USOO8673964B2

(12) United States Patent (10) Patent No.: US 8,673,964 B2 Lautt (45) Date of Patent: Mar. 18, 2014

(54) USE OF DRUG COMBINATIONS FOR OTHER PUBLICATIONS TREATING INSULIN RESISTANCE Yki-Jarvinen (Cobination Therapies with Insulin in Type 2 diabetes, Deabetes care, vol. 24, No. 4, pp. 758-767).* (75) Inventor: Wilfred Wayne Lautt, Winnipeg (CA) Holz (Glucagol-Like Peptide-1 Synthetic Analogs: New Therapeutic Agents for Use in the Treatment of Diabetes Mellitus, Curr Med (73) Assignee: DiaMedica Inc. (CA) Chem, Nov. 2003, 10(22): pp. 2471-2483).* Nodari (Efficacy and tolerability of the long-term administration of (*) Notice: Subject to any disclaimer, the term of this carvedilol in patients with chronic heart failure with and without patent is extended or adjusted under 35 concomitant diabetes mellitus, The European Journal of Heart Fail ure, 2003, vol. 4, pp. 803-809).* U.S.C. 154(b) by 971 days. Beyer et al. "Assessment of Insulin Needs in Insulin-Dependent Diabetics and Healthy Volunteers under Fasting Conditions' Horm (21) Appl. No.: 11/597,032 Metab Res Suppl 1990; vol. 24 p.71-77. Brownlee “Biochemistry and molecular cell biology of diabetic com (22) PCT Filed: May 20, 2005 plications' Nature vol. 414, Dec. 2001 p. 813-820. Hsu et al. “Five Cysteine-Containing Compounds Delay Diabetic Deterioration in Balb/cA Mice' J. Nutr, 2004 134:3245-3249. (86). PCT No.: PCT/CA2005/000775 Lautt “Hepatic Parasympathetic Neuropathy as cause of Maturity S371 (c)(1), Onset Diabetes?” Gen. Pharmac. vol. 11 p. 343-345. Xie et al. “Induction of insulin resistance by cholinergic blockade (2), (4) Date: May 7, 2008 with atropine in the cat' J. Auton. Pharmacol. (1995) 15, p. 361-369. Lautt et al. “Hepatic parasympathetic (HISS) control of insulin sen (87) PCT Pub. No.: WO2005/112949 sitivity determined by feeding and fasting” American Journal of Physiology—Gastrointestinal and Liver Physiology 281: p. G29 PCT Pub. Date: Dec. 1, 2005 G36 (2001). Lautt “The HISS story overview: a novel hepatic neurohumoral regu (65) Prior Publication Data lation of peripheral insulin sensitivity in health and diabetes' Cana US 2009/O233995 A1 Sep. 17, 2009 dian Journal of Physiology and Pharmacology 77: p. 553-562 (1999). Ling et al. “Hyperglycemia Induced by Glucose Infusion Causes Hepatic Oxidative Stress and Systemic Inflammation, But Not STAT3 or MAP Kinase Activation in Liver in Rats' Metabolism vol. Related U.S. Application Data 52, No. 7 p. 868-874 (2003). (60) Provisional application No. 60/572,486, filed on May Moore et al. “Effect of hepatic denervation on peripheral insulin sensitivity in conscious dogs' American Journal of Physiology— 20, 2004. Endocrinology and Metabolism 282: p. E286-E296, (2002). Lautt “New Paradigm for Insulin Resistance: The HISS Story” Ath (51) Int. Cl. erosclerosis, Hypertension and Diabetes, Kluwer Academic Publish A6 IK3I/385 (2006.01) ers, Boston 2003. p. 263-276. A6 IK3I/27 (2006.01) PorSZaSZ et al. “The sensory nitrergic nature of the hepatic insulin A6 IK3I/98 (2006.01) sensitizing Substance mechanism in conscious rabbits' European A6 IK3I/55 (2006.01) Journal of Pharmacology 443 (2002) p. 211-212. A6 IK3I/4439 (2006.01) Latour et al. "A Rapid Insulin Sensitivity Test (RIST) in the Anes A6 IK3I/7036 (2006.01) thetized Mice” Diabetes 51 p. A422, 1734-P. A6 IK3I/4965 (2006.01) Definition of “cholinergic', www.merriam-Webster.com/dictionmy? cholinergic, 1 page (Nov. 10, 2010). A6IP3/10 (2006.01) Stedman's Medical Dictionary, 26' Edition (Williams & Wilkins, (52) U.S. Cl. 1995), pp. 38 and 461–462. USPC ...... 514/440; 514/479:514/562; 514/635; 514/342; 514/255.06; 514/42 * cited by examiner (58) Field of Classification Search USPC ...... 514/440, 479, 562, 635, 342, 42, Primary Examiner — San-Ming Hui 514/255.06 Assistant Examiner — Kathrien Cruz See application file for complete search history. (57) ABSTRACT (56) References Cited The present invention provides pharmaceutical compositions U.S. PATENT DOCUMENTS comprising: (a) a modulator of hepatic parasympathetic tone, (b) at least one diabetes drug, and (c) a pharmaceutically 5,561,165 A * 10/1996 Lautt et al...... 514,667 acceptable carrier. The present invention includes methods 5,762.922 A * 6/1998 Noble et al. ... 424/854 for the treatment and/or prevention of insulin resistance, type 2003/0235609 A1* 12/2003 Lautt ...... 424/450 2 diabetes, impaired glucose intolerance, and other associated disorders with pharmaceutical compositions described FOREIGN PATENT DOCUMENTS herein. The invention also provides for a kit comprising a CA 2415392 1, 2002 pharmaceutical composition and instructions for its use. CA 2465 632 5, 2003 WO OOf 19992 4/2000 18 Claims, 7 Drawing Sheets U.S. Patent Mar. 18, 2014 Sheet 1 of 7 US 8,673,964 B2

