skilled artisan, including but not limited to chemical or enzymatic synthesis of the terminal repeats based upon the published sequence of AAV. The ordinarily skilled artisan can determine, by well-known methods such as deletion analysis, the minimum sequence or part of the AAV ITRs which is required to allow function, i.e., stable and site-specific integration. The ordinarily skilled artisan also can determine which minor modifications of the sequence can be tolerated while maintaining the ability of the terminal repeats to direct stable, site-specific integration.

(iv) Other Viral Vectors as Expression Constructs.

Other viral vectors may be employed as expression constructs in the present invention.

Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden,

1986; Coupar et al, 1988) and herpes viruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988: Horwich et al, 1990).

(v) Non-viral vectors.

Several non-viral methods for the transfer of expression constructs into cultured mammalian cells are contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb. 1973; Chen and Okayama, 1987; Rippe et al. 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986; Potter et al, 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al, 1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al, 1987), gene bombardment using high velocity microprojectiles (Yang et al, 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.

Once the expression construct has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.

In one embodiment of the invention, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al, (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.

Another embodiment of the invention for transferring naked DNA expression constructs into cells may involve particle bombardment. This method depends on the ability to accelerate

DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al. 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

In a further embodiment of the invention, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. Wong et al, (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells. Nicolau et al, (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection.

In certain embodiments of the invention, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989). In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non- histone chromosomal proteins (HMG-1) (Kato et al, 1991). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.

Other expression constructs which can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al, 1990). Recently, a synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al, 1993; Perales et al. 1994) and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085).

G. Monitoring Ceramide Composition of Individuals

In certain instances, it may be necessary to monitor the levels of ceramides present in an individuals circulation and/or cells. The present invention encompasses methods for the determination of the ceramide content of cells. Thus, in particular aspects of the present invention, it will be necessary to monitor the levels of ceramides in diabetic individuals in order to determine baseline levels of the ceramides as well as to periodically monitor the ceramides throughout treatment to track the course of the diabetes therapy.

Briefly, one generally will isolate ceramides from the lipid components of a cell using for example a Bligh and Dyer extraction (Bligh and Dyer, 1959). This will involve the initial separation of lipid components of a cell from (i) non-lipid components and (ii) separation of each lipid species from the other will then permit quantitation of the each lipid species (for example, ceramides). Quantitation of separated components may be achieved by any standard methodology, that would include photodensitometric scanning of TLC plates or scintillation counting of membrane bound or liquid samples separated by various chromatographic techniques.

Any of a wide variety of chromatographic procedures may be employed. For example, thin layer chromatography, gas chromatography, high performance liquid chromatography, paper chromatography, affinity chromatography or supercritical flow chromatography may be employed. See Freifelder, 1982. Partition-chromatography is based on the theory that, if two phases are in contact with one another, and if one or both phases constitute a solute, the solute will distribute itself between the two phases. Usually, partition chromatography employs a column which is filled with a sorbent and a solvent. The solution containing the solute is layered on top of the column. The solvent is then passed through the column, continuously, which permits movement of the solute through the column material. The solute can then be collected based on is movement rate. The two most common types of partition chromatography are paper chromatography and thin-layer chromatography (TLC); together these are called adsorption chromatography. In both cases, the matrix contains a bound liquid. Other examples of partition chromatography as gas-liquid and gel chromatography.

Paper chromatography is a variant of partition chromatography that is performed on cellulose columns in the form of a paper sheet. This technique may be useful in identifying and characterizing the lipid content of a particular sample. Cellulose contains a large amount of bound water even when extensively dried. Partitioning occurs between the bound water and the developing solvent. Frequently, the solvent used is water. Usually, very small volumes of the solution mixture to be separated is placed at top of the paper and allowed to dry. Capillarity draws the solvent through the paper, dissolves the sample, and moves the components in the direction of flow. Paper chromatograms may be developed for either ascending or descending solvent flow. Two dimensional separations are permitted by changing the axis of migration 90° after the first run.

Thin-layer-chromatography (TLC) is very commonly used to separate lipids and, therefore, is considered a preferred embodiment of the present invention. TLC has the advantages of paper chromatography, but allows the use of any substance that can be finely divided and formed into a uniform layer. In TLC, the stationary phase is a layer of sorbent spread uniformly over the surface of a glass or plastic plate. The plates are usually made by forming a slurry of sorbent that is poured onto the surface of the gel after creating a well by placing tape at a selected height along the perimeter of the plate. After the sorbent dries, the tape is removed and the plate is treated just as paper in paper chromatography. The sample is applied and the plate is contacted with a solvent. Once the solvent has almost reached the end of the plate, the plate is removed and dried. Spots can then be identified by fluorescence, lmmunologic identification, counting of radioactivity, or by spraying varying reagents onto the surface to produce a color change

