METHODS OF USE OF ADENINE DERIVATIVES FOR THE TREATMENT OF DIABETES AND OTHER DISORDERS

BACKGROUND OF THE INVENTION

The present application claims the benefit of priority to U.S. Provisional Application

No. 61/048,813, filed April 29, 2008, the entire contents of this application being incorporated by reference. The invention was made with government support under grants

DK47919 and DK50610 awarded by the National Institute of Health. The government has certain rights in the invention.

I. Field of the Invention

The present invention relates generally to the treatment and prevention of diabetes. The methods and compositions of this invention comprise the use of small molecules.

II. Description of Related Art Type 1 diabetes is known to be caused by the selective autoimmune destruction of pancreatic β -cells that leads to a severe state of insulin insufficiency requiring insulin injection therapy and close to constant monitoring to avoid complications setting in. However, insulin therapy is not a cure for diabetes, which would require the replenishment of a functional β-cell mass. One very active area of type 1 diabetes research is to search for a pharmacological means to promote β-cell growth, regeneration and/or survival.

There is also a continued need for the treatment of type 2 diabetes mellitus (T2DM) which has reached almost epidemic proportions in the U.S. T2DM is initially characterized by insulin resistance and hyperinsulinemia which is eventually followed by the development of insulin secretory dysfunction and a reduction in the population of beta cells. It is predicted by the WHO that unless drastic action is taken 350 million people will have T2D by 2030.

Failure to control β-cell mass is pivotal to most forms of diabetes. Pancreatic β-cell mass is flexible and can adjust to compensate for a rise in metabolic load (as in pregnancy or obesity) (Rhodes, 2005; Sorenson and Brelje, 1997), in recovery from pancreatic injury (Smith et ah, 1991), or after surgical removal of an insulinoma (Chick et ah, 1977). Conversely, it can decrease postpartum (Scaglia et ah, 1995), or with controlled weight loss in treatment of obesity (Ferrannini et ah, 2004). Signal transduction mechanisms that control pancreatic β-cell growth, regeneration and survival at the molecular level have fundamental relevance for type 1 diabetes.

Soon after birth there is a neonatal burst of β-cell replication and neogenesis that declines after weaning (Rhodes, 2005). This sets the baseline β-cell mass in an individual. Under normal conditions in lean individuals there is a slow turnover of β-cells so that the β- cell mass stays relatively constant throughout adult life (Rhodes, 2005). However, the β-cell mass is plastic. Changes in net β-cell growth are set by the balance between the sum of β-cell hypertrophy (β-cell size) and hyperplasia (β-cell replication and neogenesis), minus the incidence of β-cell death (either via apoptosis (Butler et al., 2003), necrosis (Olejnicka et al, 1999) and/or autophagic mediated cell death (Marsh et al, 2007; Rhodes, 2005)). Many peptide growth factors, certain nutrients, peptide hormones and neural connections have been implicated in regulating β-cell growth (Lingohr et al, 2002). However, few of these have been shown to have physiological relevance.

There are several drugs on the market that target the β-cell to secrete more insulin as a therapy to treat type 2 diabetes: (1) Sulphonylureas: these target the ATP-sensitive K- channel to trigger insulin secretion. Use of sulphonylureas is problematic since their actions are not glucose dependent, which can cause unwanted and potentially dangerous hypoglycemic episodes for the patient. In addition, sulphonylureas do not upregulate insulin production (to replenish secreted insulin) and the secretory capacity of the β-cell is reduced leading to β-cell exhaustion. This can result in acceleration of the patient towards insulin injection therapy. As a consequence the clinical use of sulphonylureas is in fast decline. (2) GLP-I analogues (Byetta™): this is a stable form of the endogenous Glucagon-Like Peptide (GLP)-I incretin peptide that potentiates insulin secretion (via a specific G-protein coupled receptor and elevation of [CAMP] ₁ ) in a glucose dependent manner. The glucose dependency means that far fewer instances of hypoglycemia are caused by GLP-I analogs in comparison to sulphonylureas. GLP-I analogs may also have beneficial effects on promoting β-cell growth and survival. In addition, GLP-I can lead to a beneficial gradual weight loss in obese type 2 diabetics. However, GLP-I needs to be injected and there are significant side effects of which nausea is the most common. (3) DPP-IV inhibitors (Januvia™): This is a new class of drugs that delays the degradation of endogenous GLP-I and has modest effects on potentiating glucose-induced insulin secretion. DPP-IV inhibitors appear safe, have relatively few side effects and can be administered orally. However, the effects of DPP-IV inhibitors effects are quite weak and they need to be combined with other type 2 diabetes drugs such as Metformin or TZDs.

There is, therefore, a continued need for the treatment and prevention of diabetes. Methods to promote β-cell growth, regeneration and/or survival would be desirable, as would methods to potentiate the increase in insulin production in response to glucose.

SUMMARY OF THE INVENTION

Thus, in order to overcome deficiencies in the prior art, this invention provides methods of treating and preventing diabetes and other diseases using small molecules.

I. Indications and Methods of Treatment and Prevention

In some of its aspects, the present invention provide methods of treating and/or preventing glucose-associated disorders, such as diabetes and insulin secretory dysfunction. Also provided are methods of stimulating and/or producing pancreatic β-cells. Also provided are methods of prolonging the honey-moon period in the pathogenesis of type 1 diabetes in a subject. These methods all comprise administering an effective amount of an adenine derivative to a subject and/or contacting a pancreatic β-cell with an adenine derivative.

In some embodiments, the diabetes is type 1, in others it is type 2. In further embodiments, the glucose-dependent stimulation of insulin and/or proinsulin secretion, biosynthesis and/or processing is potentiated in the subject. In still other embodiments, the regeneration, growth, survival and/or neogenesis of pancreatic β-cells in the subject is promoted. In some of these embodiments, the promotion occurs through the induction of anti-apoptotic regulatory gene expression in these cells. For example, the anti-apoptotic regulatory gene can be IRS-2 or CREB.

In some embodiments, the subject in which the glucose-associated disorder is to be treated or prevented can be a primate, such as a human. In some embodiments, the method can further comprise identifying a subject in need of treatment or prevention. Examples of identifying subjects includes those having a family or patient history of diabetes, or those having symptoms of diabetes. In some embodiments, the method further comprises testing the subject for symptoms of diabetes and/or monitoring the level of sugar in the subject's blood. In some embodiments, the method of simulating and/or producing pancreatic β-cells takes place ex vivo. In some variations, the method includes culturing the pancreatic β-cells.

In further variations, the pancreatic β-cell can be cultured with a growth factor {e.g., TGF-βl,

TGF-β2, TGF-βl.2, VEGF, insulin-like growth factor I or II, BMP2, BMP4, BMP7, GLP-I analog, a GIP analog, a DPP-IV inhibitor, a GPRl 19 agonist, a GPR40 agonist, gastrin, EGF, betacellulin, KGF, NGF, insulin, growth hormone, HGF, FGFs and FGF homologues, PDGF,

Leptin, prolactin, placental lactogen, PTHrP, activin, inhibin, INGAP, a parathyroid hormone, calcitonin, interleukin-6, or interleukin-11). In still further variations, the pancreatic β-cell is purified after being stimulated, produced and/or cultured. Some variation provide for implanting said cell in vivo after one or more of the above steps.

In some embodiments, the pancreatic β-cell is stimulated to (1) secrete insulin, (2) potentiate the increase in insulin production in response to glucose, (3) increase the expression of pro-survival genes, and/or (4) increase the level of cAMP in the pancreatic β- cell. In some embodiments, the pancreatic β-cell is of human, bovine, equine, canine, feline, murine, rat or chick origin.

II. Adenine Derivatives

In the context of the present application, adenine derivatives are defined as compounds of the formula:

wherein the groups R ₁ and R ₂ are independently attached to either carbon or nitrogen atoms of either ring shown above; further wherein R ₁ and R ₂ are independently hydrogen, hydroxy, amino, cyano, halo, nitro, mercapto, or a heteroatom-substituted or heteroatom-unsubstituted alkyl( _cl- c8), alkenyl( _cl-C 8), alkynyl( _cl- c8), aryl( _cl-C 8), aralkyl( _C2-C 8), acyl( _cl- c8), alkoxyfci-cs), alkenyloxyfci-cs), alkynyloxyfci-cs), aryloxyfci-cs), aralkyloxy( _C2- c8), acyloxy( _cl- c8), alkylamino( _cl- c8), alkenylamino( _cl- c8), alkynylamino^i-cs), arylamino(ci-c8), aralkylamino(ci-c8) or amido(ci-c8); provided that when R ₁ is attached to a nitrogen atom, R ₁ is not hydroxy, amino, cyano, halo, nitro, mercapto, or a heteroatom-substituted or heteroatom-unsubstituted alkoxy( _cl- c8), alkenyloxy( _cl- c8), alkynyloxy( _cl- c8), aryloxy( _cl- c8), aralkyloxy(c2-c8), acyloxy(ci-c8), alkylamino(ci-c8), alkenylamino(ci-c8), alkynylaminofci-cs), arylaminofci-cs), aralkylaminofci-cs) or amidofci-cs); further provided that when R ₂ is attached to a nitrogen atom, R ₂ is not hydroxy, amino, cyano, halo, nitro, mercapto, or a heteroatom-substituted or heteroatom-unsubstituted alkoxy(ci-c8), alkenyloxy(ci-c8), alkynyloxy(ci-c8), aryloxy( _cl- c8), aralkyloxy( _C2 -c8), acyloxy( _cl- c8), alkylamino( _cl- c8), alkenylamino(ci-c8), alkynylamino(c _1- c8), arylamino(ci-c8), aralkylamino(ci-c8) or amido(ci-c8); also provided that R ₁ and R ₂ are not both hydrogen; or pharmaceutically acceptable salts, hydrates, solvates, tautomers, or optical isomers thereof.

The invention specifically contemplates that a subgenus of the above formula may be excluded.

In some embodiments, R ₂ is hydrogen and/or R ₁ is heteroatom-substituted or heteroatom-unsubstituted alkyl(ci-c4), such as a heteroatom-unsubstituted alkyl(ci-c3).

In certain embodiments, the adenine derivative is one of the following:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, or optical isomers thereof.

In further embodiments, the adenine derivative is soluble in either water or bodily fluids.

III. Methods of Administration

In some embodiments, the adenine derivative can be administered systemically. For example, it can be administered intravenously, intra-arterially, intra-muscularly, intra- peritoneally, subcutaneously or orally. In some variations, the effective amount is less than or equal to 500 mg daily. In other variations, the effective amount is from about 1 mg/kg of body weight to about 15 mg/kg of body weight, for example about 7 mg/kg of body weight. In other embodiments, the adenine derivative can be administered by contacting a pancreatic β-cell during ex vivo purging.