FIGURE

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FIGURE 2

Nr. -N 1. Mc COS; N-Et S. Ms s N n S NY S1 OCH N N N N C M Me Ms c C O Arcohns Karine alvain elitic Fictic (Li25.109 (c.979) RJ A7213 - s CN s COR

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FIGURE 3

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caeecael©© Control BChNAC NACalone Control BENAC NACalone Bethanecholalone Bethanecholalone

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FIGURE 6 U.S. Patent Mar. 18, 2014 Sheet 7 Of 7 US 8,673,964 B2

FIGURE 7

GD Prevention and reversal of GD parasympathetic neuropathy

Ach -- Chase (2) (3) Cholinesterase antagonists (3) Cholinergic agonists N (3) (3) NOS stimulators NOS (5) NO donors p (5) (6) Phosphodiesterase antagonists cGMP--- PDE (i) Glutathione replenishers l? Yao HiSS assay / GSSG administrationdiagnostic HISS US 8,673,964 B2 1. 2 USE OF DRUG COMBINATIONS FOR HDIR is produced physiologically in response to fasting, TREATING INSULIN RESISTANCE these interventions do not produce any further decrease in insulin action. This application is a national stage entry under 35 U.S.C. HDIR is a normal and essential response to fasting. Insulin S371 of PCT/CA2005/000775, filed May 20, 2005, which 5 release occurs even in the fasted State and performs a number claims benefit of priority under 35 U.S.C. S 119(e) of the U.S. of growth regulating functions. Insulin is released in a pulsa Provisional Application No. 60/572,486, filed on May 20, tile manner throughout the day with only approximately 50% 2004, the entire disclosures of which are hereby incorporated of insulin release being regulated by food ingestion (Beyer et by reference. al., 1990). In the fasting state, it would be disadvantageous for 10 insulin to cause a massive shifting of glucose from blood to FIELD OF INVENTION skeletal muscle glycogen stores. The glucose disposal action in response to an injection of insulin decreases progressively The present invention relates to combination drug therapy to insignificance by 24 hours of fasting. This decrease in for the treatment of insulin resistance, and in particular com response to insulin represents a physiologically adjusted bination therapies which modulate hepatic sympathetic and 15 decrease in the HISS-dependent component as demonstrated parasympathetic action. by the observation that the HISS-independent (post-atropine or post-hepatic denervation) component of insulin action is BACKGROUND similar in fed and 24-hour fasted rats. In the immediate postprandial state, approximately 55% of Following a meal, hepatic parasympathetic nerves provide 20 the total glucose disposal effect of a bolus administration of a permissive signal to the liver that regulates the ability of insulin over a wide physiological range (5-100 mu?kg) is insulin to stimulate the release of a hormone, HISS, from the accounted for by HISS. By 18 hours of fasting, Sprague liver. HISS selectively stimulates glucose uptake and storage Dawley rats show HISS-dependent insulin action that as glycogen in skeletal muscle and accounts for over one-half accounts for only 26% of total insulin action (Lautt et al., of the whole body glucose disposal that has previously been 25 2001). The proportion of insulin action accounted for by assumed to be a direct effect of insulin. Hepatic sympathetic HISS action remaining after 18 hours of fasting in cats is 35% nerves block the parasympathetic signal thus preventing the (Xie & Lautt, 1995) and 25% in dogs (Moore et al., 2002). release of HISS and resulting in a 50% reduction in the HISS action in rabbits accounts for approximately 44% of glucose disposal effect of insulin. This condition is referred to insulin action although the time since feeding was not stated as HISS-dependent insulin resistance (HDIR). 