In Gas-Liquid-Chromatography (GLC), the mobile phase is a gas and the stationary phase is a liquid adsorbed either to the inner surface of a tube or column or to a solid support The liquid usually is applied as a solid dissolved in a volatile solvent such as ether The sample, which may be any sample that can be volatized, is introduced as a liquid with an inert gas, such as helium, argon or nitrogen, and then heated This gaseous mixture passes through the tubing The vaporized compounds continually redistribute themselves between the gaseous mobile phase and the liquid stationary phase, according to their partition coefficients

The advantage of GLC is in the separation of small molecules Sensitivity and speed are quite good, with speeds that approach 1000 times that of standard liquid chromatography By using a non-destructive detector, GLC can be used preparatively to purify grams quantities of material The principal use of GLC has been in the separation of alcohols, esters, fatty acids and amines

High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks This is achieved by the use of very fine particles and high pressure to maintain and adequate flow rate Separation can be accomplished in a matter of minutes, or a most an hour Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample

Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to This is a receptor-hgand type interaction The column material is synthesized by covalentl) coupling one of the binding partners to an insoluble matrix The column material is then able to specifically adsorb the substance from the solution Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc )

The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability The ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.

H. Cell-Based Delivery and Devices

In particular embodiments, leptin expressing cells or leptin-receptor expressing cells may be introduced into animals, including humans, having an NIDDM phenotype, so that ceramide lowering capacity may be provided to the individual. Thus, cells that have been engineered to expressing leptin or the β-cells that have been engineered to express leptin receptor that will decrease or diminish the ceramide content in a obesity related NIDDM may be introduced to an individual manifesting such a state.

In order for the cells to be used in regulating such a state, ideally the cells may be engineered to sense the plasma glucose concentration. However, other cells will also achieve advantages in accordance with the invention. It should be pointed out that the studies of Madsen and coworkers have shown that implantation of poorly differentiated rat insulinoma cells into animals results in a return to a more differentiated state, marked by enhanced insulin secretion in response to metabolic fuels (Madsen et al, 1988). These studies suggest that exposure of engineered cell lines to the in vivo milieu may have some effects on their response(s) to secretagogues.

A preferred method of providing the cells to an animal involves the encapsulation of the engineered cells in a biocompatible coating. In this approach, the cells are entrapped in a capsular coating that protects the contents from immunological responses. One preferred encapsulation technique involves encapsulation with alginate-polylysine-alginate. Capsules made employing this technique generally have a diameter of approximately 1 mm and should contain several hundred cells.

Cells may thus be implanted using the alginate-polylysine encapsulation technique of O'Shea and Sun (1986), with modifications, as later described by Fritschy et al, (1991 ; both references incorporated herein by reference). The engineered cells are suspended in 1.3% sodium alginate and encapsulated by extrusion of drops of the cell/alginate suspension through a syringe into CaCl₂. After several washing steps, the droplets are suspended in polylysine and rewashed. The alginate within the capsules is then reliquified by suspension in 1 mM EGTA and then rewashed with Krebs balanced salt buffer.

An alternative approach is to seed Amicon fibers with stable cells of the present invention. The cells become enmeshed in the fibers, which are semipermeable, and are thus protected in a manner similar to the micro encapsulates (Altman et al, 1986; incorporated herein by reference). After successful encapsulation or fiber seeding, the cells may be implanted intraperitoneally, usually by injection into the peritoneal cavity through a large gauge needle (23 gauge).

A variety of other encapsulation technologies have been developed that are applicable to the practice of the present invention (see, e.g., Lacy et al, 1991; Sullivan et al, 1991 ; WO 91/10470; WO 91/10425; WO 90/15637; WO 90/02580; U.S. Patent 5,011,472; U.S. Patent 4,892,538; and WO 89/01967; each of the foregoing being incorporated by reference).

Lacy et. al. (1991) encapsulated rat islets in hollow acrylic fibers and immobilized these in alginate hydrogel. Following intraperitoneal transplantation of the encapsulated islets into diabetic mice, normoglycemia was reportedly restored. Similar results were also obtained using subcutaneous implants that had an appropriately constructed outer surface on the fibers. It is therefore contemplated that engineered cells of the present invention may also be straightforwardly "transplanted" into a mammal by similar subcutaneous injection.

