WO2008014299A2

WO2008014299A2 - Use of an alpha2-agonist composition for the treatment of hyperlipidemia

Info

Publication number: WO2008014299A2
Application number: PCT/US2007/074284
Authority: WO
Inventors: Jyotirmoy X. Kusari; Daniel W. Gil; John E. Donello
Original assignee: Allergan Inc
Current assignee: Allergan Inc
Priority date: 2006-07-27
Filing date: 2007-07-25
Publication date: 2008-01-31
Anticipated expiration: 2009-01-27
Also published as: WO2008014299A3

Abstract

Disclosed is the use of an agonist of the alpha2B and/or alρha2C adrenergic receptor subtypes that lacks (a) significant alpha2A adrenergic receptor activity or (b) significant alphalA adrenergic receptor activity, or that lacks both (a) and (b) for treating hyperglycemia or hyperlipidemia.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF HYPERLIPIDEMIA

The present invention is directed to methods of treating hyperlipidemia using an agonist of the α2B and/or α2C adrenergic receptor subtypes that lacks (a) significant α2A adrenergic receptor activity or (b) significant α1A adrenergic receptor activity, or that lacks both (a) and (b).

Diabetes mellitus is a condition involving the presence of abnormally high levels of glucose in the blood (hyperglycemia). A normal range of glucose in the blood is considered between about 70 mg/dl and about 110 mg/dl. Hyperglycemia would comprise a blood glucose level above 110 at a time greater than about 2-3 hours after eating. This condition arises due to reduced or absent production or secretion of insulin (Type 1 or insulin-dependent diabetes), or to a cell's lack of response to the presence of insulin in the extracellular milieu (Type 2 or insulin independent diabetes).

Although hyperlipidemia (such as hypertriglyceremia) is often observed in diabetic patients, existing anti-diabetic agents able to decrease serum glucose may not have a significant effect on the level of triglycerides in the blood. The most common oral hypoglycemic (antidiabetic) agents, for example, are generally described only with regard to their ability to lower blood glucose levels. By contrast, those agents used to lower serum triglycerides, such as statins, may have little or no effect on blood glucose levels. Thus, simply because an agent is reported to be useful for the treatment of diabetes, such as type Il diabetes, does not mean that the agent is also effective at lowering the level of serum triglycerides.

Non-sedating α2 receptor agonist compositions contain agents that have already been characterized in the imidazole, thiourea, imidazoline, and imidazole thione, amino imidazoline, amino oxazoline and amino thiazoline chemical classes. It is to be expected that future non-sedating α2 agents (or combinations of agents) will be found in additional chemical classes including phenethylamine, amino thiazine, benzazepine, quinazoline, guanidine, piperazine, yohimbine alkaloid, and phenoxypropanolamine chemical classes. In particular, it has been found that non-sedating α2 adrenergic agonist compositions have certain biochemical properties in common, regardless of the chemical structure of the agents contained in the compositions. For example, in one embodiment such compounds, in addition to having α2 adrenergic agonist activity, particularly but not necessarily exclusively, α2B and or α2C adrenoreceptor activity, also lack significant α1 adrenoreceptor activity. However, in another embodiment, a therapeutic composition comprising a nonsedating α2 adrenergic agonist may comprise a combination of an α2 adrenergic agonist with an α1 adrenergic antagonist. In each case, the reduced or absent α1 adrenergic activity results in a significant increase in the potency of the α2 adrenergic agonist activity with no significant increase in the potency of the sedative activity. Thus, at therapeutically effective concentrations, the α2 adrenergic agonist has little or no sedative effect, particularly as compared to a composition comprising an α2 adrenergic agonist at a dosage conferring the same therapeutic effect, but lacking significant α1 A inhibitory activity.

Potency, as used here, refers to the concentration of an agonist required to produce a therapeutic effect. Potency is quantified by EC50, the concentration at which half of the maximum therapeutic effect of the agonist is seen. Change in potency, therefore, is quantified by a change in EC50: an increase in potency, for example, results in a decrease in EC50.

Efficacy, as used here, refers to maximum effect of an agonist. Percent efficacy (% E) is determined by comparing the maximum effect of each agonist to the maximum effect of a standard full agonist (phenylephrine for α-1 receptors and bhmonidine for α-2 receptors. By "lacking significant α1A activity" is meant having an α1 A/α2A EC₅₀ ratio greater than that of bhmonidine (for which this ratio is greater than about 25). In preferred embodiments the ratio is at least 20% greater, or at least 40% greater, or at least 50% greater, or at least 70% greater, or at least 80% greater, or at least 100% greater, or at least 200% greater, or at least 500% greater than that of bhmonidine.

In another embodiment, the non-sedating α2 adrenergic agonist may comprise a adrenergic agonist having selective α2B and/or α2C agonist activity, but lacking significant alpha 2A activity. An "α2 agonist lacking significant α2A activity" is an α2 agonist that has less than 40% of the efficacy of bhmonidine at the α2A receptor and has the ability to produce a therapeutic effect without concomitant sedation upon peripheral administration in genetically unaltered animals. It will be understood that such a characterization includes α2B selective agonists lacking significant α2A activity, α2C selective agonists lacking significant α2A activity, and α2B/α2C agonists lacking significant α2A activity. Such agonists have an EC50 of less than 1000 nM at the indicated receptor subtype(s)(α2B, α2C, or α2B and α2C), or at least 100-fold greater activity at the indicated receptor subtype(s) than at the α2A receptor. Preferably, the agonists have an EC50 value of less than 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 10 nM or 1 nM at the indicated receptor subtype(s).