The invention provides in some embodiments that the effective amount of adenine derivative is administered as a single dose per day, at two of more doses per day. The effective amount may also be calculated based on the level of sugar in the subject's blood.

IV. Combination Therapies

In some embodiments, the methods of the invention further comprise administering immunosuppressive therapy, wherein the adenine derivative and the immunosuppressive therapy are provided to the subject in an effective amount to treat or prevent diabetes in the subject. In some variations, the immunosuppressive therapy comprises administering an anti- CD3 monoclonal antibody to the subject. In some embodiments, the invention provides methods comprising administering to the subject a second drug, wherein the adenine derivative and the second drug are provided in an effective amount to treat diabetes in the subject. Examples of second drugs include dipeptidyl peptidase-4 (DPP-4) inhibitors (e.g., sitagliptin, vildagliptin, SYR-322, BMS 477118 and GSK 823093), biguanides {e.g., metformin), thiazolidinedione (TZD) or thiazolidinedione derivatives (e.g., pioglitazone, rosiglitazone and troglitazone), sulfonylurea derivative (e.g., tolbutamide, acetohexamide, tolazamide, chlorpropamide, glipizide, glyburide, glimepiride and gliclazide), meglitinide (e.g., repaglinide, mitiglinide and nateglinide), insulin, alpha-glucosidase inhibitors (e.g., acarbose, miglitol and voglibose), glucagon-like peptide- 1 analogs (e.g., exenatide and liraglutide), gastric inhibitory peptide analogs, GPR40 agonists, GPRl 19 agonists, GPR30 agonists, glucokinase activators, glucagon receptor antagonists, amylin analogs (e.g., pramlintide), IL- lβ receptor antagonists (e.g., anakinra), endocannabinoid receptor antagonists or inverse agonists (e.g., rimonabant), Orlistat, Sibutramine, and growth factors (e.g., TGF-βl, TGF-β2, TGF-βl.2, VEGF, insulin- like growth factor I or II, BMP2, BMP4, BMP7, GLP-I analogs, GIP analogs, DPP-IV inhibitors, GPR40 agonists, gastrin, EGF, betacellulin, KGF, NGF, insulin, growth hormone, HGF, FGFs or FGF homologues, PDGF, Leptin, prolactin, placental lactogen, PTHrP, activin, inhibin, INGAP, parathyroid hormone, calcitonin, interleukin-6, or interleukin-11). In further embodiments, the method further comprises administering to the subject a second drug and a third drug, wherein the adenine derivative, the second drug and the third drug are provided in an effective amount to treat diabetes in the subject. For example, the second and third drugs can be independently selected from the group recited above. A non- limiting example of a possible combination includes using an adenine derivative with a DPP- 4 inhibitor and a biguanide, such as metformin. Another non-limiting example includes using an adenine derivative with an amylin analog and a leptin analog.

V. Further Embodiments

Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well. 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 specific 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 of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGS. IA & B. The glucose-dependent potentiation of insulin secretion by certain methylated adenine analogs. FIG. IA shows a preliminary study in isolated rat islets (mean ± SE of > 3 experiments), where 250μM 3-MA, M-methyl adenine (M)-MA) or 9-methyl adenine (9-MA) significantly potentiated glucose-induced insulin secretion. In contrast, 250μM 1-methyl adenine (1-MA), 7-methyl adenine (7-MA), adenosine, adenine, cytosine, guanine or thymine had no effect on glucose-induced insulin secretion. As only some methylated forms of adenine (i.e. 3-MA, M)-MA and 9-MA, not 1-MA or 7-MA) potentiated glucose-induced insulin secretion this indicates that the effect is specific. There was no effect of these compounds on basal insulin secretion at 3mM glucose (the left bar of each pair shown). FIG. IB shows the glucose-dependence of 250μM 3-MA, M-MA or 9-MA on potentiating insulin secretion that is equivalent, if not better, than the effect of the GLP-I analog exendin-4 (5nM). FIG. 2. The effect of 3-MA (30μg/g oral dose) in an IPGTT in normal adult rats. Oral administration of either 3-MA or M)-MA (at a dose of 30-50μg/g body weight (bw)) given 15-20 minutes prior to an intraperitoneal glucose tolerance test (IPGTT; 1 mg glucose/g bw) in overnight fasted normal lean rats, significantly lowers the excursion in blood glucose over a subsequent 120 minute period without notable hypoglycemia. 3-MA data shown as mean ± SE (n > 4).

FIG. 3. M)-MA and 9-MA potentiate glucose-induced rat islet β-cell replication. In both INS-I cells and isolated rat islets, M)-MA and 9-MA (250μM) significantly potentiate β- cell proliferation at glucose concentrations >8mM when assessed by [ ³ H]thymidine incorporation over a 48h period. This figure shows such an effect at a stimulatory HmM glucose (data are a mean ± SE (n > 3)).

FIG. 4. 3-MA, M-MA and 9-MA protect rat islet β-cells against cvtokine-induced apoptosis. Isolated rat islets were exposed to a cytokine cocktail of interleukin-lβ (IL- lβ, 10ng/ml), tumor necrosis factor-α (TNF-α, 50ng/ml) and interferon-γ (IFN-γ, 50ng/ml), at a basal 3mM glucose in the presence or absence of 3MA, M-MA or 9-MA (250μM) for 24h. It can be seen that 3MA, M-MA and 9-MA are quite protective against cytokine-induced apoptosis as shown by reduced caspase-3 activation, an indicator of apoptosis.

FIG. 5. 3-MA, M-MA and 9-MA increase IRS-2 expression and decrease pro- apoptotic gene expression in isolated rat islets. This figure shows that 250μM 3-MA, M-MA, and 9-MA, each augment a 15mM glucose-induced IRS-2 protein expression in isolated rat islets within 6h. The 3MA/M-MA/9-MA augmented increase in IRS-2 expression in rat islet β-cells is associated with decreased expression of the pro-apoptotic genes, Bax, Bad and Bak, but not the anti-apoptotic genes Bcl-2 or BCI-X _L , which are unchanged relative to a loading control protein, the 85kD subunit of PI3K. FIGS. 6A & B. 3-MA increases glucose-induced insulin secretion and improve glucose tolerance in normal rats. Normal Sprague-Dawley rats, -200 g body weight (bw), were fasted overnight, then subjected to an intraperitoneal glucose tolerance test (IPGTT), using a lmg glucose/g bw dose of glucose. These IPGTTs were conducted with 3-MA (30 μg/g bw) or vehicle control, given IP, 15 minutes prior to the glucose dose. Tail blood samples were collected at the indicated time pints and glucose (FIG. 6A) and insulin levels (FIG. 6B) were subsequently measured. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

I. The Present Invention

The present invention relates generally to the prevention and treatment of glucose- associated diseases such as diabetes using adenine derivatives having the formula:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, or optical isomers thereof, wherein the variables are as defined herein.

IL Definitions

The use of the word "a" or "an," when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. The terms "comprise," "have" and "include" are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as "comprises," "comprising," "has,"

"having," "includes" and "including," are also open-ended. For example, any method that

"comprises," "has" or "includes" one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps. As used herein, the term "IC ₅₀ " refers to an inhibitory dose which results in 50% of the maximum response obtained.

The terms "inhibiting," "reducing," or "prevention," or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result. The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dogs, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human subjects are adults, juveniles, infants and fetuses. As used herein, "predominantly one enantiomer" means that the compound contains at least 85% of one enantiomer, or more preferably at least 90% of one enantiomer, or even more preferably at least 95% of one enantiomer, or most preferably at least 99% of one enantiomer. Similarly, the phrase "substantially free from other optical isomers" means that the composition contains at most 5% of another enantiomer or diastereomer, more preferably 2% of another enantiomer or diastereomer, and most preferably 1% of another enantiomer or diastereomer.

As used herein, the term "water soluble" means that the compound dissolves in water at least to the extent of 0.010 mole/liter or is classified as soluble according to literature precedence. As used herein, the term "amino" means -NH ₂ ; the term "nitro" means -NO ₂ ; the term "halo" designates -F, -Cl, -Br or -I; the term "mercapto" means -SH; the term "cyano" means -CN; the term "azido" means -N ₃ ; the term "silyl" means -SiH ₃ , and the term "hydroxy" means -OH.

The term "alkyl" includes straight-chain alkyl, branched-chain alkyl, cycloalkyl (alicyclic), cyclic alkyl, heteroatom-unsubstituted alkyl, heteroatom-substituted alkyl, heteroatom-unsubstituted alkyl(c _n ), and heteroatom-substituted alkyl(c _n )- The term "heteroatom-unsubstituted alkyl(c _n )" refers to a radical, having a linear or branched, cyclic or acyclic structure, further having no carbon-carbon double or triple bonds, further having a total of n carbon atoms, all of which are nonaromatic, 3 or more hydrogen atoms, and no heteroatoms. For example, a heteroatom-unsubstituted alkyl(ci-cio) has 1 to 10 carbon atoms. The groups, -CH ₃ (Me), -CH ₂ CH ₃ (Et), -CH ₂ CH ₂ CH ₃ (n-Pτ), -CH(CH ₃ ) ₂ (iso-Pτ), -CH(CH ₂ ) ₂ (cyclopropyl), -CH ₂ CH ₂ CH ₂ CH ₃ (n-Bu), -CH(CH ₃ )CH ₂ CH ₃ (sec-butyl), -CH ₂ CH(CH ₃ ) ₂ (wo-butyl), -C(CH ₃ ) ₃ (tert-butyϊ), -CH ₂ C(CH ₃ ) ₃ (weo-pentyl), cyclobutyl, cyclopentyl, and cyclohexyl, are all non-limiting examples of heteroatom-unsubstituted alkyl groups. The term "heteroatom-substituted alkyl(c _n )" refers to a radical, having a single saturated carbon atom as the point of attachment, no carbon-carbon double or triple bonds, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, all of which are nonaromatic, 0, 1 , or more than one hydrogen atom, at least one heteroatom, wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted alkyl(ci-cio) has 1 to 10 carbon atoms. The following groups are all non- limiting examples of heteroatom- substituted alkyl groups: trifluoromethyl, -CH ₂ F, -CH ₂ Cl, -CH ₂ Br, -CH ₂ OH, -CH ₂ OCH ₃ , -CH ₂ OCH ₂ CF ₃ , -CH ₂ OC(O)CH ₃ , -CH ₂ NH ₂ , -CH ₂ NHCH ₃ , -CH ₂ N(CH ₃ ) ₂ , -CH ₂ CH ₂ Cl, -CH ₂ CH ₂ OH, CH ₂ CH ₂ OC(O)CH ₃ , -CH ₂ CH ₂ NHCO ₂ C(CH ₃ ) ₃ , and -CH ₂ Si(CH ₃ ) ₃ .