30 (Porszasz et al., 2002). Fasting induces a 45% reduction in HISS action can be clinically diagnosed by determining the insulin action in mice (Latour & Chan, 2002). Preliminary response to insulin in the fasted state and following re-feed results indicate that 62% of insulin action in the fed state is ing. The difference in the glucose disposal effect of an injec accounted for by HISS action in humans. This physiological tion of insulin determined in the fed and fasted state repre regulation of HDIR is an appropriate response to fasting and, sents the HISS-dependent component of insulin action. The 35 as such HDIR is a useful physiological state. glucose disposal produced in the fasted State is independent of While HDIR is a useful physiological state in the fasted HISS whereas the approximately doubled effect of insulin condition, failure to release HISS and the resultant HDIR in following a meal is due to both the HISS-dependent and the fed State is suggested to account for the major metabolic HISS-independent component of insulin action with the dif disturbance seen in type 2 diabetes and many other conditions ference between the two states being defined as the HISS- 40 of insulin resistance. According to this model, post-meal dependent component of insulin action. nutrient processing normally results in approximately 80% of HISS-dependent and HISS-independent insulin action can the glucose absorbed from a meal being stored in the large be most readily quantitated using the rapid insulin sensitivity skeletal muscle mass of the body. Although HISS is released test (RIST) which is a transienteuglycemic clamp in response from the liver, it selectively stimulates glucose uptake into to a bolus administration of insulin. Normally insulin injec- 45 glycogen stores in skeletal muscle. Lack of HISS action tion stimulates removal of glucose from the blood into storage results in a greatly impaired glucose disposal effect of insulin sites with a resultant decrease in blood glucose level occur thus resulting in postprandial hyperglycemia. Additional ring. The RIST method uses variable glucose infusion rates to insulin is released in response to the elevated glucose thus maintain the blood glucose level constant. The amount of accounting for postprandial hyperinsulinemia in the type 2 glucose required to be administered in order to maintain the 50 diabetic. Insulin stimulates glucose uptake into adipose tissue glycemic baseline is the index of insulin sensitivity and is and into the limited stores of the liver. When the glycogen referred to as the RIST index. The RIST index produced by stores in the liver are Saturated, the remaining glucose is this procedure consists of a HISS-dependent component and converted to lipid thus accounting for postprandial hyperlipi a HISS-independent component that can be readily differen demia in the type 2 diabetic. The biochemical effects of tiated by testing in the control fed state and then repeating the 55 hyperglycemia including the generation of free radicals has test after blockade of HISS release by any of a number of been Suggested to account for the major non-metabolic means including Surgical denervation of the liver, blockade of pathologies common to diabetics including endothelial cell hepatic muscarinic receptors, blockade of hepatic nitric oxide dysfunction, deposition ofatherosclerotic plaques, blindness, production, or blockade of hepatic cyclooxygenase. Elimi renal failure, nerve damage, stroke, and hindlimb amputation nating HISS action by any of these procedures results in a 60 (Brownlee, 2001). HDIR has been shown to occur in chronic reduction of the RIST index, in the fed state, of approximately liver disease, fetal alcohol exposed adults, obesity, Sucrose 55%. That is, the glucose disposal effect that has been previ fed rats, hypertension, pregnancy and trauma. ously attributed to the direct action of insulin on a variety of The present inventors propose that HDIR is the main cause tissues is actually mediated to a large extent by a hepatic for type 2 diabetes, impaired glucose tolerance, impaired insulin sensitizing process that has previously been