Agonist selectivity can be characterized using any of a variety of routine functional assays, for example, in vitro cell-based assays which measure the response of an agent proximal to receptor activation. Useful assays include, without limitation, in vitro assays such as cyclic AMP assays or GTPγS incorporation assays for analyzing function proximal to α2 receptor activation (Shimizu et al., J. NEUROCHEM. 16:1609-1619 (1969); Jasper et al., BIOCHEM. PHARMACOL. 55: 1035-1043 (1998); and intracellular calcium assays such as FLIPR assays and detection of calcium pulses by Ca⁺⁺-sensitive fluorescent dyes such as fluo-3 for analyzing function proximal to α1 receptor activation (Sullivan et al., METHODS MOL. BIOL. 1 14:125-133 (1999); Kao et al., J. BIOL. CHEM. 264:8179-8184 (1989)). α2A selectivity assays based on inhibition of forskolin- induced cAMP accumulation in PC12 cells stably expressing an α2A receptor, and increases in intracellular calcium in HEK293 cells stably expressing an α2A receptor are known and have been described in, for example, U.S. Patent Application Publication No. 2005/0059664, which is incorporated by reference as part of this disclosure in its entirety. Additional useful assays include, without limitation, inositol phosphate assays such as scintillation proximity assays (Brandish et al., ANAL. BIOCHEM. 313:31 1-318 (2003)); assays for β-arrestin

GPCR sequestration such as bioluminescence resonance energy transfer assays (Bertrand et al., J. RECEPTOR SIGNAL TRANSDUC. RES. 22:533-541 (2002)); and cytosensor microphysiometry assays (Neve et al., J. BIOL. CHEM. 267:25748- 25753 (1992)). These and additional assays for α2 and α1 (for example α1A) receptor function are routine and well known in the art and are hereby incorporated by reference as part of this specification in their entirety.

As a non-limiting example, a GTPγS assay is an assay useful for determining, for instance, the functional selectivity of an agent for activating an α2A receptor as compared to an α1A receptor in the methods of the invention. α2 adrenergic receptors mediate incorporation of guanosine 5'-0-(γ-thio)- triphosphate ([³⁵S]GTPyS) into G-proteins in isolated membranes via receptor- catalyzed exchange of [³⁵S]GTPyS for GDP. An assay based on [³⁵S]GTPyS incorporation can be performed essentially as described in Jasper et al., supra, 1998. Briefly, confluent cells treated with an agent to be tested are harvested from tissue culture plates in phosphate buffered saline before centhfuging at 300 x g for five minutes at 4^°C. The cell pellet is resuspended in cold lysis buffer (5 mM Tris/HCI, 5 mM EDTA, 5 mM EGTA, 0.1 mM PMSF, pH 7.5) using a Polytron Disrupter (setting #6, five seconds), and centhfuged at 34,000 x g for 15 minutes at 4^°C before being resuspended in cold lysis buffer and centhfuged again as above. Following the second wash step, aliquots of the membrane preparation are placed in membrane buffer (50 mM Tris/HCI, 1 mM EDTA, 5 mM MgCI₂, and 0.1 mM PMSF, pH 7.4) and frozen at -70°C until used in the binding assay. GTPγS incorporation is assayed using [³⁵S]GTPyS at a specific activity of

1250 Ci/mmol. Frozen membrane aliquots are thawed and diluted in incubation buffer (50 mM Tris/HCI, 5 mM MgCI₂, 100 mM NaCI, 1 mM EDTA, 1 mM DTT, 1 mM propranolol, 2 μM GDP, pH 7.4) and incubated with radioligand at a final concentration of 0.3 nM at 25°C for 60 minutes. After incubation, samples are filtered through glass fiber filters (Whatman GF/B, pretreated with 0.5% bovine serum albumin) in a 96-well cell harvester and rapidly washed four times with four ml of ice-cold wash buffer (50 mM Tris/HCI, 5 mM MgCI₂, 100 mM NaCI, pH 7.5). After being oven dried, the filters are transferred to scintillation vials containing five ml of Beckman's Ready Protein® scintillation cocktail for counting. The EC50 and maximal effect (efficacy) of the agent to be tested are then determined for the α2A receptor.