The term "alkanediyl" includes straight-chain alkanediyl, branched-chain alkanediyl, cycloalkanediyl, cyclic alkanediyl, heteroatom-unsubstituted alkanediyl, heteroatom- substituted alkanediyl, heteroatom-unsubstituted alkanediyl(cn), and heteroatom-substituted alkanediyl(cn)- The term "heteroatom-unsubstituted alkanediyl (c _n )" refers to a diradical, having a linear or branched, cyclic or acyclic structure, further having no carbon-carbon double or triple bonds, further having a total of n carbon atoms, all of which are nonaromatic, 2 or more hydrogen atoms, and no heteroatoms. For example, a heteroatom-unsubstituted alkanediyl(ci-cio) has 1 to 10 carbon atoms. The groups, -CH ₂ - (methylene), -CH ₂ CH ₂ -, and -CH ₂ CH ₂ CH ₂ -, are all non-limiting examples of heteroatom-unsubstituted alkanediyl groups. The term "heteroatom-substituted alkanediyl(cn)" refers to a radical, having two points of attachment to one or two saturated carbon atoms, no carbon-carbon double or triple bonds, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, all of which are nonaromatic, O, 1, or more than one hydrogen atom, at least one heteroatom, wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted alkanediyl(ci-cio) has 1 to 10 carbon atoms. The following groups are all non-limiting examples of heteroatom- substituted alkanediyl groups: -CH(F)-, -CF ₂ -, -CH(Cl)-, -CH(OH)-, -CH(OCH ₃ )-, and -CH ₂ CH(Cl)-.

The term "alkenyl" includes straight-chain alkenyl, branched-chain alkenyl, cycloalkenyl, cyclic alkenyl, heteroatom-unsubstituted alkenyl, heteroatom-substituted alkenyl, heteroatom-unsubstituted alkenyl(c _n ), and heteroatom-substituted alkenyl(cn)- The term "heteroatom-unsubstituted alkenyl(c _n )" refers to a radical, having a linear or branched, cyclic or acyclic structure, further having at least one nonaromatic carbon-carbon double bond, but no carbon-carbon triple bonds, a total of n carbon atoms, three or more hydrogen atoms, and no heteroatoms. For example, a heteroatom-unsubstituted alkenyl(c _2- cio) has 2 to 10 carbon atoms. Heteroatom-unsubstituted alkenyl groups include: -CH=CH ₂ (vinyl), -CH=CHCH ₃ , -CH=CHCH ₂ CH ₃ , -CH ₂ CH=CH ₂ (allyl), -CH ₂ CH=CHCH ₃ , and -CH=CH-C _O H ₅ . The term "heteroatom-substituted alkenyl(c _n )" refers to a radical, having a single nonaromatic carbon atom as the point of attachment and at least one nonaromatic carbon-carbon double bond, but no carbon-carbon triple bonds, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, and at least one heteroatom, wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted alkenyl(c2-cio) has 2 to 10 carbon atoms. The groups, -CH=CHF, -CH=CHCl and -CH=CHBr, are non-limiting examples of heteroatom- substituted alkenyl groups.

The term "alkynyl" includes straight-chain alkynyl, branched-chain alkynyl, cycloalkynyl, cyclic alkynyl, heteroatom-unsubstituted alkynyl, heteroatom-substituted alkynyl, heteroatom-unsubstituted alkynyl(cn), and heteroatom-substituted alkynyl(cn)- The term "heteroatom-unsubstituted alkynyl(cn)" refers to a radical, having a linear or branched, cyclic or acyclic structure, further having at least one carbon-carbon triple bond, a total of n carbon atoms, at least one hydrogen atom, and no heteroatoms. For example, a heteroatom- unsubstituted alkynyl(c2-cio) has 2 to 10 carbon atoms. The groups, -C≡CH, -C=CCH ₃ , and -C=CC ₆ Hs are non-limiting examples of heteroatom-unsubstituted alkynyl groups. The term "heteroatom-substituted alkynyl(cn)" refers to a radical, having a single nonaromatic carbon atom as the point of attachment and at least one carbon-carbon triple bond, further having a linear or branched, cyclic or acyclic structure, and having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, and at least one heteroatom, wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted alkynyl(c2-cio) has 2 to 10 carbon atoms. The group, -C=CSi(CHs) ₃ , is a non-limiting example of a heteroatom-substituted alkynyl group.

The term "aryl" includes heteroatom-unsubstituted aryl, heteroatom-substituted aryl, heteroatom-unsubstituted aryl( _Cn ), heteroatom-substituted aryl( _Cn ), heteroaryl, heterocyclic aryl groups, carbocyclic aryl groups, biaryl groups, and single-valent radicals derived from polycyclic fused hydrocarbons (PAHs). The term "heteroatom-unsubstituted aryl(cn)" refers to a radical, having a single carbon atom as a point of attachment, wherein the carbon atom is part of an aromatic ring structure containing only carbon atoms, further having a total of n carbon atoms, 5 or more hydrogen atoms, and no heteroatoms. For example, a heteroatom- unsubstituted aryl(c6-cio) has 6 to 10 carbon atoms. Non-limiting examples of heteroatom- unsubstituted aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C ₆ H ₄ CH ₂ CH ₃ , -C ₆ H ₄ CH ₂ CH ₂ CH ₃ , -C ₆ H ₄ CH(CH ₃ ) ₂ , -C ₆ H ₄ CH(CH ₂ ) ₂ ,

-C ₆ H ₃ (CH ₃ )CH ₂ CH ₃ , -C ₆ H ₄ CH=CH ₂ , -C ₆ H ₄ CH=CHCH ₃ , -C ₆ H ₄ C=CH, -C ₆ H ₄ C=CCH ₃ , naphthyl, and the radical derived from biphenyl. The term "heteroatom-substituted aryl(c _n )" refers to a radical, having either a single aromatic carbon atom or a single aromatic heteroatom as the point of attachment, further having a total of n carbon atoms, at least one hydrogen atom, and at least one heteroatom, further wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-unsubstituted heteroaryl(ci-cio) has 1 to 10 carbon atoms. Non- limiting examples of heteroatom-substituted aryl groups include the groups: -C ₆ H ₄ F, -C ₆ H ₄ Cl, -C ₆ H ₄ Br, -C ₆ H ₄ I, -C ₆ H ₄ OH, -C ₆ H ₄ OCH ₃ , -C ₆ H ₄ OCH ₂ CH ₃ , -C ₆ H ₄ OC(O)CH ₃ , -C ₆ H ₄ NH ₂ , -C ₆ H ₄ NHCH ₃ , -C ₆ H ₄ N(CH ₃ ) ₂ , -C ₆ H ₄ CH ₂ OH, -C ₆ H ₄ CH ₂ OC(O)CH ₃ , -C ₆ H ₄ CH ₂ NH ₂ , -C ₆ H ₄ CF ₃ , -C ₆ H ₄ CN, -C ₆ H ₄ CHO, -C ₆ H ₄ CHO, -C ₆ H ₄ C(O)CH ₃ , -C ₆ H ₄ C(O)C ₆ H ₅ , -C ₆ H ₄ CO ₂ H, -C ₆ H ₄ CO ₂ CH ₃ , -C ₆ H ₄ CONH ₂ , -C ₆ H ₄ CONHCH ₃ , -C ₆ H ₄ CON(CH ₃ ) ₂ , furanyl, thienyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, quinolyl, indolyl, and imidazoyl.

The term "aralkyl" includes heteroatom-unsubstituted aralkyl, heteroatom-substituted aralkyl, heteroatom-unsubstituted aralkyl(c _n ), heteroatom-substituted aralkyl(c _n ), heteroaralkyl, and heterocyclic aralkyl groups. The term "heteroatom-unsubstituted aralkyl(cn)" refers to a radical, having a single saturated carbon atom as the point of attachment, further having a total of n carbon atoms, wherein at least 6 of the carbon atoms form an aromatic ring structure containing only carbon atoms, 7 or more hydrogen atoms, and no heteroatoms. For example, a heteroatom-unsubstituted aralkyl(c7-cio) has 7 to 10 carbon atoms. Non-limiting examples of heteroatom-unsubstituted aralkyls are: phenylmethyl (benzyl, Bn) and phenylethyl. The term "heteroatom-substituted aralkyl(c _n )" refers to a radical, having a single saturated carbon atom as the point of attachment, further having a total of n carbon atoms, O, 1, or more than one hydrogen atom, and at least one heteroatom, wherein at least one of the carbon atoms is incorporated an aromatic ring structures, further wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted heteroaralkyl(c _2- cio) has 2 to 10 carbon atoms.