unrecog- 65 fasting glucose, hyperinsulinemia, hyperlipidemia, obesity, nized. This area has recently been reviewed (Lautt, 1999; postprandial hyperglycemia and other insulin resistant states. Lautt, 2003). Blockade of HISS release results in HDIR. If For patients suffering from these disorders, the only approved US 8,673,964 B2 3 4 form of treatment currently available is insulin and certain endothelial dysfunction, congestive heart failure, angina, oral medications. The oral drugs fall into five main classes: peripheral arterial disease, chronic renal failure, peripheral Sulfonylureas, biguanides, alpha-glucosidase inhibitors, artery disease and acute renal failure. meglitinide agents and thiazolidinedione agents. In a fifth aspect, the present invention provides a method of These medications only achieve the best results when com treating or preventing a disorder selected from a group con bined with a restricted diet and regular exercise. However, sisting of type II diabetes, insulin resistance, impaired glu even then the treatment is not successful for all patients. cose intolerance, hyperglycemia, hyperlipideamia, hyperin Blood glucose levels drop but many never see a decrease to Sulinaemia, impaired glucose metabolism, obesity, diabetic levels that are within the normal range and oral medications retinopathy, diabetic nephropathy, glomerulosclerosis, Syn are known to spontaneously stop working for unknown rea 10 drome X, hypertension, heart disease, cardiovascular disease, sons. In addition, success rate for individuals who have had stroke, endothelial dysfunction, congestive heart failure, type 2 diabetes for more than 10 years is very low. Oral angina, peripheral arterial disease, chronic renal failure, acute medications are usually successful for the first three years of renal failure and peripheral artery disease, comprising admin treatment, but at this point 50% of people with type 2 diabetes istering a therapeutically effective amount of the pharmaceu need additional therapy. After 9 years, 75% of people need 15 tical composition according to the invention. combination treatment to keep their diabetes under control In a sixth aspect, the present invention provides a method (Turner et al., JAMA 281:2005-2012, 1999) of treating or preventing a disorder selected from a group Combination therapy comprising of two diabetes medica consisting of type II diabetes, insulin resistance, impaired tions is prescribed in some cases when the single therapy glucose intolerance, hyperglycemia, hyperlipideamia, hyper proves to be ineffective. However, the combination of oral insulinaemia, impaired glucose metabolism, obesity, diabetic therapies is limited, and only certain combinations can be retinopathy, diabetic nephropathy, glomerulosclerosis, Syn given simultaneously. Sulfonylureas and meglitinide agents drome X, hypertension, heart disease, cardiovascular disease, can be administered together, but can cause hypoglycemia. stroke, endothelial dysfunction, congestive heart failure, Biguanide agents and thiazolidinedione agents cannot be angina, chronic renal failure, acute renal failure and periph taken withinsulin secreting agents and acarbose, a commonly 25 eral artery disease, comprising administering a therapeuti prescribed alpha glucosidase inhibitor cannot be combined cally effective amount of a modulator of hepatic sympathetic with any otherantidiabetic agent. The most common result of tone and a therapeutically effective amount at least one dia these combinations is hypoglycemia and weight gain. betes drug. Thus, there is a need for more effective and safer combi In a seventh aspect, the present invention provides a nation therapies for the treatment of diabetes and other insulin 30 method of treating or preventing a disorder selected from a resistant states. Also, there is a need for treatments that group consisting of type II diabetes, insulin resistance, address the specific mechanism involved in post-prandial impaired