Various other methods can be used to assay receptor selectivity. For example, a method for measuring alpha agonist activity and selectivity comprises the RSAT (Receptor Selection and Amplification Technology) assay as reported in Messier et al., High Throughput Assays Of Cloned Adrenergic, Muscarinic, Neurokinin And Neurotrophin Receptors In Living Mammalian Cells, PHARMACOL. TOXICOL. 76:308-1 1 (1995), which has been adapted for use with α1 and α2 receptors. The assay measures a receptor-mediated loss of contact inhibition that results in selective proliferation of receptor-containing cells in a mixed population of confluent cells. The increase in cell number is assessed with an appropriate transfected marker gene such as β-galactosidase, the activity of which can be easily measured in a 96-well format. Receptors that activate the G protein, G_q, elicit this response. Alpha 2 receptors, which normally couple to G,, activate the RSAT response when coexpressed with a hybrid Gq protein that has a Gi receptor recognition domain, called Gq/i5. See Conklin et al., Substitution Of Three Amino Acids Switches Receptor Specificity Of G_qa To That Of G,a, NATURE 363:274-6. (1993). Using assay systems such as these, or other generally known methods, the person of ordinary skill in the art can screen drug libraries such as commercial drug libraries available from companies such as, without limitation, Sigma Aldrich, TimTec, Novascreen and the like to select compounds having α2 agonist activity, but lacking significant sedative activity at therapeutic concentrations of the drug.

Alternatively, known or unknown α2 agonists (such as the α2 pan agonist brimonidine) may be used in a non-sedating α2 agonist therapeutic composition comprising an α1 (preferably an α1 A) antagonist to provide a therapeutic effect, wherein the dosage of the α2 agonist necessary to provide a therapeutic effect is substantially lowered in such composition relative to a second composition comprising only the α2 agonist as the sole active agent. Due to this increase in potency, the amount of sedation and cardiovascular depression experienced by a mammal to whom said agent is administered, either peripherally or non- peripherally, is greatly decreased at a therapeutically effective dose of the 2 agonist.

In specific embodiments of this non-sedating α2 adrenergic agonist composition, the α1 adrenergic receptor antagonist is selected from the group consisting of prazosin, terazosin, doxazonine, urapidil and 5-methylurapadil. The former two compounds and their syntheses are described in U.S. Patents 3,51 1 ,836, and 4,026,894, respectively; the latter compound is an easily synthesized derivative of urapidil, whose synthesis is described in U.S. Patent 3,957,786. These and all other references cited in this patent application are hereby incorporated by reference herein. Additionally, other α1 receptor antagonists (including α1A receptor antagonists) are well known in the art; many such compounds have been clinically approved. See also Lagu, 26 DRUGS OF THE FUTURE 757-765 (2001 ) and Forray et al., 8 EXP. OPIN. INVEST. DRUGS 2073 (1999), hereby incorporated by reference herein, which provide examples of numerous α1 antagonists.

The present invention is based in part on the surprising finding that α2- receptor agonist compositions are useful in treating hyperglycemia and hyperlipidemia and raising blood insulin levels, rather than in maintaining or causing hyperglycemia and hyperlipidemia, as has previously been observed in studies using α2 receptor agonist compounds having sedative activity. This effect is seen using non-sedating α2B selective receptor agonists compositions but is also observed using non-sedating α2 pan-agonist compositions as well. By "pan-agonist" is meant that the agonist is α2 receptor agonist able to stimulate the α2A, α2B and α2C receptor subtypes. By "α2 agonist composition" is meant that the composition comprises an α2 agonist having activity at the α2B and/or α2C adrenergic receptor subtypes, and either a) lacking significant α2A activity, b) lacking significant α1A activity, or both a) and b). In one embodiment the α2 agonist composition may comprise a non-sedating α2 receptor agonist, such as an α2 agonist lacking substantial α1 A activity or an α2 agonist lacking significant α2A activity. In another embodiment the α2 agonist composition may comprise an α2 agonist (either an α2B or 2C selective agonist or an α2 pan-agonist) having activity at the α2B and/or α2C adrenergic receptor subtypes plus comprising an additional component selected from the group consisting of an α1 receptor antagonist (such as an α1 A receptor antagonist) or an alpha 2A receptor antagonist or both. The term "treat" means to deal with medically. It includes, for example, preventing the onset of a disease, alleviating its symptoms, or slowing its progression.

By a "therapeutically effective" amount, concentration, or dosage is meant an amount, concentration or dosage that is capable of treating at least one symptom of the indicated medical condition.

Thus, in one aspect the present invention is drawn to a method for the treatment of a patient having hyperglycemia or hypertriglyceremia and/or elevated levels of blood insulin comprising administering to said patient a therapeutically effective amount of an α2 agonist composition comprising an α2 receptor subtype agonist. In a preferred aspect, the invention comprises administering to a patient a therapeutically effective amount of a non-sedating α2 agonist composition comprising a α2 agonist lacking significant α2A activity.