The term "acyl" includes straight-chain acyl, branched-chain acyl, cycloacyl, cyclic acyl, heteroatom-unsubstituted acyl, heteroatom-substituted acyl, heteroatom-unsubstituted acyl(cn), heteroatom-substituted acyl(cn), alkylcarbonyl, alkoxycarbonyl and aminocarbonyl groups. The term "heteroatom-unsubstituted acyl(cn)" refers to a radical, having a single carbon atom of a carbonyl group as the point of attachment, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 1 or more hydrogen atoms, a total of one oxygen atom, and no additional heteroatoms. For example, a heteroatom-unsubstituted acyl(ci-cio) has 1 to 10 carbon atoms. The groups, -CHO, -C(O)CH ₃ , -C(O)CH ₂ CH ₃ , -C(O)CH ₂ CH ₂ CH ₃ , -C(O)CH(CH ₃ ) ₂ , -C(O)CH(CH ₂ ) ₂ , -C(O)C ₆ H ₅ , -C(O)C ₆ H ₄ CH ₃ , -C(O)C ₆ H ₄ CH ₂ CH ₃ , and -COC ₆ H ₃ (CH ₃ ) ₂ , are non-limiting examples of heteroatom-unsubstituted acyl groups. The term "heteroatom-substituted acyl(c _n )" refers to a radical, having a single carbon atom as the point of attachment, the carbon atom being part of a carbonyl group, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, O, 1, or more than one hydrogen atom, at least one additional heteroatom, in addition to the oxygen of the carbonyl group, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted acyl(ci-cio) has 1 to 10 carbon atoms.. The groups, -C(O)CH ₂ CF ₃ , -CO ₂ H, -CO ₂ CH ₃ , -CO ₂ CH ₂ CH ₃ , -CO ₂ CH ₂ CH ₂ CH ₃ , -CO ₂ CH(CH ₃ ) ₂ , -CO ₂ CH(CH ₂ ) ₂ , -C(O)NH ₂ (carbamoyl), -C(O)NHCH ₃ , -C(O)NHCH ₂ CH ₃ , -CONHCH(CH ₃ ) ₂ , -CONHCH(CH ₂ ) ₂ , -CON(CH ₃ ) ₂ , and -CONHCH ₂ CF ₃ , are non-limiting examples of heteroatom-substituted acyl groups. The term "alkoxy" includes straight-chain alkoxy, branched-chain alkoxy, cycloalkoxy, cyclic alkoxy, heteroatom-unsubstituted alkoxy, heteroatom-substituted alkoxy, heteroatom-unsubstituted alkoxy(c _n ), and heteroatom-substituted alkoxy(c _n )- The term "heteroatom-unsubstituted alkoxy(cn)" refers to a group, having the structure -OR, in which R is a heteroatom-unsubstituted alkyl(c _n ), as that term is defined above. Heteroatom- unsubstituted alkoxy groups include: -OCH ₃ , -OCH ₂ CH ₃ , -OCH ₂ CH ₂ CH ₃ , -OCH(CH ₃ ) ₂ , and -OCH(CH ₂ ) ₂ . The term "heteroatom-substituted alkoxy(cn)" refers to a group, having the structure -OR, in which R is a heteroatom-substituted alkyl(c _n ), as that term is defined above. For example, -OCH ₂ CF ₃ is a heteroatom-substituted alkoxy group.

The term "alkenyloxy" includes straight-chain alkenyloxy, branched-chain alkenyloxy, cycloalkenyloxy, cyclic alkenyloxy, heteroatom-unsubstituted alkenyloxy, heteroatom-substituted alkenyloxy, heteroatom-unsubstituted alkenyloxy(cn), and heteroatom- substituted alkenyloxy(cn)- The term "heteroatom-unsubstituted alkenyloxy(c _n )" refers to a group, having the structure -OR, in which R is a heteroatom-unsubstituted alkenyl(c _n ), as that term is defined above. The term "heteroatom-substituted alkenyloxy(cn)" refers to a group, having the structure -OR, in which R is a heteroatom-substituted alkenyl(c _n ), as that term is defined above.

The term "alkynyloxy" includes straight-chain alkynyloxy, branched-chain alkynyloxy, cycloalkynyloxy, cyclic alkynyloxy, heteroatom-unsubstituted alkynyloxy, heteroatom-substituted alkynyloxy, heteroatom-unsubstituted alkynyloxy(cn), and heteroatom-substituted alkynyloxy(cn)- The term "heteroatom-unsubstituted alkynyloxy(cn)" refers to a group, having the structure -OR, in which R is a heteroatom-unsubstituted alkynyl(cn), as that term is defined above. The term "heteroatom-substituted alkynyloxy(cn)" refers to a group, having the structure -OR, in which R is a heteroatom-substituted alkynyl(cn), as that term is defined above.

The term "aryloxy" includes heteroatom-unsubstituted aryloxy, heteroatom- substituted aryloxy, heteroatom-unsubstituted aryloxy(cn), heteroatom-substituted aryloxy(cn), heteroaryloxy, and heterocyclic aryloxy groups. The term "heteroatom- unsubstituted aryloxy(cn)" refers to a group, having the structure -OAr, in which Ar is a heteroatom-unsubstituted aryl(c _n ), as that term is defined above. A non-limiting example of a heteroatom-unsubstituted aryloxy group is -OC _O H _S . The term "heteroatom-substituted aryloxy(cn)" refers to a group, having the structure -OAr, in which Ar is a heteroatom- substituted aryl(cn), as that term is defined above.

The term "aralkyloxy" includes heteroatom-unsubstituted aralkyloxy, heteroatom- substituted aralkyloxy, heteroatom-unsubstituted aralkyloxy(cn), heteroatom-substituted aralkyloxy(cn), heteroaralkyloxy, and heterocyclic aralkyloxy groups. The term "heteroatom- unsubstituted aralkyloxy(cn)" refers to a group, having the structure -OAr, in which Ar is a heteroatom-unsubstituted aralkyl(c _n ), as that term is defined above. The term "heteroatom- substituted aralkyloxy(cn)" refers to a group, having the structure -OAr, in which Ar is a heteroatom-substituted aralkyl(c _n ), as that term is defined above.

The term "acyloxy" includes straight-chain acyloxy, branched-chain acyloxy, cycloacyloxy, cyclic acyloxy, heteroatom-unsubstituted acyloxy, heteroatom-substituted acyloxy, heteroatom-unsubstituted acyloxy(c _n ), heteroatom-substituted acyloxy(c _n ), alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, and carboxylate groups. The term "heteroatom-unsubstituted acyloxy(cn)" refers to a group, having the structure -OAc, in which Ac is a heteroatom-unsubstituted acyl(cn), as that term is defined above. For example, -OC(O)CH ₃ is a non-limiting example of a heteroatom-unsubstituted acyloxy group. The term "heteroatom-substituted acyloxy(c _n )" refers to a group, having the structure -OAc, in which Ac is a heteroatom-substituted acyl(cn), as that term is defined above. For example, -OC(O)OCH ₃ and -OC(O)NHCH ₃ are non-limiting examples of heteroatom-unsubstituted acyloxy groups.

The term "alkylamino" includes straight-chain alkylamino, branched-chain alkylamino, cycloalkylamino, cyclic alkylamino, heteroatom-unsubstituted alkylamino, heteroatom-substituted alkylamino, heteroatom-unsubstituted alkylamino(cn), and heteroatom-substituted alkylamino(cn)- The term "heteroatom-unsubstituted alkylamino(cn)" refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two saturated carbon atoms attached to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, containing a total of n carbon atoms, all of which are nonaromatic, 4 or more hydrogen atoms, a total of 1 nitrogen atom, and no additional heteroatoms. For example, a heteroatom-unsubstituted alkylamino(ci-cio) has 1 to 10 carbon atoms. The term "heteroatom-unsubstituted alkylamino(cn)" includes groups, having the structure -NHR, in which R is a heteroatom-unsubstituted alkyl(c _n ), as that term is defined above. A heteroatom-unsubstituted alkylamino group would include -NHCH ₃ , -NHCH ₂ CH ₃ , -NHCH ₂ CH ₂ CH ₃ , -NHCH(CH ₃ ) ₂ , -NHCH(CH ₂ ) ₂ , -NHCH ₂ CH ₂ CH ₂ CH ₃ , -NHCH(CH ₃ )CH ₂ CH ₃ , -NHCH ₂ CH(CH ₃ ) ₂ , -NHC(CH ₃ ) ₃ , -N(CH ₃ ) ₂ , -N(CH ₃ )CH ₂ CH ₃ , -N(CH ₂ CH ₃ ) ₂ , 7V-pyrrolidinyl, and TV-piperidinyl. The term "heteroatom-substituted alkylamino(cn)" refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two saturated carbon atoms attached to the nitrogen atom, no carbon- carbon double or triple bonds, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, all of which are nonaromatic, 0, 1, or more than one hydrogen atom, and at least one additional heteroatom, that is, in addition to the nitrogen atom at the point of attachment, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom- substituted alkylamino(ci-cio) has 1 to 10 carbon atoms. The term "heteroatom-substituted alkylamino(cn)" includes groups, having the structure -NHR, in which R is a heteroatom- substituted alkyl(cn), as that term is defined above.

The term "alkenylamino" includes straight-chain alkenylamino, branched-chain alkenylamino, cycloalkenylamino, cyclic alkenylamino, heteroatom-unsubstituted alkenylamino, heteroatom-substituted alkenylamino, heteroatom-unsubstituted alkenylamino(cn), heteroatom-substituted alkenylamino(cn), dialkenylamino, and alkyl(alkenyl)amino groups. The term "heteroatom-unsubstituted alkenylamino(cn)" refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two carbon atoms attached to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, containing at least one nonaromatic carbon-carbon double bond, a total of n carbon atoms, 4 or more hydrogen atoms, a total of one nitrogen atom, and no additional heteroatoms. For example, a heteroatom-unsubstituted alkenylamino(c _2- cio) has 2 to 10 carbon atoms. The term "heteroatom-unsubstituted alkenylamino(cn)" includes groups, having the structure -NHR, in which R is a heteroatom-unsubstituted alkenyl(c _n ), as that term is defined above. The term "heteroatom-substituted alkenylamino(cn)" refers to a radical, having a single nitrogen atom as the point of attachment and at least one nonaromatic carbon- carbon double bond, but no carbon-carbon triple bonds, further having one or two carbon atoms attached to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, and at least one additional heteroatom, that is, in addition to the nitrogen atom at the point of attachment, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted alkenylamino(c2-cio) has 2 to 10 carbon atoms. The term "heteroatom-substituted alkenylamino(cn)" includes groups, having the structure -NHR, in which R is a heteroatom- substituted alkenyl(cn), as that term is defined above.