glucose intolerance, hyperglycemia, hyperlipide hyperglycemia, that is, HDIR. amia, hyperinsulinaemia, impaired glucose metabolism, obe sity, diabetic retinopathy, diabetic nephropathy, glomerulo SUMMARY OF THE INVENTION 35 Sclerosis, syndrome X, hypertension, heart disease, cardiovascular disease, stroke, endothelial dysfunction, con In a first aspect, the present invention provides a pharma gestive heart failure, angina, peripheral arterial disease, ceutical composition comprising: (a) a modulator of hepatic chronic renal failure, acute renal failure and peripheral artery parasympathetic tone, (b) at least one diabetes drug, and (c) a disease, comprising administering a therapeutically effective pharmaceutically acceptable carrier. 40 amount of an acetylcholinesterase antagonist and a therapeu In an embodiment of the pharmaceutical composition tically effective amount of a glutathione increasing com according to the invention, the modulator of hepatic parasym pound. pathetic tone is an NO donor. In an eighth aspect, the present invention provides a In a further embodiment of the pharmaceutical composi method of treating or preventing a disorder selected from a tion according to the invention, the modulator of hepatic 45 group consisting of type II diabetes, insulin resistance, parasympathetic tone is a cholinergic agonist and the at least impaired glucose intolerance, hyperglycemia, hyperlipide one diabetes drug is a glutathione increasing compound. amia, hyperinsulinaemia, impaired glucose metabolism, obe In a still further embodiment of the invention, the pharma sity, diabetic retinopathy, diabetic nephropathy, glomerulo ceutical composition according to the invention, the modula Sclerosis, syndrome X, hypertension, heart disease, tor of hepatic parasympathetic tone is abethanecholand theat 50 cardiovascular disease, stroke, endothelial dysfunction, con least one diabetes drug is N-acetylcysteine or C-lipoic acid. gestive heart failure, angina, chronic renal failure, acute renal In a second aspect, the present invention provides a phar failure and peripheral artery disease, comprising administer maceutical composition comprising: (a) an acetylcholinest ing a therapeutically effective amount of an phosphodi erase antagonist, (b) a glutathione increasing compound and esterase antagonist with a glutathione increasing compound. (c) a pharmaceutically acceptable carrier. 55 In a ninth aspect, the present invention provides a kitcom In a third aspect, the present invention provides a pharma prising in combination: the pharmaceutical composition ceutical composition comprising: (a) phosphodiesterase according to any one of claims 1 to 30 and instructions for the antagonist, (b) a glutathione increasing compound and (c) a dosage regimen for administration of said composition to pharmaceutically acceptable carrier. ameliorate the symptoms a disorder selected from a group In a fourth aspect, the present invention provides a use of a 60 consisting of type II diabetes, insulin resistance, impaired pharmaceutical composition according to the invention to glucose intolerance, hyperglycemia, hyperlipideamia, hyper treat or prevent a disorder selected from a group consisting of insulinaemia, impaired glucose metabolism, obesity, diabetic type II diabetes, insulin resistance, impaired glucose intoler retinopathy, diabetic nephropathy, glomerulosclerosis, Syn ance, hyperglycemia, hyperlipideamia, hyperinsulinaemia, drome X, hypertension, heart disease, cardiovascular disease, impaired glucose metabolism, obesity, diabetic retinopathy, 65 stroke, endothelial dysfunction, congestive heart failure, diabetic nephropathy, glomerulosclerosis, syndrome X, angina, chronic renal failure, peripheral artery disease and hypertension heart disease, cardiovascular disease, stroke, acute renal failure. US 8,673,964 B2 5 6 In an embodiment of the invention, the modulator of response to feeding results in the release of acetylcholine hepatic parasympathetic