Compound 1 , illustrated below, is an α2 agonist composition that may be used according to the method of the invention:

In another aspect the alpha 2-receptor agonist composition comprises a non- sedating α2B selective agonist. By "alpha 2B selective agonist" is meant that i) the efficacy relative to a standard full agonist at the α2B receptor subtype is greater than its efficacy relative to a standard full agonist at the α2A or α2C receptor subtypes and that the relative efficacy at the α2A or α2C receptor subtypes is < 0.4; or ii) the potency of the compound at the α2B receptor subtype is at least 10 fold greater than at the α2A or α2C receptor subtypes under the same experimental conditions. In another embodiment of the invention, the non-sedating α2B selective agonist has a chemical structure chosen from:

Compound 2 and

Compound 3. and

Compound 4

It is important to note that compounds 1 , 2, and 4 are of the imidazole-2- thione class of compounds, while compound 3 belongs to the thiourea chemical class; thus the methods and compositions of the present invention are not limited by structure, but apply equally to all alpha 2 non-sedating compounds. Such compounds have now been characterized in the imidazole, thiourea, imidazoline, and imidazole thione chemical classes. Additional chemical classes which comprise non-sedating α2 receptor agonists may include, without limitation, the phenethylamine, amino thiazine, amino imidazoline, benzazepine, amino oxazoline, amino thiazoline, quinazoline, guanidine, piperazine, yohimbine alkaloid, and phenoxypropanolamine chemical classes.

As is well known in the art, sedation is a term that means a reduction in motor activity. The phrase "without concomitant sedation", or "non-sedating" as used herein in reference to a α2 selective or α2 pan agonist, means that, upon administration, the agonist produces less than about 30% sedation at a dose at least 10-fold greater than the dose of selective agonist required to reduce blood glucose in a hyperglycemic mammal by 20% or more. For example, an α2- selective agonist is administered to a mammal at a dose of 2 mg/kg and reduces blood glucose from 250 mg/dl to 200 mg/dl; the α2-selective agonist is "nonsedating" if it produces less than about 30% sedation when administered to the mammal at a dose of at least about 20 mg/kg. The amount of α2 receptor agonist required to reduce blood glucose by 20% or more will generally be a "therapeutically effective dose," although in certain circumstances a lower reduction (e.g., 10%) may be desirable.

Thus as used herein the term "non-sedating" or "without concomitant sedation" does not mean that the indicated compound lacks sedative activity at any dosage; rather it is always an indication of lack of sedation relative to a therapeutically effective dose.

As non-limiting examples, the dose of the non-sedating α2 agonist required to produce about 30% sedation (reduction in motor activity) can be at least 25-fold greater than, 50-fold greater than, 100-fold greater than, 250-fold greater than, 500-fold greater than, 1000-fold greater than, 2500-fold greater than, 5000-fold greater than, or 10, 000-fold greater than less than the dose of the same α2 agonist required to produce a reduction of blood glucose in a hyperglycemic mammal to 1 10 mg/dl or less. Methods for determining the extent of a reduction in blood glucose, as well as the extent of sedation are described herein and further are well known in the art.

In additional embodiments, the present invention may comprise a composition having anti-hyperglycemic activity comprising a non-sedating α2- receptor agonist present at a dosage effective to deliver a therapeutically effective dosage of said agent when administered to a mammal in need thereof.

Generally, methods of administering a drug may include any means sufficient to deliver an effective dose of the agent. Thus, preferred routes of administration for the α2 agonist composition of the invention may be peripheral or non-peripheral and include oral, intravenous, intrathecal and epidural administration. Other possible means of administration of the non α2 agonist composition include, without limitation, by intrathecal pump, subcutaneous pump, dermal patch, intravenous injection, subcutaneous injection, intramuscular injection, and an oral pill, or a combination of such methods. While peripheral means of administration of the non-sedating α2 agonist composition are not currently preferred in the treatment of hyperglycemia or hyperlipidemia, the advantages of the instantly claimed methods may be observed in such cases as well, depending at least in part on the bioavailability of the agent or agents comprised in the α2 agonist composition.

It is understood that the pharmaceutical compositions comprising the α2 agonist composition useful in the present invention optionally (but preferably) includes an excipient such as a pharmaceutically acceptable carrier or a diluent, which is any carrier or diluent that has substantially no long term or permanent detrimental effect when administered to a subject. An excipient generally is mixed with the active compound(s), or permitted to dilute or enclose the active compound(s). A carrier can be a solid, semi-solid, or liquid agent that acts as an excipient or vehicle for the active compound. Examples of solid carriers include, without limitation, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, polyalkylene glycols, talcum, cellulose, glucose, sucrose and magnesium carbonate. Suppository formulations can include, for example, propylene glycol as a carrier. Examples of pharmaceutically acceptable carriers and diluents include, without limitation, water, such as distilled or deionized water; saline; aqueous dextrose, glycerol, ethanol and the like. It is understood that the active ingredients can be soluble or can be delivered as a suspension in the desired carrier or diluent, depending upon the means of administration.

The α2 agonist compositions may also optionally include one or more agents such as, without limitation, emulsifying agents, wetting agents, sweetening or flavoring agents, tonicity adjusters, preservatives, buffers or antioxidants. Tonicity adjustors useful in a pharmaceutical composition include, but are not limited to, salts such as sodium acetate, sodium chloride, potassium chloride, mannitol or glycerin and other pharmaceutically acceptable tonicity adjustors. Preservatives useful in pharmaceutical compositions include, without limitation, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuhc acetate, and phenylmercuric nitrate. Various buffers and means for adjusting pH can be used to prepare a pharmaceutical composition, including, but not limited to, acetate buffers, citrate buffers, phosphate buffers and borate buffers. Similarly, anti-oxidants useful in pharmaceutical compositions are well known in the art and include, for example, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene. It is understood that these and other substances known in the art of pharmacology can be included in a pharmaceutical composition useful in the methods of the invention. See, for example, Remington's Pharmaceutical Sciences Mack Publishing Company, Easton, Pa. 16.sup.th Edition 1980. Furthermore, an α2 agonist composition may be administered in conjunction with one or more other therapeutic substances, in the same or different pharmaceutical composition and by the same or different routes of administration.