The term "alkynylamino" includes straight-chain alkynylamino, branched-chain alkynylamino, cycloalkynylamino, cyclic alkynylamino, heteroatom-unsubstituted alkynylamino, heteroatom-substituted alkynylamino, heteroatom-unsubstituted alkynylamino(cn), heteroatom-substituted alkynylamino(cn), dialkynylamino, alkyl(alkynyl)amino, and alkenyl(alkynyl)amino groups. The term "heteroatom-unsubstituted alkynylamino(cn)" refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two carbon atoms attached to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, containing at least one carbon-carbon triple bond, a total of n carbon atoms, at least one hydrogen atoms, a total of one nitrogen atom, and no additional heteroatoms. For example, a heteroatom-unsubstituted alkynylamino(c2-cio) has 2 to 10 carbon atoms. The term "heteroatom-unsubstituted alkynylamino(cn)" includes groups, having the structure -NHR, in which R is a heteroatom- unsubstituted alkynyl(cn), as that term is defined above. The term "heteroatom-substituted alkynylamino(cn)" refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two carbon atoms attached to the nitrogen atom, further having at least one nonaromatic carbon-carbon triple bond, further having a linear or branched, cyclic or acyclic structure, and further having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, and at least one additional heteroatom, that is, in addition to the nitrogen atom at the point of attachment, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted alkynylamino(c2-cio) has 2 to 10 carbon atoms. The term "heteroatom-substituted alkynylamino(cn)" includes groups, having the structure -NHR, in which R is a heteroatom-substituted alkynyl(cn), as that term is defined above. The term "arylamino" includes heteroatom-unsubstituted arylamino, heteroatom- substituted arylamino, heteroatom-unsubstituted arylamino(cn), heteroatom-substituted arylamino(cn), heteroarylamino, heterocyclic arylamino, and alkyl(aryl)amino groups. The term "heteroatom-unsubstituted arylamino(cn)" refers to a radical, having a single nitrogen atom as the point of attachment, further having at least one aromatic ring structure attached to the nitrogen atom, wherein the aromatic ring structure contains only carbon atoms, further having a total of n carbon atoms, 6 or more hydrogen atoms, a total of one nitrogen atom, and no additional heteroatoms. For example, a heteroatom-unsubstituted arylamino(c6-cio) has 6 to 10 carbon atoms. The term "heteroatom-unsubstituted arylamino(cn)" includes groups, having the structure -NHR, in which R is a heteroatom-unsubstituted aryl(c _n ), as that term is defined above. The term "heteroatom-substituted arylamino(cn)" refers to a radical, having a single nitrogen atom as the point of attachment, further having a total of n carbon atoms, at least one hydrogen atom, at least one additional heteroatoms, that is, in addition to the nitrogen atom at the point of attachment, wherein at least one of the carbon atoms is incorporated into one or more aromatic ring structures, further wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted arylamino(c6-cio) has 6 to 10 carbon atoms. The term "heteroatom-substituted arylamino(cn)" includes groups, having the structure -NHR, in which R is a heteroatom-substituted aryl(c _n ), as that term is defined above. The term "aralkylamino" includes heteroatom-unsubstituted aralkylamino, heteroatom-substituted aralkylamino, heteroatom-unsubstituted aralkylamino(cn), heteroatom- substituted aralkylamino(cn), heteroaralkylamino, heterocyclic aralkylamino groups, and diaralkylamino groups. The term "heteroatom-unsubstituted aralkylamino(cn)" refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two saturated carbon atoms attached to the nitrogen atom, further having a total of n carbon atoms, wherein at least 6 of the carbon atoms form an aromatic ring structure containing only carbon atoms, 8 or more hydrogen atoms, a total of one nitrogen atom, and no additional heteroatoms. For example, a heteroatom-unsubstituted aralkylamino(c7-cio) has 7 to 10 carbon atoms. The term "heteroatom-unsubstituted aralkylamino(cn)" includes groups, having the structure -NHR, in which R is a heteroatom-unsubstituted aralkyl(c _n ), as that term is defined above. The term "heteroatom-substituted aralkylamino(cn)" refers to a radical, having a single nitrogen atom as the point of attachment, further having at least one or two saturated carbon atoms attached to the nitrogen atom, further having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, at least one additional heteroatom, that is, in addition to the nitrogen atom at the point of attachment, wherein at least one of the carbon atom incorporated into an aromatic ring, further wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted aralkylamino(c7-cio) has 7 to 10 carbon atoms. The term "heteroatom- substituted aralkylamino(cn)" includes groups, having the structure -NHR, in which R is a heteroatom-substituted aralkyl(c _n ), as that term is defined above.

The term "amido" includes straight-chain amido, branched-chain amido, cycloamido, cyclic amido, heteroatom-unsubstituted amido, heteroatom-substituted amido, heteroatom- unsubstituted amido(c _n ), heteroatom-substituted amido(cn), alkylcarbonylamino, arylcarbonylamino, alkoxycarbonylamino, aryloxycarbonylamino, acylamino, alkylaminocarbonylamino, arylaminocarbonylamino, and ureido groups. The term "heteroatom-unsubstituted amido(cn)" refers to a radical, having a single nitrogen atom as the point of attachment, further having a carbonyl group attached via its carbon atom to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 1 or more hydrogen atoms, a total of one oxygen atom, a total of one nitrogen atom, and no additional heteroatoms. For example, a heteroatom-unsubstituted amido(ci-cio) has 1 to 10 carbon atoms. The term "heteroatom-unsubstituted amido(cn)" includes groups, having the structure -NHR, in which R is a heteroatom-unsubstituted acyl(c _n ), as that term is defined above. The group, -NHC(O)CH ₃ , is a non-limiting example of a heteroatom-unsubstituted amido group. The term "heteroatom-substituted amido(cn)" refers to a radical, having a single nitrogen atom as the point of attachment, further having a carbonyl group attached via its carbon atom to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, further having a total of n aromatic or nonaromatic carbon atoms, 0, 1, or more than one hydrogen atom, at least one additional heteroatom in addition to the oxygen of the carbonyl group, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted amido(ci-cio) has 1 to 10 carbon atoms. The term "heteroatom-substituted amido(c _n )" includes groups, having the structure -NHR, in which R is a heteroatom-unsubstituted acyl(cn), as that term is defined above. The group, -NHCO2CH3, is a non-limiting example of a heteroatom-substituted amido group.

As used herein, a "chiral auxiliary" refers to a removable chiral group that is capable of influencing the stereoselectivity of a reaction. Persons of skill in the art are familiar with such compounds, and many are commercially available. The term "pharmaceutically acceptable salts," as used herein, refers to salts of compounds of this invention that are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of a compound of this invention with an inorganic or organic acid, or an organic base, depending on the substituents present on the compounds of the invention.

Non-limiting examples of inorganic acids which may be used to prepare pharmaceutically acceptable salts include: hydrochloric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid and the like. Examples of organic acids which may be used to prepare pharmaceutically acceptable salts include: aliphatic mono- and dicarboxylic acids, such as oxalic acid, carbonic acid, citric acid, succinic acid, phenyl- heteroatom-substituted alkanoic acids, aliphatic and aromatic sulfuric acids and the like. Pharmaceutically acceptable salts prepared from inorganic or organic acids thus include hydrochloride, hydrobromide, nitrate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, hydroiodide, hydro fluoride, acetate, propionate, formate, oxalate, citrate, lactate, p- toluenesulfonate, methanesulfonate, maleate, and the like.

Suitable pharmaceutically acceptable salts may also be formed by reacting the agents of the invention with an organic base such as methylamine, ethylamine, ethanolamine, lysine, ornithine and the like. Pharmaceutically acceptable salts include the salts formed between carboxylate or sulfonate groups found on some of the compounds of this invention and inorganic cations, such as sodium, potassium, ammonium, or calcium, or such organic cations as isopropylammonium, trimethylammonium, tetramethylammonium, and imidazolium.

It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable.

Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, Selection and Use

(2002), which is incorporated herein by reference.

An "isomer" of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the configuration of those atoms in three dimensions differs.

A "stereoisomer" is an isomer in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs. "Enantiomers" are stereoisomers that are mirror images of each other, like left and right hands.

"Diastereomers" are stereoisomers that are not enantiomers.

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

III. Treatment and Prevention of Diabetes and Other Glucose-Associated Conditions

The methods of the present invention have utility for treating and/or preventing glucose-associated conditions, such as diabetes. Non-limiting aspects of the invention include ameliorating disease severity, disease symptoms, and/or the periodicity of recurrence of the disease.

Glucose-associated conditions include, but are not limited to, type 1 diabetes (insulin dependent diabetes mellitus or IDDM), type 2 diabetes (non-insulin dependent diabetes mellitus or NIDDM), maturity onset diabetes of the youg (MODY), gestational diabetes, diabetic complications such as metabolic acidoses (e.g., diabetic ketoacidosis (DKA)), carbohydrate and lipid metabolism abnormalities, glucosuria, micro- and macrovascular disease, polyneuropathy and diabetic retinopathy, diabetic nephropathy, insulin resistance, impaired glucose tolerance (or glucose intolerance), obesity, hyperglycemia (elevated blood glucose concentration), hyperinsulinemia, hyperlipidemia, hyperlipoproteinemia, atherosclerosis and hypertension (high blood pressure) related thereto, and various metabolic syndromes. Metabolic syndromes include digestive tract diseases such as ulceric or inflammatory disease; congenital or acquired digestion and absorption disorder including malabsorption syndrome; disease caused by loss of a mucosal barrier function of the gut; and protein-losing gastroenteropathy. Ulceric diseases include gastric ulcer, duodenal ulcer, small intestinal ulcer, colonic ulcer and rectal ulcer. Inflammatory diseases include esophagitis, gastritis, duodenitis, enteritis, colitis, Crohn's disease, proctitis, gastrointestinal Behcet, radiation enteritis, radiation colitis, radiation proctitis, enteritis and medicamentosa. Malabsorption syndrome includes essential malabsorption syndromes such as disaccharide- decomposing enzyme deficiency, glucose-galactose malabsorption, fructose malabsorption; secondary malabsorption syndrome, short gut syndrome, cul-de-sac syndrome; and indigestible malabsorption syndromes such as syndromes associated with resection of the stomach, e.g., dumping syndrome. Other conditions associated with above-normal blood glucose concentration either in an acute or chronic form are also embraced by the invention. The invention also intends to embrace treatment of conditions which would benefit from β- cell preservation, reduced glucagon levels or increased insulin availability.

Diabetes is generally a disease in which the body is unable to produce insulin or does not adequately utilize the insulin it does produce. Insulin is a hormone that facilitates entry of sugars, starches and the like into cells, thereby allowing their conversion into useable energy for the body. In diabetes, therefore, there is a buildup of glucose in the blood due to the inefficient or nonexistent cellular uptake of sugar, starches and the like. Type 2 diabetes is also characterized by progressive beta-cell failure. Type 2 diabetes is also referred to as adult onset diabetes or non-insulin-dependent diabetes (NIDDM).

A. Treatment and/or Prevention of Diabetes

Type 1 diabetes is known to be caused by the selective autoimmune destruction of pancreatic β-cells, triggered by environmental factors, that leads to a severe state of insulin insufficiency requiring insulin injection therapy and close to constant monitoring to avoid complications. In certain embodiments, this invention improves upon this type of treatment by replenishing functional β-cell mass. In other embodiments, by promoting β-cell survival, the methods of this invention may prolong the 'honeymoon period' in the pathogenesis of type-1 diabetes and/or reduce the severity of the disease. In certain embodiments, if used together with a regime that reduces autoimmune mediated destruction of β-cells (e.g. anti- CD3 monoclonal therapy (Chatenoud and Bluestone, 2007)). Such a strategy could reverse the type-1 diabetes disease process by allowing natural spontaneous β-cell regeneration (Nir et aL, 2007).