Sullivan et. al (1991) reported the development of a biohybrid perfused "artificial pancreas," which encapsulates islet tissue in a selectively permeable membrane. In these studies, a tubular semi-permeable membrane was coiled inside a protective housing to provide a compartment for the islet cells. Each end of the membrane was then connected to an arterial polytetrafluoroethylene (PTFE) graft that extended beyond the housing and joined the device to the vascular system as an arteriovenous shunt. The implantation of such a device containing islet allografts into pancreatectomized dogs was reported to result in the control of fasting glucose levels in 6/10 animals. Grafts of this type encapsulating engineered cells could also be used in accordance with the present invention. The company Cytotherapeutics has developed encapsulation technologies that are now commercially available that are envisioned for use in the application of the present invention. A vascular device has also been developed by Biohybrid, of Shrewsbury, Mass., that can be used with the technology of the present invention. U.S. Patent No. 5,626,561, specifically incorporated herein by reference, describes the implantation devices for delivery of therapeutic compositions, the methods and compositions therein will be useful in conjunction with the instant invention.

Implantation employing such encapsulation techniques are preferred for a variety of reasons. For example, transplantation of islets into animal models of diabetes by this method has been shown to significantly increase the period of normal glycemic control, by prolonging xenograft survival compared to unencapsulated islets (O'Shea and Sun, 1986; Fritschy et al, 1991). Also, encapsulation will prevent uncontrolled proliferation of clonal cells. Capsules containing cells are implanted (approximately 1,000-10, 000/animal) intraperitoneally and blood samples taken daily for monitoring of blood glucose and insulin.

An alternate approach to encapsulation is to simply inject glucose-sensing cells into the scapular region or peritoneal cavity of diabetic mice or rats, where these cells are reported to form tumors (Sato et al, 1962). Implantation by this approach may circumvent problems with viability or function, at least for the short term, that may be encountered with the encapsulation strategy. This approach will allow testing of the function of the cells in experimental animals, which is a viable use of the present invention, but certainly is not applicable as an ultimate strategy for treating human diabetes. Nonetheless, as a pre-clinical test, this will be understood to have significant utility.

In summary, biohybrid artificial organs encompass all devices which substitute for an organ or tissue function and incorporate both synthetic materials and living cells. Implantable immunoisolation devices will preferably be used in forms in which the tissue is protected from immune rejection by enclosure within a semipermeable membrane. Those of skill in the art will understand device design and performance, as it relates to maintenance of cell viability and function. Attention is to be focused on oxygen supply, tissue density and the development of materials that induce neovascularization at the host tissue-membrane interface; and also on protection from immune rejection. Membrane properties may even be further adapted to prevent immune rejection, thus creating clinically useful implantable immunoisolation devices.

An effective amount of the cells to be delivered is determined based on the intended goal. The term "unit dose" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired response in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the subject to be treated, the state of the subject, and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.

I. Pharmaceutical Compositions

Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions in a form appropriate for the intended application. The pharmaceutical compositions of the present invention will have an effective amount of an agent that is capable of decreasing the ceramide content of a cell. Also, the present invention contemplates the use of agents to inhibit NO production in a cell. In certain embodiments, the pharmaceutical composition may further comprise delivery vectors and/or recombinant cells for the production of leptin and other ceramide-lowering peptides to ameliorate the deleterious effects of β-cell destruction caused by excess ceramides.

Generally, the compositions are prepared such that they are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals. One will generally desire to employ appropriate salts and buffers to render the compositions stable and allow for uptake by target cells. Buffers also will be employed when the compositions are introduced into a patient.

Aqueous compositions of the present invention are used to deliver an effective amount of the therapeutic composition to the target cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when admimstered to an animal or a human As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like The use of such media and agents for pharmaceutically active substances is well know in the art Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated Supplementary active ingredients also can be incorporated into the compositions

Solutions of the active ingredients as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with surfactant, such as hydroxypropylcellulose Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils Under ordinary conditions of storage and use, these preparations contain a preservative to prevent growth of microorganisms

The therapeutic compositions of the present invention may include classic pharmaceutical preparations Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route This includes oral, nasal, buccal, rectal, vaginal or topical Alternatively, administration may be by orthotopic, intradermal subcutaneous, intramuscular, intraperitoneal or intravenous injection Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra

The compositions of the present invention advantageously can be administered in the form of injectable compositions either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection also may be prepared These preparations also may be emulsified A typical composition for such purposes comprises a 50 mg or up to about 100 mg of human serum albumin per milhhter of phosphate-buffered saline Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients including salts, preservatives, buffers and the like Examples of non-aqueous solvents are propylene glycol, polyethylene glvcol, vegetable oil and injectable organic esters such as ethyloleate Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc Intravenous vehicles include fluid and nutrient replenishers Preservatives include antimicrobial agents anti- oxidants, chelating agents and inert gases. The pH and exact concentration of the various components in the pharmaceutical are adjusted according to well-known parameters.

Orally administratable formulations may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and a suitable carrier. In general, the formulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet may be prepared by compressing or molding a powder or granules containing the active agent, optionally with one or more accessory ingredients such as an immune adjuvant.