The active agents in the α2 agonist composition are administered in an effective amount. Such an effective amount generally is the minimum dose necessary to achieve the desired prevention or reduction in severity of hyperglycemia or hyperlipidemia. Such a dose generally is in the range of 0.1- 1000 mg/day and can be, for example, in the range of 0.1-500 mg/day, 0.5-500 mg/day, 0.5-100 mg/day, 0.5-50 mg/day, 0.5-20 mg/day, 0.5-10 mg/day or 0.5-5 mg/day, with the actual amount to be administered determined by a physician taking into account the relevant circumstances including the severity and type of stress-associated condition, the age and weight of the patient, the patient's general physical condition, and the pharmaceutical formulation and route of administration. Suppositories and extended release formulations also can be useful in the methods of the invention, including, for example, dermal patches, formulations for deposit on or under the skin and formulations for intramuscular injection. A pharmaceutical composition useful in the methods of the invention can be administered to a subject by a variety of means depending, for example, on the type of condition to be treated, the pharmaceutical formulation, and the history, risk factors and symptoms of the subject. Routes of administration suitable for the methods of the invention include both systemic and local administration. As non-limiting examples, a pharmaceutical composition useful in the method of the invention can be administered orally; parenterally; by pump, for example a subcutaneous pump; by dermal patch; by intravenous, intra-articular, subcutaneous or intramuscular injection; by topical drops, creams, gels or ointments; as an implanted or injected extended release formulation; by subcutaneous minipump or other implanted device; by intrathecal pump or injection; or by epidural injection. Depending on the mode of administration, the α2 agonist composition can be incorporated in any pharmaceutically acceptable dosage form such as, without limitation, a tablet, pill, capsule, suppository, powder, liquid, suspension, emulsion, aerosol or the like, and can optionally be packaged in unit dosage form suitable for single administration of precise dosages, or sustained release dosage forms for continuous controlled administration. A method of the invention can be practiced by peripheral administration of the α2 agonist composition. As used herein, the term "peripheral administration" or "administered peripherally" means introducing the α2 agonist composition into a subject outside of the central nervous system. Peripheral administration encompasses any route of administration other than direct administration to the spine or brain.

Peripheral administration can be local or systemic. Local administration results in significantly more of a pharmaceutical composition being delivered to and about the site of local administration than to regions distal to the site of administration. Systemic administration results in delivery of a pharmaceutical composition essentially throughout at least the entire peripheral system of the subject.

Routes of peripheral administration useful in the methods of the invention encompass, without limitation, oral administration, topical administration, intravenous or other injection, and implanted minipumps or other extended release devices or formulations. A pharmaceutical composition useful in the invention can be peripherally administered, for example, orally in any acceptable form such as in a tablet, liquid, capsule, powder, or the like; by intravenous, intraperitoneal, intramuscular, subcutaneous or parenteral injection; by transdermal diffusion or electrophoresis; topically in any acceptable form such as in drops, creams, gels or ointments; and by minipump or other implanted extended release device or formulation.

Each and every published patent, patent application publication and other reference cited in the present application are hereby incorporated by reference as part of this specification.

While this invention has been described with respect to various specific examples and embodiments, it is to be understood that the invention is not limited thereto and that it can be variously practiced within the scope of the following claims.

Brief Description of the Drawings

Figure 1 is a graph showing an increase in body weight of prediabetic female Zucker rats given vehicle or selected non-sedating α2 agonist compositions from initiation (week 7) to end of the study (week 15). At the 8th week, animals were given high fat diet to raise blood glucose.

Figure 2 is a graph showing the effects on blood glucose levels (at weeks 7, 12 and 15) of chronic treatment of prediabetic Zucker rats with either vehicle or non-sedating α2 agonist compositions.

Figure 3 is a graph showing the effects on blood triglyceride levels (at weeks 7, 12 and 15) of chronic treatment of prediabetic Zucker rats with either vehicle or non-sedating α2 agonist compositions.

Figure 4 is a graph showing an increase in body weight of prediabetic female db/db mice given vehicle or selected non-sedating α2 agonist compositions from age = week 6 to the end of the study (week 1 1 ).

Figure 5 is a graph showing the effects on blood glucose levels (at weeks 6, 7, 9 and 1 1 ) of chronic treatment of prediabetic db/db mice with either vehicle or non-sedating α2 agonist compositions. Figure 6 is a graph showing an increase in body weight of prediabetic female Zucker rats given vehicle or selected non-sedating α2 agonist compositions different from those in Example 1 , from initiation (week 8) to end of the study (week 14). At the 9th week, animals were given high fat diet.