One category of subjects to be treated according to the invention are those that demonstrate impaired glucose tolerance (or glucose intolerance), such as but not limited to subjects having or at risk of developing type 2 diabetes. These subjects generally demonstrate an inability to control glucose levels upon eating in comparison to a non-diabetic or non- prediabetic "normal" subject. Subjects at risk of developing type 2 diabetes who demonstrate impaired glucose tolerance are considered to be in a prediabetic state. Glucose tolerance can be measured using glucose challenge tests. There are at least two such tests currently available: the Fasting Plasma Glucose Test (FPG) and the Oral Glucose Tolerance Test (OGTT). In human subjects, a FPG blood glucose level between 100-125 mg/dl of blood is indicative of a prediabetic state and an FPG blood glucose level equal to or greater than 126 mg/dl of blood is indicative of diabetes. OGTT measures blood glucose levels two hours after ingestion of a glucose-rich drink following a fasting period. An OGTT blood glucose level between 140-199 mg/dl is indicative of prediabetes, and a level equal to or greater than 200 mg/dl is indicative of diabetes. The presence of glycosylated hemoglobin at levels equal to or greater than 7.0% is also considered an early indicator of the onset of diabetes.

Risk factors for type 2 diabetes include obesity, family history of diabetes, prior history of gestational diabetes, impaired glucose tolerance (as discussed above), physical inactivity, and race/ethnicity. African Americans, Hispanic/Latino Americans, American Indians, and some Asian Americans and Pacific Islanders are at particularly high risk for type 2 diabetes.

Subjects at risk of developing diabetes also may be overweight to the point of being obese. The state of being overweight or obese is defined in terms of the medically recognized body mass index (BMI). BMI equal to a person's body weight (kg) divided by the square of his or her height in meters (i.e., wt/(ht) ). A subject having a BMI of 25 to 29.9 is considered overweight. A subject having a BMI of 30 or more is considered obese.

Symptoms associated with diabetes include but are not limited to frequent urination, excessive thirst, extreme hunger, unusual weight loss, increased fatigue, irritability and blurred vision. Diabetes is also associated with other conditions, many of which result from a diabetic state. These include acute metabolic complications such as diabetic ketoacidosis and hyperosmolar coma, and late complications such as circulatory abnormalities, retinopathy, nephropathy, neuropathy and foot ulcers. A more detailed description of the foregoing terms can be obtained from a number of sources known in the art (see, e.g., Harrison's Principles of Experimental Medicine). Thus, the methods of the invention also embrace ameliorating or resolving diabetes-associated conditions such as but not including those recited above.

B. Adenine Derivatives

The invention provides novel methods of treating and/or preventing diabetes using adenine derivatives. In the context of the present invention adenine derivatives refers to a class of compounds of the structure: or pharmaceutically acceptable salts, hydrates, solvates, tautomers, or optical isomers thereof, wherein the variables are as defined in the summary section above.

Examples of adenine derivatives that may be used in accordance with the invention are provided throughout this application. For example, in certain embodiments the adenine derivative is 3-methyladenine (3-MA), M)-methyladenine (M5-MA), or 9-methyladenine (9- MA). i. Modes of Action

The effects of methylated adenine analogs on isolated rat islets (see Example 1) are consistent with many of these compounds potentiating glucose-induced insulin secretion (see also FIG. IA). The affect appears to be compound specific and there was no effect of these compounds on basal insulin secretion at 3mM glucose. The results shown in FIG. IB are consistent with glucose-dependence of 250μM 3-MA, M5-MA or 9-MA on potentiating insulin secretion that is equivalent, if not better, than the effect of the GLP-I analog exendin- 4 (5nM).

In rat islet perifusion studies (see Example 1), 3-MA was found to potentiate both the 1 ^st and 2 ^nd phases of glucose-induced insulin secretion, similar to exendin-4. A dose response of 3-MA, M5-MA or 9-MA in isolated rat islet experiments has been conducted and found to be similar. As well as potentiating glucose-induced insulin secretion, adenine derivatives were found to significantly potentiate glucose-induced proinsulin biosynthesis at the translational level in rat islets, without affect on basal proinsulin biosynthesis at 3mM glucose (see Example 1). These derivatives did not affect preproinsulin mRNA expression in rat islet β-cells. The data on (pro)insulin production is similar to that found for GLP-I analogs (Alarcόn et α/., 2006). The results of intraperitoneal glucose tolerance tests (see Example 2) of orally administered adenine derivatives on rats was found to significantly lower the excursion in blood glucose over a subsequent time period without notable hypoglycemia (see FIG. 2). This is consistent with an in vivo effect of adenine derivatives that may occur through potentiation of insulin secretion in an analogous way to GLP-I analogs (Drucker, 2006), and is further supportive of the therapeutic potential of these compounds.

Example 3 shows results related to how adenine derivatives exert their glucose dependent effects on β-cells. Results using real time imaging approaches as described Landa et al. (2005), which is incorporated by reference herein, have found in INS-I cells and rat islet monolayers that 3 -MA rapidly evokes a glucose-dependent increase in cytosolic [CAMP] ₁ without significantly affecting cytosolic [Ca ²⁺ J ₁ levels. As such, like GLP-I, 3-MA may operate via elevation of cytosolic [CAMP] ₁ . The intracellular [CAMP] ₁ can be elevated in β- cells either (i) via GPCR/Gαs coupling to activate adenylyl cyclase to catalyze the conversion of ATP to [CAMP] ₁ and pyrophosphate or (ii) via inhibition of a phosphodiesterase (PDE) that mediates the degradation of [CAMP] ₁ . As presented in Example 3, it was found that inhibition of adenylate cyclase with 2',5'-dideoxyadenosine (DDA) decreased 3-MA mediated potentiation of glucose-induced insulin secretion in rat islets by -70%. In parallel studies, the isoform specific PDE inhibitors, 8-Methoxymethyl-3-isobutyl-l-methylxanthine (8MM- IBMX; for PDEl), milrinone (a PDE3 inhibitor) or rolipram (a PDE4 inhibitor) either had no effect, or, in the case of milrinone, further increased 3-MA mediated potentiation of glucose- induced insulin secretion. These observations suggest that further studies to identify the islet- relevant enzymes responsible for the insulin-secretion effect is warranted. This can be accomplished using an expanded collection of PDE isoform inhibitors. Suramin (lOOμM), a non-selective purine receptor antagonist, or pyridoxal phosphate-6-azaphenyl-2',4'-disulfonic acid (PPADS; 50μM), a more selective inhibitor of P2X, P2Y1 and 4 & 6 purine/pyrimidine receptors, appeared to have no effect on 3-MA mediated potentiation of glucose-induced insulin secretion (data not shown). Also, the effect of 3-MA (250μM) on potentiation of glucose-induced insulin secretion and proinsulin synthesis is additive to the effects of exendin-4 (5nM).

The invention also provides methods of using adenine derivatives for the promotion of β-cell growth and/or survival. Example 4 shows that 3-MA, M)-MA and 9-MA can each promote β-cell growth and/or survival. These results are consistent with 3-MA, M)-MA and 9-MA each augmenting glucose-induced IRS-2 protein expression in isolated rat islets within 6h (FIG. 5). This is preceded by a correlating increase in phosphorylation of the cAMP- activated transcription factor, CREB, which has been previously shown to be involved in GLP-I mediated regulation of IRS-2 expression in β-cells (Jhala et al, 2003). This specific increase in the ratio of anti- versus pro-apoptotic gene expression, together with increased IRS-2 expression, is consistent with 3MA, M)-MA or 9-MA promoting β-cell survival. Increased IRS-2 expression has also been shown to augment growth factor and nutrient induced β-cell replication (Lingohr et al, 2002; Lingohr et al, 2003; Hugl et al, 1998).

Since it is known that not all β-cells are destroyed in type-1 diabetes (Meier et al, 2006; Meier et al, 2005), one aspect of this invention provides a pharmacological means to promote β-cell growth and/or survival. The invention also provides methods for accelerating the regeneration β-cells from endogenous β-cells. In further embodiments, this invention provides for β-cell neogenesis. In some embodiments, this strategy provides methods for increasing the number of β-cells in vitro that are available for transplantation and derived from isolated human islets, and/or enhance protection of surrogate islet β-cells during the transplantation process thereby improving graft survival.

In one aspect, the treatment in accordance with the methods of this invention provides one or more of the following:- (i) an increase in pancreatic insulin levels relative to the levels measured in the absence of an adenine derivative after administration to a subject with symptoms of diabetes. In some embodiments, the compound induces at least about a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% increase in pancreatic insulin levels in a subject, (ii) A reduction or an absence of symptoms of islet inflammation after administration of an adenine derivative to a subject with symptoms of diabetes, (iii) A decrease in blood glucose levels relative to the levels measured in the absence of an adenine derivative in subjects with symptoms of diabetes. In some embodiments, the compounds induce at least about a 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decrease in blood glucose levels. In other embodiments, the compounds yield blood glucose levels about or close to the levels commonly observed in a normal subject, (iv) An improvement in glucose tolerance after administration of an adenine derivative. In particular, at least about a 5-95%, 10-90%, 10- 80%, 10-70%, 10-60%, improvement in glucose tolerance, (v) An increase in C-peptide levels relative to the levels measured in the absence of adenine derivatives in subjects with symptoms of diabetes. In some embodiments, the compounds induce at least about a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% increase in C-peptide levels, (vi) Maintenance of normal blood glucose levels for a prolonged period of time following administration of an adenine derivative, in particular for at least 1 week, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 20 weeks, 24 weeks, 30 weeks, 40 weeks, 52 weeks, or 78 weeks, more particularly, 2 to 4 weeks, 2 to 5 weeks, 3 to 5 weeks, 2 to 6 weeks, 2 to 8 weeks, 2 to 10 weeks, 2 to 12 weeks, 2 to 16 weeks, 2 to 20 weeks, 2 to 24 weeks, 2 weeks to 12 months, or 2 weeks to 18 months, (vii) A reduction, prevention, or slowing of the rate of disease progression in a subject with diabetes, (viii) A reduction or prevention of the development of severe hyperglycemia and ketoacidosis with symptoms of diabetes, (ix) An increase in survival in a subject with symptoms of diabetes, (x) A decrease in requirement for insulin injection/intake by at least 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%; 10- 30%, or 10-20%. ii. Methods of Making Some adenine derivatives may be obtained commercially. Others can be made using the principles and techniques of organic chemistry as applied by a person skilled in the art. Such principles and techniques are taught, for example, in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (March's Advanced Organic Chemistry) by Michael B. Smith and Jerry March (Hardcover - Jan 16, 2007), which is incorporated by reference herein.