The pharmaceutical compositions may also be formulated for parenteral systemic administration to the host. The compositions for parenteral administration, may be subcutaneously, intramuscularly, or intravenously administered. Thus, the present invention provides compositions for administration to a host, where the compositions comprise a pharmaceutically acceptable solution of the identified compound in an acceptable carrier, as described above. Typically, injectibles are prepared either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.

The pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectible solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringabihty exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Sterile injectible solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectible solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly, concentrated solutions for local injection also is contemplated. In this regard, the use of DMSO as solvent is preferred as this will result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms

An effective amount of the therapeutic agent is determined based on the intended goal.

The term "unit dose" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired response in association with its administration, i.e. , the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the subject to be treated, the state of the subject, and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.

In certain embodiments, the present invention contemplates the use of varying doses of fumonisin Bl, aminoguanidine, troglitazone and triacsin C in the therapeutic intervention of diabetes. Troglitazone may be administered in doses ranging from about 100 to about 1000 mg per day. Thus it is contemplated that 100 mg/day, 200 mg/day, 300 mg/day, 400 mg/day, 500 mg/day, 600 mg/day, 700 mg/day, 800 mg/day, 900 mg/day or 1000 mg/day dosages may be administered to an individual. This troglitazone may be administered as one, two, three, four or more doses per day. In therapeutic applications in which fumonisin B,, B₂ or an analog with a similar activity is utilized, the amount of fumonisin, or an analog thereof, which is administered can vary. Preferably the amount is between about 5 and about 500 mg, especially between about 25 mg and 75 mg when fumonisin B, or B₂ is utilized or an analog with a similar activity. In particular embodiments, it is contemplated that 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 100 mg, 200 mg, 300 mg, 400 mg or 500 mg may be administered in daily doses. These doses may be administered one, two, three, four or more times a day.

Similarly, doses of aminoguanidine or an analog thereof may be administered to an individual. Analogs of aminoguanidine include but are not limited to N,N'-diaminoguanidine, 1,1-dimethylguanidine and methylguanidine. Compositions of aminoguanidine comprising a daily dose of 5 mg/kg body weight, 10 mg/kg body weight, 20 mg/kg body weight, 30 mg/kg body weight. 40 mg/kg body weight, 50 mg/kg body weight, 60 mg/kg body weight, 70 mg/kg body weight, 80 mg/kg body weight, or 100 mg/kg body weight, are contemplated. Such compositions may comprise formulations containing 50 mg, 60 mg. 70 mg, 80 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 1000 mg, or 2000 mg may be administered as a daily dose. These doses may be administered one, two, three, four or more times a day as deemed necessary by the clinician.

In those embodiments in which an NO synthase inhibitor is administered orally or via injection, the dose may be from about 1 mg to 100 mg/kg body weight per day. When the NO synthase inhibitors are given by injection, this will normally be in the form of an intravenous bolus or by infusion, preferably the latter. The dose range for adult humans is generally from 70 mg to 2.5 g/day and preferably 150 mg to 2 g/day. Tablets or other forms of presentation provided in discrete units may conveniently contain an amount of compound of the invention which is effective at such dosage or as a multiple of the same, for instance, units containing 5 mg to 500 mg, usually around 10 mg to 200 mg. Thus, it is contemplated that doses of 5 mg/day, 10 mg/day, 20 mg/day, 40 mg/day, 60 mg/day, 80 mg/day. 100 mg/day, 200 mg/day, 400 mg/day. 600 mg/day, 800 mg/day and 1000 mg/day will be useful.

Of course it is understood that the formulations and compositions described herein above are only exemplary in each category and that these compositions may be varied by the clinician on an individual basis according to the physical and physiological characteristics of the individual being treated.

The compositions of the present invention may be advantageously packaged into a kit comprising the active reagent(s) a suitable container means and even instructions for use of said kit. The reagent(s) of the kit can be provided as a liquid solution, attached to a solid support or as a dried powder. Preferably, when the reagent is provided in a liquid solution, the liquid solution is an aqueous solution. Preferably, when the reagent provided is attached to a solid support, the solid support can be chromatograph media, a test plate having a plurality of wells, or a microscope slide. When the reagent provided is a dry powder, the powder can be reconstituted by the addition of a suitable solvent, that may be provided.

The container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the reagents may be placed, and preferably suitably aliquoted. Where a second reagent is provided, the kit will also generally contain a second vial or other container into which this additional reagent may be placed. The kits of the present invention will also typically include a means for containing the reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

J. Examples

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. EXAMPLE 1

Fatty Acid-Induced β Cell Apoptosis

Materials and Methods

Animals. Lean wild-type (+/+) male ZDF rats and obese homozygous (fa/fa) male ZDF rats were bred in the inventors' laboratory from [ZDF/Drt-fa(F10)] rats purchased from R. Peterson (University of Indiana School of Medicine, Indianapolis).