Figure 7 is a graph showing the effects on blood glucose levels (at weeks 8, 12 and 14) of chronic treatment of prediabetic Zucker rats with either vehicle or non-sedating α2 agonist compositions different from those in Example 1. Figure 8 is a graph showing the effects on blood triglyceride levels (at weeks 8, 12, and 14) of chronic treatment of prediabetic Zucker rats with either vehicle or non-sedating α2 agonist compositions different from those in Example 1. Figure 9 is a graph showing the effects on blood glucose levels (at weeks

8, 9, 10, 12 and 14) of chronic treatment of diabetic Zucker rats with either vehicle or non-sedating α2 agonist compositions (Compound 1 ).

Figure 10 is a graph showing the effects on blood triglyceride levels (at weeks 8, 10, 12 and 14) of chronic treatment of diabetic Zucker rats with either vehicle or non-sedating α2 agonist compositions (Compound 1 ).

Figure 1 1 A shows a comparison of insulin levels of female Zucker rats given vehicle control versus those given Compound 1 on week 14.

Figure 1 1 B shows a comparison of cholesterol levels of female Zucker rats given vehicle control versus those given Compound 1 on week 14. Figure 1 1 C shows a comparison of low density lipoprotein (LDL) levels of female Zucker rats given vehicle control versus those given Compound 1 on week 14.

Figure 1 1 D shows a comparison of glucose levels of female Zucker rats given vehicle control versus those given Compound 1 on week 14. Figure 1 1 E shows a comparison of high density lipoprotein (HDL) levels of female Zucker rats given vehicle control versus those given Compound 1 on week 14.

Figure 1 1 F shows a comparison of FFA (free fatty acid) levels of female Zucker rats given vehicle control versus those given Compound 1 on week 14. Figure 12A shows a line graph showing a comparison of blood glucose levels of female Zucker diabetic fatty rats following a single injection of either vehicle or Compound 3.

Figure 12B shows a line graph showing a comparison of blood glucose levels of female Zucker diabetic fatty rats following a single injection of either vehicle or Compound 1 . Figure 12C shows a line graph showing a comparison of blood glucose levels of female diabetic Zucker rats following a single injection of either vehicle or Compound 2.

Examples

Example 1 : Chronic Study

Female Zucker rats are animal models for Type Il diabetes, developing hyperglycemia and hyperthglyceremia after 1 to 2 weeks of being placed on the high fat diet.

Female Zucker fatty rats (Charles River Laboratories) between 6-7 weeks old were acclimated to the animal research facilities for at least one week. Animals were housed and maintained on a normal diet during the acclimation period.

After acclimation, the rats were weighed and tail-snip glucose and triglyceride levels were determined using a One Touch Ultra® Blood Glucose Monitoring system (LIFESCAN, Milpitas, CA) and CardioChek® A analyzer (Polymer Technology Systems, Inc, Indianapolis, IN), respectively. The resulting data were used as a baseline for comparison with later treatment results. The animals were randomized to various treatment groups based on blood glucose, triglycerides and body weight.

Vehicle (60% Polyethylene Glycol 300; hereinafter "PEG 300") or the indicated doses of the tested non-sedating α2 agonists (Compound 1 , Compound 2 or Compound 3 in 60% PEG 300) was administered continuously in the experimental rats using an osmotic pump (Alzet Osmotic Pumps, Model 2ML2 (5 μl/hr) Duret Corp., Cupertino, CA), which was inserted subcutaneously on back of the animals. Rats were anesthetized by isoflurane inhalation (using 5% isoflurane for induction and 2-3% isoflurane for maintenance of anesthesia by nose cone). An area of approximately six inches² located on the back of each rat was shaved, rinsed with saline solution, cleaned with antiseptic soap solution and wiped with 70% ethanol. A single 1 inch incision was made perpendicular to the long axis of the animal in the skin covering the lumbar region of the back. Using blunt scissors, a subcutaneous pocket was made toward the head of the animal. A sterile osmotic pump filled with 2 ml of the vehicle or non-sedating α2 agonist composition containing from 0.13 to 6.6 μg/μl of Compound 1 , Compound 2, or Compound 3 was placed into the subcutaneous pocket, and the incision was closed using surgical clips.

Compound 1 was administered at 100 μg or 2.4 mg/kg/day. Compound 2 and Compound 3 were administered at 240 μg or 2.4 mg/kg/day. Non-sedating α2 agonists were administered one week prior to the initiation of a high fat diet to the rats, simulating a "pre-diabetic" condition; this diet was continued until the end of the study.

In the second set of experiments (see Example 4), Compound 1 was first administered 1 to 2 weeks after the introduction of the high fat diet to the animals, which continued until end of the study. After 1 or two weeks on the high fat diet, the female Zucker rats become diabetic, with blood glucose at or above 200 mg/dl in the absence of any added therapeutic agent.

For pre-diabetic animals, body weight, blood glucose and triglycerides of the animals were measured as described above at different times after treatment with agonists, high fat diet or both.

Data were compiled and analyzed using Microsoft Excel. Data are expressed as mean +/- standard error of the mean. Comparisons between groups were made using two-tailed, 2-sample equal variance (homoscedastic) student's t-test. The significance values were set at p<0.05 and p<0.01 as indicated by ^* and ^**, respectively.