Examples of synthetic approaches that can be taken for the synthesis of adenine derivatives include, (i) starting from a substituted pyrimidine nucleus followed by the imidazole ring construction, (ii) starting from the corresponding imidazole ring, then constructing the pyrimidine ring, and (iii) directing substitution of a preformed purine ring. Direct alkylation of the purine ring can be done using different experimental conditions. One example would be the alkylation of chloropurines with an alkyl halide and sodium hydride and dimethylformamide to obtain a position 9 adenine derivative (Raboisson et ah, 2003, which is incorporated herein by reference). Other methods to produce different adenine derivatives have been reported. Some examples can be found in: Fujii et ah, (1979) and Ukena et ah, (1987), both of which are incorporated by reference herein. iii. Administration Methods

Adenine derivatives may be administered, e.g., orally or by injection {e.g. subcutaneous, intravenous, intraperitoneal, intramuscularly, intravenously, intra-arterially, intra-muscularly, etc.) Depending on the route of administration, an adenine derivative may be coated in a material to protect it from the action of acids and other natural conditions which may inactivate it. Pharmaceutically-acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials which are well-known in the art. Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic or prophylactic agents. Adenine derivatives may be orally administered, for example, with an inert diluent or an assimilable edible carrier. Adenine derivatives and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, an adenine derivative may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of an adenine derivative in the compositions and preparations may be varied. The amount of an adenine derivative in such therapeutically useful compositions is such that a suitable dosage will be obtained. The invention also embraces pharmaceutical compositions which are formulated for local administration, such as by implants.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

Sterile injectable solutions can be prepared by incorporating an adenine derivative in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating an adenine derivative into a sterile carrier which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (i.e., an adenine derivative ) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

When used in vivo, the agent(s), such as an adenine derivative, may be formulated as a pharmaceutical composition or preparation. In general, a pharmaceutical composition comprises the agent(s) and a pharmaceutically-acceptable carrier. As used herein, a pharmaceutically-acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the agents of the invention.

Compounds of the invention may also be formulated for local administration, e.g., for topical administration to the skin or mucosa, for topical administration to the eye, for delivery to the lungs by inhalation, or by incorporation into a biocompatible matrix for controlled release to a specified site over an extended period of time {e.g. , as an active ingredient in a drug-eluting cardiac stent). In certain cases significant systemic concentrations may also be achieved by these routes of administration {e.g., via pulmonary or transmucosal delivery).

In another aspect of the invention, pancreatic islet cells are treated ex vivo with a sufficient amount of an adenine derivative to increase the number of precursor pancreatic β- cells in the islets prior to implantation into the diabetic or prediabetic patient. In some embodiments, following expansion ex vivo the population of precursor pancreatic β-cells is differentiated in culture prior to implantation by contacting them with at least an adenine derivative. Methods for expanding a population of pancreatic β-cells in vitro are known in the art (see U.S. 2006/0234373, which is incorporated by reference herein). Upon isolation of the pancreas from a suitable donor, cells are isolated and grown in vitro. The cells which are employed are obtained from tissue samples from mammalian donors including human cadavers, porcine fetuses or another suitable source of pancreatic cells. The donor cells are major histocompatibility matched with the recipient for compatibility whenever human cells are used. Purification of the cells can be accomplished by gradient separation after enzymatic {e.g., collagenase) digestion of the isolated pancreas. The purified cells may be grown in media containing sufficient nutrients and gastrin/CCK receptor ligand and EGF receptor ligand to permit β-cell survival and proliferation , thus allowing formation of insulin secreting pancreatic β-cells. In certain embodiments, following stimulation the insulin secreting pancreatic β-cells may be directly expanded in culture prior to being transplanted into a patient in need thereof, or can be transplanted directly following treatment with an adenine derivative

Methods of transplantation include transplanting insulin secreting pancreatic β-cells obtained into a patient in need thereof are also known in the art. For example, certain such methods are described in U.S. 2006/0234373, which is incorporated by reference herein. Such methods include encapsulating the insulin producing cells in a semi-permeable membrane prior to transplantation. Such membranes permit insulin secretion from the encapsulated cells while protecting the cells from immune attack. The optimum number of cells to be transplanted is estimated to be between 10,000 and 20,000 insulin producing β-cells per kg of the patient. Repeated transplants may be required as necessary to maintain an effective therapeutic number of insulin secreting cells. The transplant recipient can also, according to the invention, be provided with a sufficient amount of an adenine derivative to promote growth, neogenesis, survival, and/or to induce proliferation of the transplanted insulin secreting β-cells. iv. Dosing

Adenine derivatives are administered in effective amounts. Generally, an effective amount may vary with the subject's age, condition, and sex, as well as the extent of the disease in the subject (e.g., whether the subject is diabetic or prediabetic) and can be determined by one of skill in the art. The dosage may be adjusted by the individual physician in the event of any complication.

An effective amount typically will vary from about 0.001 μg/kg to about 1000 μg/kg, from about 0.01 μg/kg to about 750 μg/kg, from about 100 μg/kg to about 500 μg/kg, from about 1.0 μg/kg to about 250 μg/kg, from about 10.0 μg/kg to about 150 μg/kg in one or more dose administrations daily, for one or several days (depending of course of the mode of administration and the factors discussed above). Other suitable dose ranges include 1 μg to

10000 μg per day, 100 μg to 10000 μg per day, 500 μg to 10000 μg per day, and 500 μg to

1000 μg per day. In some particular embodiments, the amount is less than 10,000 μg per day with a range of 750 μg to 9000 μg per day.

The effective amount that is less than 1 mg/kg/day, less than 500 μg/kg/day, less than

250 μg/kg/day, less than 100 μg/kg/day, less than 50 μg/kg/day, less than 25 μg/kg/day or less than 10 μg/kg/day. It may alternatively be in the range of 1 μg/kg/day to 200 μg/kg/day.

In another embodiment, the unit dosage is an amount that reduces blood glucose by at least 40% as compared to an untreated subject. In another embodiment, the unit dosage is an amount that reduces blood glucose to a level that is ± 10% of the blood glucose level of a non-diabetic subject.

Single or multiple doses of the agents are contemplated. Desired time intervals for delivery of multiple doses can be determined by one of ordinary skill in the art employing no more than routine experimentation. As an example, subjects may be administered two doses daily at approximately 12 hour intervals. In some embodiments, the agent is administered once a day. The adenine derivatives may be administered on a routine schedule. As used herein a routine schedule refers to a predetermined designated period of time. The routine schedule may encompass periods of time which are identical or which differ in length, as long as the schedule is predetermined. For instance, the routine schedule may involve administration twice a day, every day, every two days, every three days, every four days, every five days, every six days, a weekly basis, a monthly basis or any set number of days or weeks therebetween. Alternatively, the predetermined routine schedule may involve administration on a twice daily basis for the first week, followed by a daily basis for several months, etc. In other embodiments, the invention provides that the agent(s) may taken orally and that the timing of which is or is not dependent upon food intake. Thus, for example, the agent can be taken every morning and/or every evening, regardless of when the subject has eaten or will eat.

Clinical efficacy of the treatment may be evaluated by administering an escalating dose of single or combinatorial formulations of the invention to a population of patients suffering from hyperglycemia. Subsequently, patients may be evaluated for the following criteria: fasting and postprandial plasma glucose concentration, postprandial glucose excursion, insulin secretion rate, glycated hemoglobin (HbAIc) levels, oral glucose tolerance test (OGTT).

Improvement in one or more of these clinical parameters would constitute one methods for showing the efficacy of the therapy. For example, an escalating dosage study would identify the optimal dosage required per Kg of subject weight balanced against elevation of cardiac and hepatotoxicity biomarkers.

IV. Combination Therapy

In addition to being used as a monotherapy, the adenine derivatives of the present invention may also be used in combination therapies. Such a combination therapy would comprise treating a subject with adenine derivatives and one or more other therapies. Non limiting examples of such other therapies include immunosuppressive therapy, hormonal therapy, or one or more additional drug(s) or agent(s) (e.g., other anti-diabetic drugs). Suitable additional drugs or agents that may be used in accordance with this invention are provided throughout this application. In some embodiments, the nature of the additional drug or agent will depend on which of the glucose-associated conditions the subject has or is at risk of developing. Examples of such additional drugs or agents are given in the summary section above and in U.S. Patents 6,121,282, 6,057,343, 6,048,842, 6,037,359, 6,030,990, 5,990,139, 5,981,510, 5,980,902, 5,955,481, 5,929,055, 5,925,656, 5,925,647, 5,916,555, 5,900,240, 5,885,980, 5,849,989,

5.837.255, 5,830,873, 5,830,434, 5,817,634, 5,783,556, 5,756,513, 5,753,790, 5,747,527, 5,731,292, 5,728,720, 5,708,012, 5,691,386, 5,681,958, 5,677,342, 5,674,900, 5,545,672,

5.532.256, 5,531,991, 5,510,360, 5,480,896, 5,468,762, 5,444,086, 5,424,406, 5,420,146, RE34,878, 5,294,708, 5,268,373, 5,258,382, 5,019,580, 4,968,707, 4,845,231, 4,845,094,

4,816,484, 4,812,471, 4,740,521, 4,716,163, 4,695,634, 4,681,898, 4,622,406, 4,499,279,

4,467,681, 4,448,971, 4,430,337, 4,421,752, 4,419,353, 4,405,625, 4,374,148, 4,336,391,

4,336,379, 4,305,955, 4,262,018, 4,220,650, 4,207,330, 4,195,094, 4,172,835, 4,164,573,

4,163,745, 4,141,898, 4,129,567, 4,093,616, 4,073,910, 4,052,507, 4,044,015, 4,042,583, 4,008,245, 3,992,388, 3,987,172, 3,961,065, 3,954,784, 3,950,518, 3,933,830, the disclosures of which are incorporated herein by reference.

In embodiments involving contacting cells with an adenine derivative and a second agent, the adenine derivative and the second agent may contact the cells at the same time.

Alternatively, contact by an adenine derivative may precede or follow contact by a second agent by intervals ranging from hours (e.g., 1, 2, 3, 4, 5, 6, 8, 12, 15, or 18), days (e.g., 1, 2, 3,

4, 5, 6 or 7) to weeks (e.g., 1, 2, 3, 4, 5, 6, 7, or 8).