Islet Isolation and Culture. Pancreatic islets were isolated by the method of Naber et al. (1980) with modifications (Milburn et al , 1994). Isolated islets were cultured as described (Milburn et al, 1994; Shimabukuro et al. 1997a). In some studies, islets were cultured with or without 1 mM long-chain FFAs (2: 1 oleate/palmitate; Sigma Chemical Co.) in the absence and presence of 15 μM Fumonisin Bj 15 μM C₂-ceramide, 0.5 mM aminoguanidine (Sigma), 10 μM triacsin C (Biomol, Plymouth Meeting, PA), and 10 μM troglitazone (Sankyo, Tokyo, Japan).

DNA Fragmentation Assay. DNA fragmentation was assayed by a modification of the method of Duke and Sellins (1989). Groups of freshly isolated or cultured islets were washed twice with ice-cold PBS and suspended in 100 μl of lysis buffer (10 mM Tris-HCl/10 mM

EDTA/0.5%) Triton X-100, pH 8.0). vortex-mixed, sonicated, and incubated on ice for 20 min.

After centrifugation for 20 min at 4°C (14,000 x g), the supernatant containing fragmented

(soluble) DNA was transferred to another tube. Lysis buffer (100 μl) was added to the pellet containing insoluble DNA. Both samples were treated with RNase A (0.5 mg/ml) for 1 h at

37°C and then with proteinase K (Sigma, 0.4 mg/ml) for 1 h at 37°C. After adding 20 μl of 5

M NaCl and 120 μl of isopropanol. the samples were incubated overnight at -20°C, and the

DNA concentrations were measured by the method of Hopcroft et al. (1985). Fragmented

DNA was calculated as 100% x soluble DNA/(soluble + insoluble DNA). The soluble fraction of DNA was determined by electrophoresis on 1.5% agarose gel and has a ladder-like appearance.

Ceramide Determination. Ceramide concentrations were measured in freshly isolated or cultured islets by a modification of the diacylglycerol kinase assay (Preiss et al . 1986; Okazaki et al, 1990). Islets were washed twice with ice-cold PBS and lipids were extracted by the method of Bligh and Dyer (1959). The dried lipid was solubilized in 20 μl of detergent solution (7.5% actyl β-D-glucopyranosice/5 mM cardiolipin in 1 mM (diethylenetriamine pentaacetic acid (DETAPAC). After adding 50 μl of assay buffer (100 mM midazole hydrochloride, pH 6.6/100 mM NaCl/25 mM MgCl₂/2 mM EGTA), 20 μl of 10 mM DDT, and 10 μl of 1 :1 diluted diacylglycerol kinase (Calbiochem,), the reaction was started by adding 10 μl of 10 mM/ [γ³²P]ATP (specific activity, 30,000-40,000 cpm/mmol) prepared in 100 mM imidazole/1 mM DETAPAC, pH 6.6. After mixing, the reaction was continued for 45 min at 27°C. Lipids were extracted again and the lower chloroform phase was washed twice with 2 ml of 1% perchloric acid (PCA), and dried under N₂. The sample was resuspended in 80 μl of chloroform, spotted onto a Silica Gel 60 (Merck) HPTLC plate, and developed with chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1). The radioactive spot corresponding to ceramide- 1 -phosphate was quantified by using the Molecular Imager (BioRad).

De Novo Synthesis of ϋJCeramide and Oxidation from [³ HJPalmitate. Freshly isolated islets were labeled for 0.5-4 h with [Ηjpalmitate (Amersham) in the absence and presence of the ceramide synthase inhibitor fumonisin B, (Sigma). Lipids were extracted as described above. Lipid extracts dissolved in 80 μl of chloroform were spotted onto high-performance thin-layer chromatography plate and developed with CHCl₂/CH₂OH/7 M NH₄OH/H₂O (85:15, 0.5:0.5). The radioactive spot corresponding to [Ηjpalmitate was counted as described (Unger, 1995; Lee et al. , 1997). [^JH]Palmitate oxidation were determined as [ H]H₂O production in medium as described (Lee et al, 1997; Milburn et al, 1994).