For prediabetic animals, Figure 1 shows that the body weight of the Zucker rats fed the high fat diet increased with time over the period of the study (from age 7 weeks to age 15 weeks), and that the administration of the non-sedating α2 agonists and vehicle control had no effect on this increase in body weight. Also as expected, the increase in body weight correlated with an approximately four-fold increase in blood glucose levels (from about 100 mg/dl to about 400 mg/dl by week 12 in rats given vehicle alone, with even higher levels seen at week 15 (Figure 2). Figure 2 also demonstrates that among prediabetic Zucker rats given non-sedating α2 agonist compositions (Compound 2, an α2B selective agonist lacking substantial α2A activity, at 240 μg/kg/day and 2.4 mg/kg/day, and Compound 1 , an α2 pan-agonist lacking substantial α1 activity, at 100 μg/kg/day and 2.4 mg/kg/day, all showed a significant inhibition in the increase in blood glucose relative to the untreated group, with the higher dose of Compound 1 showing the best activity among the groups, lowering blood glucose to about 300 mg/dl at 12 weeks.

A similar trend was seen in triglyceride levels. Figure 3 shows that blood triglycerides increased approximately eight-fold in the untreated rats by 12 weeks. All of the dosages of both non-sedating α2 agonist compositions tested (Compound 1 and Compound 2) significantly prevented in increase in serum triglyceride levels in the prediabetic Zucker rats, with the higher dosage of Compound 1 again showing the best activity among those therapeutic compositions tested.

Example 2: Prediabetic db/db Mice

Female db/db mice are considered to be animal models for Type Il diabetes and hypertriglyeremia. These animals are identifiably obese at around 3 to 4 weeks of age. Elevation in plasma insulin occurs at about 10 to 14 days of age and elevation of blood sugar at about 4-8 weeks. These animals carry the db gene, which contains a G to T point mutation for the leptin receptor.

Five-week-old female db/db mice (Jackson Laboratories) were acclimated to the animal research facilities for one week and housed and maintained on a normal diet until the initiation of the experiment.

For the study of prediabetic db/db mice after acclimation for one week, the mice (6 weeks old) were weighed and tail-snip glucose levels were determined using One Touch Ultra Blood Glucose Monitoring system (LIFESCAN, Milpitas, CA). The animals were randomized into vehicle, clonidine and Compound 2 groups based on blood glucose and body weight. Body weight and blood glucose of db/db mice at week 6 were considered as base-line values. Once the blood glucose levels of the experimental mice mice were above 150 mg/dL in week 6, vehicle (60% Polyethylene Glycol 300, PEG 300), clonidine or Compound 2 in 60% PEG 300 was administered continuously using osmotic pumps (Alzet mini-osmotic pumps Model 2002 (0.5 μl/hr), Duret Corp., Cupertino, CA), which were inserted subcutaneously on back of the animals. The mice were anesthetized by isoflurane inhalation (5% induction and 2-3% maintenance by nose cone). An area of approximately 1 inch by 1 inch located in the back of the mice was shaved, rinsed with saline solution, cleaned with antiseptic soap solution and wiped with 70% ethanol. A single 0.5 inch incision was made perpendicular to the long axis of the animal in the skin covering the lumbar region of the back. Using blunt scissors, a subcutaneous pocket was made toward the head of the animal. The sterile osmotic pump filled with 0.5 ml vehicle, clonidine (0.21-0.29 μg/ul) or Compound 2 (5-7 μg/ul) was placed into the subcutaneous pocket, and the incision was closed with surgical clips. Clonidine and Compound 2 were administered at 100 μg/kg/day and 2.4 mg/kg/day, respectively. At different times after administration of vehicle, clonidine or Compound 2, body weight and blood glucose of the animals were measured as described above. Every two weeks the pumps were replaced with fresh ones and dosing was continued through week 1 1. Data were compiled and analyzed using Microsoft Excel. Data are expressed as mean +/- standard error of the mean. Comparisons between groups were made using two-tailed, 2-sample equal variance (homoscedastic) student's t-test. The significance value was set at p<0.05 as indicated by ^*.

Figure 4 shows that the body weight of the db/db mice increased steadily from week 6 to week 8 in both control and experimental groups, and similar to the results seen for prediabetic Zucker rats, the increase in body weight of prediabetic db/db mice was unaffected by the administration of 100μg/kg/day clonidine or 2.4 mg/kg/day of Compound 2. Clonidine, a sedating α2 agonist and 11 imidizole receptor agonist, is known to have hypoglycemic activity and was used as a reference.

Figure 5 shows that both clonidine and Compound 2 decreased blood glucose in db/db mice beginning at week 9, and that this trend remained until week 1 1 , the end of the study. These results and the Zucker rat results (Example 1 ) indicate that pre- treatment with α2 agonists attenuates the spontaneous increase in blood glucose in pre-diabetic db/db mice, and also in Zucker rats subsequently given a high fat diet. Thus the non-sedating α2 agonist compositions are effective in preventing or lessening the extent of hyperglycemia and hypertriglycermia in prediabetic animals.

Example 3: Prediabetic Zucker Rats and Compound 3

Female Zucker rats were handled essentially as described in Example 1.