Where two therapies are combined, various combinations may be employed. In the non-limiting example below, the adenine derivative therapy is "A" and the secondary therapy is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of the adenine derivatives of the present invention to a patient will follow general protocols for the administration of pharmaceuticals, taking into account the toxicity, if any, of the drug. It is expected that the treatment cycles would be repeated as necessary.

V. Examples The following examples as well as the figures 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 or figures represent techniques discovered by the inventors 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 - Effects of methylated adenine analogs on isolated rat islets.

The results of a preliminary study in isolated rat islets (mean ± SE of > 3 experiments), where 250μM 3-MA, N6-methyl adenine (M5-MA) or 9-methyl adenine (9- MA) potentiated glucose-induced insulin secretion, are shown in FIG. IA. In contrast, 250μM 1 -methyl adenine (1-MA), 7-methyl adenine (7-MA), adenosine, adenine, cytosine, guanine or thymine had no effect on glucose-induced insulin secretion. There was no effect of these compounds on basal insulin secretion at 3mM glucose. FIG. IB shows the glucose- dependence of 250μM 3-MA, M5-MA or 9-MA on potentiating insulin secretion, as compared to the GLP-I analog exendin-4 (5nM). For insulin secretion studies, isolated rat islets were preincubated in Krebs-Ringer bicarbonate buffer, pH 7.4, containing 20 mM hepes and 0.1% bovine serum albumin (KRBH buffer), at basal 2.8 mM glucose, for 1 h at 37 C. Islets were then incubated for 1 h at 37 C in KRBH buffer supplemented as indicated. Insulin secretion was analyzed by radioimmunoassay (RIA) in an aliquot of the incubation medium. Islets were lysed in a 50 mM hepes/ 1% Triton X-100 buffer, pH 8. Islets lysates were also analyzed for insulin content by RIA. Insulin secretion was normalized by islets insulin content, and expressed as the percentage of content.

In rat islet perifusion studies, 3-MA (250μM) was found to potentiate both the 1 ^st and 2 ^nd phases of 15mM glucose-induced insulin secretion, similar to exendin-4 (5nM). A dose response of 3-MA, M)-MA or 9-MA in isolated rat islet experiments has been conducted and found to be similar, with a threshold of potentiating 15mM glucose-induced insulin secretion between 20-30μM that reaches a maximum at >500μM. Near-maximal concentration of 250μM were used for most of the pilot in vitro studies to date. Isolated rat islets were perifused in KRBH buffer for 30 min at basal 2.8 mM glucose, followed by 40 min at stimulatory 16.7 mM glucose +/- MAs and 10 min at 2.8 mM glucose. Perifusion was at 1 ml/min rate and 1-ml fractions were collected. Insulin secretion was analyzed by RIA in the fractions.

As well as potentiating glucose-induced insulin secretion, 3-MA, M)-MA and 9-MA (250μM) were found to potentiate 15mM glucose-induced proinsulin biosynthesis at the translational level in rat islets (2-3 fold), with no affect on basal proinsulin biosynthesis at 3mM glucose. 3-MA did not affect preproinsulin mRNA expression in rat islet β-cells. For proinsulin biosynthesis studies, isolated rat islets were incubated as for the insulin secretion experiment (see above) plus an additional incubation period of 30 min in KRBH buffer at 16.7 mM glucose in the presence of 100 μCi of ³ H-Leucine. Islets were then lysed and radiolabeled pro(insulin) immunoprecipitated with an anti-insulin antibody. Immunoprecipitates were analyzed by polyacrylamide gel electrophoresis and fluorography.

Example 2 - Intraperitoneal glucose tolerance test results in rat studies.

Oral administration of either 3-MA or M)-MA (at a dose of 30-50 μg/g body weight (bw)) 15-20 minutes prior to an intraperitoneal glucose tolerance test (IPGTT; 1 mg glucose/g bw) in overnight fasted normal lean rats, was found to significantly lower the excursion in blood glucose over a subsequent 120 minute period without notable hypoglycemia (see FIG.

2).

In a further study, 3-MA was found to increase glucose-induced insulin secretion and improve glucose tolerance in normal rats. Normal Sprague-Dawley rats, -200 g bw, were fasted overnight, then subjected to an intraperitoneal glucose tolerance test (IPGTT) (Yaekura et al, 2003), using a 1 mg glucose/g bw dose of glucose. These IPGTTs were conducted with 3-MA (30 μg/g bw) or vehicle control given IP, 15 min prior to the glucose dose. Tail blood samples were collected at the indicated time pints and glucose (FIG. 6A) and insulin levels (FIG. 6B) were subsequently measured. The IPGTT procedure used is based on Yaekura et al, 2003, which is incorporated herein by reference.

Example 3 - Studies on glucose dependent effects of 3-MA, 7V6-MA and 9-MA on β-cells.

Results using real time imaging approaches as described in Landa et al (2005), which is incorporated by reference herein, have found in INS-I cells and rat islet monolayers that 3-

MA (250μM) rapidly evokes a glucose-dependent increase in cytosolic [CAMP] ₁ without significantly affecting cytosolic [Ca ²⁺ J ₁ levels. It was found that inhibition of adenylate cyclase with 2',5'-dideoxyadenosine (DDA (100μM)) markedly decreased 3-MA mediated potentiation of glucose-induced insulin secretion in rat islets by -70%. In parallel studies, the isoform specific PDE inhibitors (lOOμM), 8-Methoxymethyl-3-isobutyl-l-methylxanthine

(8MM-IBMX; for PDEl), milrinone (a PDE3 inhibitor) or rolipram (a PDE4 inhibitor) either had no effect, or, in the case of milrinone, further increased 3 -MA mediated potentiation glucose-induced insulin secretion.

Suramin (lOOμM), a non-selective purine receptor antagonist, or pyridoxal phosphate- 6-azaphenyl-2',4'-disulfonic acid (PPADS; 50μM), a more selective inhibitor of P2X, P2Y1 and 4 & 6 purine/pyrimidine receptors, appeared to have no effect on 3 -MA mediated potentiation of glucose-induced insulin secretion. Also, the effect of 3-MA (250μM) on potentiation of glucose-induced insulin secretion and proinsulin synthesis appears additive to the effect on exendin-4 (5nM).

Example 4 - Studies on promotion of β-cell survival.

The ability of 3-MA, M)-MA and 9-MA to promote β-cell growth and survival was tested. M)-MA and 9-MA (250μM) significantly potentiates β-cell proliferation in both INS-I cells and isolated rat islets at glucose concentrations >8mM when assessed by [ ³ H] thymidine incorporation over a 48h period, as described by Lingohr et al. (2002) and Hϋgl et al. (1998), which are both incorporated by reference herein. FIG. 3 shows such an effect at a stimulatory concentration of 1 ImM glucose. No effect of M-MA or 9-MA on β-cell proliferation at basal glucose was observed. Also M-MA, 9-MA and to a lesser extent 3-MA were found to be protective against cytokine-induced apoptosis.

In FIG. 4, a preliminary experiment is shown where isolated rat islets are exposed to a cytokine cocktail of interleukin-lβ (IL- lβ, 10ng/ml), tumor necrosis factor-α (TNF-α, 50ng/ml) and interferon-γ (IFN-γ, 50ng/ml), at a basal 3mM glucose in the presence or absence of 3MA, M-MA or 9-MA (250μM) for 24h. Using activated caspase-3 as an indicator of apoptosis, it can be seen that 3MA, M-MA or 9-MA are quite protective against cytokine-induced apoptosis as indicated by reduced caspase-3 activation. How might 3MA, M-MA or 9-MA promote β-cell growth and survival? FIG 5 shows that 250μM 3MA, and especially M-MA and 9-MA, all augment 15mM glucose-induced IRS-2 protein expression in isolated rat islets within 6h. This is preceded by a correlating increase in phosphorylation of the cAMP-activated transcription factor, CREB, which has been previously shown to be involved in GLP-I mediated regulation of IRS-2 expression in β-cells (Jhala et al, 2003), which is incoporated by reference herein). The 3MA/M-MA/9-MA augmented increase in IRS-2 expression in rat islet β-cells is associated with decreased expression of the pro-apoptotic genes, Bax, Bad and Bak, but not the anti- apoptotic genes Bcl-2 or BCI-X _L (see FIG. 5), which are unchanged relative to a loading control protein, the 85kD subunit of PBK (see FIG. 5). Caspase-3, IRS-2, Bax, Bad, Bak, Bcl-2, BCI-X _L and PI3K(p85) levels and CREB phosphorylation were determined by immunoblotting analysis with specific antibodies for each protein. Isolated rat islets or INS-I cells were preincubated for 1 h at 37 C in KRBH buffer, 2.8 mM glucose and then incubated for 6 h in KRBH buffer at 15 mM glucose in the absence or presence of the MAs. Islets were lysed in 50 mM hepes, pH 8/1% Triton X-IOO lysis buffer. Islet lysates were then analyzed by polyacrylamide gel electrophoresis and immunoblotting.

All of the 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 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.

REFERENCES

The following references, and those listed in the Appendix, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

U.S. Patents 6,121,282; 6,057,343; 6,048,842; 6,037,359; 6,030,990; 5,990,139; 5,981,510;

5,980,902; 5,955,481; 5,929,055; 5,925,656; 5,925,647; 5,916,555; 5,900,240;

5,885,980; 5,849,989; 5,837,255; 5,830,873; 5,830,434; 5,817,634; 5,783,556;

5,756,513; 5,753,790; 5,747,527; 5,731,292; 5,728,720; 5,708,012; 5,691,386; 5,681,958; 5,677,342; 5,674,900; 5,545,672; 5,532,256; 5,531,991; 5,510,360;

5,480,896; 5,468,762; 5,444,086; 5,424,406; 5,420,146; RE34,878; 5,294,708;

5,268,373; 5,258,382; 5,019,580; 4,968,707; 4,845,231; 4,845,094; 4,816,484;

4,812,471; 4,740,521; 4,716,163; 4,695,634; 4,681,898; 4,622,406; 4,499,279;

4,467,681; 4,448,971; 4,430,337; 4,421,752; 4,419,353; 4,405,625; 4,374,148; 4,336,391; 4,336,379; 4,305,955; 4,262,018; 4,220,650; 4,207,330; 4,195,094;

4,172,835; 4,164,573; 4,163,745; 4,141,898; 4,129,567; 4,093,616; 4,073,910;

4,052,507; 4,044,015; 4,042,583; 4,008,245; 3,992,388; 3,987,172; 3,961,065;

3,954,784; 3,950,518; 3,933,830

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