Semiquantitation of iNOS mRNA by Reverse Transcription-Coupled PCR™. iNOS mRNA expression was analyzed in cultured islets by using reverse transcription-coupled PCR™ as described in detail (Shimabukuro et al. 1997a). Briefly, total RNA was extracted by using a TRIzol isolation kit (Life Technologies) and treated with RNase-free DNase. First-strand cDNA was obtained by using a first-strand cDNA synthesis kit (CLONTECH). iNOS and β-actin genes were amplified by PCR™ as described (Shimabukuro et al, 1997a). The products were electrophoresed on a 1.2% agarose gel. After transferring to Hybond-N Nylon membrane (Amersham). DNA samples were hybridized with [ PJATP-labeled specific probes and analyzed in the Molecular Imager (BioRad). Nitrite Determination. NO formation was determined as nitrite by a modification of the method of Green et al. (1982) with modifications (Shimabukuro et al, 1997a).

Results

Measurements of DNA Laddering and Ceramide in Prediabetic and Diabetic ZDF Islets. Initially it was necessary to determine whether apoptosis was the cause of the previously observed loss of β cells that coincided with the onset of diabetes in ZDF rats. The inventors therefore islets were isolated from obese ZDF rats in the early prediabetic stage (5 wk of age), in the late prediabetic stage (7 wk), and about 4 wk after the onset of diabetes (14 wk) and determined whether DNA fragmentation, an index of apoptosis, was present. As shown in FIG. 1A, there was a more than 7-fold increase in DNA ladder formation in freshly isolated islets from 5-, 7-, and 14-wk-old ZDF rats, whereas none was detected in lean wild-type controls (FIG. 1A). Moreover, in islets of wild-type rats apoptosis did not increase with age, whereas it increased almost 3-fold in those of the obese rats. Thus, in ZDF islets, there was evidence of a high level of apoptosis in islet cells prior to the onset of diabetes and a further increase thereafter.

To determine whether ceramide, a fatty acid-containing messenger for certain cytokines in the apoptosis pathway, might also be involved in the apoptosis of ZDF rats. It was measured in freshly isolated islets from 7-wk-old obese, prediabetic 14-wk-old obese diabetic fa/fa ZDF rats and in age-matched lean wild-type controls. As shown in FIG. IB, the levels of ceramide were slightly but significantly higher (P < 0.01) in fa/fa rats than in +/+ controls at 7 wk of age and they increased further at 14 wk.

Effect of FFA on Ceramide and DNA Laddering. To determine whether an increase in ceramide and DNA laddering are induced by FFA, the inventors measured, at various times, the effect of FFA on ceramide levels and on DNA fragmentation in cultured islets isolated from prediabetic fa/fa ZDF rats and wild-type controls. In islets from the former rats cultured in 1 mM FFA. DNA laddering rose from 6% at 0 time to 19% at 24 h, whereas in wild-type controls, it rose from 0.7% to only 4.6%o at 24 h (FIG. 2A). The peak 82% increase in ceramide in fa/ fa ZDF rats took place within 1 h (FIG. 2B), before the increase in DNA fragmentation had occurred. Source of FFA-induced Increase in Ceramide and Relation in FFA Oxidation.

Ceramide can be generated either by degradation of sphingomyelin or by ceramide synthase-catalyzed condensation of fatty acyl-CoA and sphinganine (Merrill and Jones, 1990). To determine whether the increase in ceramide reflected de novo synthesis from FFA and to provide support for the diacylglyceride kinase assay for ceramide (Watts et al, 1997), the inventors incubated islets in 1 mM [ HJpalmitate and measured [ HJceramide. In addition the inventors measured [^JH]H₂O as an index of FFA oxidation. In wild-type islets, [Ηjceramide increased 3-fold within 2 h. In islets from fa/ fa rats, [ HJceramide was at least twice that of wild-type rats at all time points (FIG. 2C), whereas [Ηjpalmitate oxidation in islets of fa/fa rats was only 23% of that of wild-type rats (FIG. 2D). Because palmitoyl CoA is rate-limiting for ceramide synthesis in other systems (Paumen et al, 1997), the reduction in FFA oxidation may have contributed increased substrate for ceramide synthesis. However the increase in [ HJceramide was greater than could be explained by even a complete block in oxidation (FIG. 2D), suggesting an intrinsic increase in ceramide synthase activity.

Effect of C -Ceramide and Ceramide Synthase Inhibition on FFA-induced

Apoptosis. If ceramide is, in fact, the cause of FFA-induced apoptosis in β cells, exogenous ceramide should induce it, and inhibition of ceramide synthesis should prevent it. In islets from prediabetic obese fa/ fa ZDF rats cultured in 15 μM C₂-ceramide without FFA, DNA fragmentation rose 2-fold (FIG. 3). By contrast, in culture medium containing 1 mM FFA, the presence of 50 μM fumonisin Bj (the ceramide synthase inhibitor) almost completely prevented the FFA-induced increase in apoptosis (FIG. 3). These results point strongly to ceramide as the mediator of FFA-induced apoptosis in these islets.