Both Compound 1 and Compound 2 belong to the imidazole-2-thione class of compounds. To determine whether the prophylactic antihyperglycemic and antihyperthglyceremic effect of the non-sedating α2 agonist compositions is limited to certain classes of compounds, Compound 3, a benzyl thiourea having α2B selective activity was tested in the same manner as Compound 1 and

Compound 2 at 0.24 mg/kg/day or 2.4 mg/kg/day. Figure 6 shows no effect on the increase in body weight; Figures 7 & 8 show a dose-dependent inhibition in the development of hyperglycemia and hypertriglyceremia, similar to that seen in the cases of Compounds 1 and 2. Thus the prophylactic effect of the non- sedating α2 agonists is unlimited by a particular class of chemical compound.

Example 4: Diabetic Zucker Rats; Compound 1

Female Zucker rats were handled essentially as described in Example 1 with the following modifications. The non-sedating α2 agonist composition

(Compound 1 at 2.4 mg/kg/day) was administered by osmotic pump at week 9, 1 week after initiation of the diabetic phenotype (week 8) by switching the Zucker rats to a high fat diet. At week 9 the Zucker rats showed a blood glucose level of over 250 mg/dl, indicating that the rats are diabetic. Administration of the drug was continued until week 14. Tail-snip glucose and triglycerides were measured at weeks 8, 9, 10, 12 and 14.

At the end of the experiment (after 14 weeks) the rats were fasted overnight, anesthetized with isofluorane and approximately 2 ml of blood was drawn from the orbital sinus using capillary tubes to measure blood glucose, insulin, triglycerides, cholesterol, HDL, LDL and free fatty acids using an automated clinical chemistry analyzer. Blood glucose, insulin, free fatty acids (FFA), cholesterol, HDL and LDL levels were measured at this point. Figure 1 1 shows that Compound 1 significantly inhibited the increase in blood glucose of treated diabetic animals relative to those treated with vehicle alone. The Compound 1 treated group also had a significantly increased level of serum insulin. However, no effect on blood cholesterol, FFA, HDL and LDL was seen. Lean Zucker rats were unaffected by the non-sedating α2 agonist compositions.

Example 5: Female Zucker Rats: Acute Treatment

The following experiment was performed to see whether chronic treatment (such as by osmotic pump) is necessary to observe the anti-diabetic effects of the non-sedating α2 agonist compositions.

Female Zucker fatty rats were fed the high fat diet for 3 to 4 weeks to raise their blood glucose levels above 300 mg/dl. The animals were then fasted overnight. The following morning blood glucose was measured; this baseline point was called 0 hr. Animals were then treated with vehicle (60% PEG 300 in water) or a single injection of a non-sedating α2 agonist composition (Compound 1 , Compound 2 and Compound 3, respectively) in the vehicle (300 μg/kg) using intraperitoneal (IP) injection. Blood glucose was measured at 1 , 3 and 5 hrs after IP injection. Figure 12A shows a time course of changes in blood glucose levels in

Zucker rats following injection of 300 μg/kg Compound 3. Figure 12B shows a time course of changes in blood glucose levels following injection of 300 μg/kg Compound 1 . Figure 12C shows a time course of changes in blood glucose levels following injection of 300 μg/kg Compound 2. In all cases injection of the non-sedating α2 agonist composition resulted in a significant decrease in blood glucose levels relative to the vehicle only control animals. While this invention has been described with respect to various specific examples and embodiments, it is to be understood that the invention is not limited thereto and that it can be variously practiced within the scope of the following claims.

Claims

CLAIMSWhat is claimed is:

1. The use of a therapeutically effective amount of an α2-agonist composition in the manufacture of a medicament for the treatment of hyperlipidemia and hyperthglyceremia.

2. The use of claim 1 , wherein the α2 agonist composition is non-sedating.

3. The use of claim 1 or 2, wherein the α2 agonist composition comprises an α2 agonist lacking significant α1 activity.

4. The use of claim 3, wherein the α2 agonist is an α2 pan-agonist.

5. The use of claim 4, wherein the α2 pan-agonist is the sole active agent in the α2 agonist composition.

6. The use of claim 3, wherein the α2 agonist lacks significant α2A activity.

7. The use of claim 1 or 2, wherein the α2 agonist composition comprises an α2 agonist lacking significant α2A activity.

8. The use of claim 7, wherein the α2 agonist is an α2B selective agonist.

9. The use of claim 8, wherein the α2 agonist comprises Compound 2.

10. The use of claim 8, wherein the α2 agonist comprises Compound 3.

1 1. The use of claim 8, wherein the α2 agonist comprises Compound 4.

12. The use of claim 1 or 2, wherein the α2 agonist composition comprises an α2 pan-agonist.

13. The use of claim 12, wherein the α2 agonist comprises Compound 4.

14. The use of claim 1 or 2, wherein the α2 agonist composition comprises an α1 antagonist.

15. The use of claim 1 or 2, wherein the α2 agonist composition comprises an alpha 2A antagonist.

16. The use of claim 1 or 2, wherein the medicament is formulated for administration by a method selected from the group consisting of injection, oral administration, via pump, and via transdermal patch.