Role of NO in FFA-induced Apoptosis. The inventors have observed an exaggeration of the normal FFA-induced increase of iNOS mRNA and NO production in fat-laden islets of fa/fa rats (Shimabukuro et al, 1997a). Because inhibitors of iNOS prevent the diabetic phenotype in islets of fa/fa rats, the inventors have hypothesized that FFA-induced β cell dysfunction and damage is mediated by NO (Shimabukuro et al, 1997a). If so, any maneuver that inhibits iNOS expression in islets should block DNA fragmentation and prevent the diabetes. Aminoguanidine is an iNOS inhibitor (Corbett and McDaniel. 1 96) that profoundly reduces NO production in vitro and effectively prevents β cell loss and diabetes when administered to ZDF rats (Shimabukuro et al, 1997a). In ZDF islets cultured in 1 mM FFA plus aminoguanidine, iNOS expression and NO production were decreased and DNA fragmentation was lowered substantially (FIG. 4A-FIG. 4C).

All of the composition and/or 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 composition and/or 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.

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Claims

CLAIMS:

1. A method of inhibiting ceramide-mediated apoptosis in a mammalian cell comprising contacting said cell with an agent that reduces ceramide levels in said cell as compared to an untreated cell.

2. The method of claim 1, wherein said agent inhibits the synthesis of ceramide in said cell.

3. The method of claim 1, wherein said agent inhibits the uptake of ceramide by said cell.

4. The method of claim 1, wherein said agent removes ceramide from said cell.

5. The method of claim 1, wherein said agent is selected from the group consisting fumonisin B_j and fuminosin B₂.

6. The method of claim 1, wherein said cell is further contacted with an agent that inhibits NO production in said cell.

7. The method of claim 1, wherein said apoptosis is cytokine-mediated.

8. The method of claim 7, wherein said cytokine is IL-1 β, IL-lα, γlFN, TNF-α, TNF-β, an IL-8, an IL-12, IL-6, IL-2, IL-3, IL-5, IL-7, IL-9, IL-14, IL-17, granulocyte- macrophage colony stimulating factor or monocyte chemoattractant protein- 1.

9. The method of claim 1, further comprising introducing into said cell a gene operatively linked to a promoter.

10. The method of claim 9, wherein said gene encodes insulin, growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone, adrenocorticotropin (ACTH), angiotensin I, angiotensin II, β-endorphin, β-melanocyte stimulating hormone (β-MSH), cholecystokinin, endothelin I, galanin, gastric inhibitory peptide (GIP), glucagon, amylin, lipotropins, neurophysins, GLP-1, leptin, leptin receptor, calcitonin and somato statin.

11. The method of claim 9, wherein said promoter is inducible.

12. The method of claim 9, wherein said promoter is selected from the group consisting of CMV IE, SV40 IE, RSV LTR, RIP, modified RIP, POMC, βgal, lac operon, ecdysone- inducible expression system, tetracyline operon, glucocorticoid response element, heat shock promoter and GHP promoter.

13. The method of claim 1, wherein the mammalian cell is a human cell.

14. The method of claim 1, wherein the mammalian cell is a non-human cell.

15. The method of claim 1, wherein said mammalian cell is a secretory cell.

16. The method of claim 1 , wherein said mammalian cell is a neuroendocrine cell.

17. The method of claim 1, wherein said mammalian cell is a pancreatic beta cell.

18. The method of claim 1, wherein said mammalian cell is a pituitary cell.

19. The method of claim 1, wherein said mammalian cell is secretagogue responsive.

20. The method of claim 1, wherein said mammalian cell is glucose responsive.

21. The method of claim 1, wherein said mammalian cell is non-glucose responsive.

22. The method of claim 1, wherein the cell is derived from a CM- 1. TRM, TRM6 or HAP5 cell.

23. The method of claim 9, wherein said gene is introduced into said mammalian cell along with a selectable marker gene.

24. The method of claim 23, wherein said selectable marker is selected from a group consisting of hygromycin resistance, neomycin resistance, puromycin resistance, zeocin, gpt, DHFR and histadinol.

25. A method of treating a subject for β cell destruction comprising contacting said subject with an agent that reduces levels of ceramide in the cells of said subject as compared to the untreated ceramide level, wherein reduction in ceramide levels protects cells of said subject against ceramide-induced lipotoxicity.

26. The method of claim 25, wherein said agent inhibits the synthesis of ceramide in said subject.

27. The method of claim 25, wherein said agent inhibits the uptake of ceramide by said subject.

28. The method of claim 25, wherein said agent removes ceramide from said subject.

29. The method of claim 25, wherein said subject is further contacted with an agent that inhibits NO production in said cell.

30. The method of claim 25, wherein said β-cell destruction is cytokine-mediated.

31. The method of claim 30, wherein said cytokine is IL-l β, IL-lα, γlFN. TNF-α, TNF-β, an IL-8, an IL-12. interleukin-6, IL-2, IL-3, IL-5, IL-7, IL-9, IL-14, IL-1 7, granulocyte- macrophage colony stimulating factor or monocyte chemoattractant protein- 1.

32. The method of claim 25, wherein said inhibitory agent is selected from the group consisting of fumonisin B, and fumonisin B₂.

33. The method of claim 25, wherein said subject exhibits at least one pathologic condition selected from the group consisting of insulin-dependent diabetes mellitus (IDDM); insulin-independent diabetes mellitus (NIDDM) and obesity.

34. The method of claim 25, further comprising introducing into said mammalian cell a gene operatively linked to a promoter.

35. The method of claim 34, wherein said gene encodes insulin, growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone, adrenocorticotropin (ACTH), angiotensin I, angiotensin II, β-endorphin, β-melanocyte stimulating hormone (β-MSH), cholecystokinin, endothelin I, galanin, gastric inhibitory peptide (GIP), glucagon, amylin, lipotropins, neurophysins, GLP-1 , leptin, leptin receptor, calcitonin and somatostatin.

36. The method of claim 34, wherein said promoter is selected from the group consisting of CMV IE, SV40 IE, RSV LTR, RIP, modified RIP, POMC and GH.

37. A method of preventing non-insulin dependent diabetes mellitus (NIDDM) in a subject comprising the steps of:

(a) identifying a subject at risk of diabetes mellitus; and (b) providing to said subject a composition comprising an agent that reduces levels of ceramide in the cells of said subject as compared to the untreated ceramide level, wherein said reduction in ceramide level protects cells of said subject against ceramide- mediated apoptosis of β-cells.

38. The method of claim 37, wherein said agent inhibits the synthesis of ceramide in said cell.

39. The method of claim 37, wherein said agent inhibits the uptake of ceramide by said cell.

40. The method of claim 37, wherein said agent removes ceramide from said cell.

41. The method of claim 37, wherein said NIDDM is NO-mediated NIDDM.

42. The method of claim 41 , wherein said subj ect is further contacted with an agent that inhibits NO production in said subject.

43. The method of claim 41, wherein said NO-mediated NIDDM is cytokine-mediated.

44. The method of claim 43, said cytokine is IL-l β, IL-lα, γlFN, TNF-α, TNF-β, an IL-8, an IL-12, interleukin-6, IL-2, IL-3, IL-5, IL-7. IL-9, IL-14, IL-17, granulocyte- macrophage colony stimulating factor or monocyte chemoattractant protein- 1.

45. The method of claim 37, further comprising providing to said subject a gene operatively linked to a promoter.

46. The method of claim 45, wherein said gene encodes insulin, growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone, adrenocorticotropin (ACTH), angiotensin I, angiotensin II, β-endorphin, β-melanocyte stimulating hormone (β-MSH), cholecystokinin, endothelin I, galanin, gastric inhibitory peptide (GIP), glucagon, amylin, lipotropins, neurophysins, GLP- 1. leptin, leptin receptor, calcitonin and somatostatin.

47. The method of claim 45, wherein said promoter is selected from the group consisting of

CMV, SV40 IE, RSV LTR, GAPHD and RIP1 .

48. A method for inhibiting ceramide-mediated lipotoxicity of a target cell comprising blocking ceramide production or accumulation in said cell.

49. The method of claim 48, wherein said blocking results in a decrease in the ceramide content of said cell when compared to the level of ceramide in said cell in the absence of said inhibitor.

50. The method of claim 49, wherein said decrease in the ceramide content of said cell is mediated by contacting said cell with leptin.

51. The method of claim 50, wherein said contacting comprises contacting said cell with an expression construct comprising a leptin encoding gene operatively linked to a promoter.

52. The method of claim 51 , wherein said promoter is selected from the group consisting of CMV, SV40 IE, RSV LTR, GAPHD and RIP1.

53. The method of claim 52, wherein said cell is a pancreatic beta cell.

54. A method of preventing non-insulin dependent diabetes mellitus (NIDDM) in a subject comprising blocking ceramide production in a pancreatic beta cell in said subject, wherein blocking ceramide production is accomplished by administering to said cell an amount of an inhibitory agent sufficient to protect said cell from ceramide-mediated lipotoxicity.

55. The method of claim 54, wherein blocking creamide production is accomplished by administering to said cell an amount of an inhibitory agent sufficient to protect said cell from NO-mediated lipotoxicity.

56. The method of claim 54, wherein said cell is engineered to secrete insulin.