tone is a cholinergic agonist selected which activates muscarinic receptors in the liver. This activa from a group consisting of bethanechol, acetylcholine, done tion leads to increased production of nitric oxide which pezil, tacrine, edrophonium, demecarium, pyridostigmine, Zanapezil, phospholine, metrifonate, neostigmine, stimulates guanyl cyclase activity, resulting in increased lev phenserine and galathamine.a cholinergicagonist, methacho els of cyclic guanosine monophosphate which acts in stimu line, BIBN 99, DIBD, SCH-57790, SCH-217443, SCH lating the release of HISS. Feeding also results in elevated 72788, arecoline, an arecoline analogue, Xanomeline, hepatic glutathione levels. Interruption of any component of alvameline, milameline, RU 47213, sabcomeline, this system can result in reduction or abolishment of the PD-151832, CDD-0034-C, CDD-0102, a spriopiperidine, a parasympathetic response to feeding. Accordingly, insulin spiroqulnuclidine, muscarine, cis-dioxblane, RS86, AF-30, 10 resistance and related disorders may be the result of not only ocvimeline, AF150(S), AF267B, SDZ 210-086, YM-796, a abnormal parasympathetic activity but also abnormal sympa rigid analogue of acetylcholine, acclidine, tasaclidine, thetic activity. Thus, the invention provides pharmaceutical oxotremorine, an oxotremorine analogue, pilocarpine, a pilo compositions and uses thereof for relieving insulin resistance carpine analogue, thiopilocarpine, and a nitrosylated deriva tive thereof. 15 and related disorders and diseases, which correct both hepatic In an embodiment of the invention, the modulator of sympathetic and parasympathetic function. hepatic parasympathetic tone is an NO donor selected from a In some instances, the parasympathetic function in group consisting of an organic nitrate, an organic nitrite, a response to feeding is impaired due to decreased acetylcho metal-NO complex, a N-nitrosamine, a N-hydroxy nitro line production or release. In other instances, the parasympa samine, a nitrosothiol, a C-nitro compound, a diazetine diox thetic function is impaired due to decreased nitric oxide pro ide, a furoxan, a benzofuroxan, a oXatriazole-6-imine, a duction. The inventors have previously disclosed the use of Sydonoimine, an oximine, a hydroxylamine, a N-hydrox cholinergic agonists (see for example, U.S. Pat. No. 5,561, yguanidine, a hydroxyurea, a nitrosylated derivative thereof, 165), the use of nitric oxide donors (see for example, WO a pharmaceutical salt thereof, and a mixture thereof. 00/19992) for the treatment of insulin resistance and diabetes. In an embodiment of the invention, the at least one diabetes 25 The inventors have now discovered that the administration drug is selected from a group consisting of a glutathione ofan agent that acts through the hepatic mechanism, as shown increasing compound, an antioxidant, an insulin oran insulin analogue, an O-adrenergic receptor antagonist, a B-adrener in the flow diagram of FIG. 7, is synergistic with existing gic receptor antagonist, a non-selective adrenergic receptor treatments for insulin resistance, diabetes, and HDIR. Since antagonist, a Sulphonylurea, a biguanide agent, a benzoic acid now we have added. For example, combination therapy com derivative, a C-glucosidase inhibitor, a thiazolidinedione, a 30 prising the co-administration of a cholinergic agonist with phosphodiesterase inhibitor, a cholinesterase antagonist, a one or more diabetes drugs or the co-administration of a nitric GLP-1 analogue and a pharmaceutical salt thereof. oxide donor with one or more diabetes drugs, or both with a In an embodiment of the invention, the acetylcholinest diabetes drug is significantly more effective as compared to erase inhibitor is is selected from a group consisting: of conventional combination therapies comprising two or more phenserine, donepezil, galanthamine, rivastigme, tacrine, 35 oral diabetes drug. The inventors have also discovered that physostigmine, neostigmine, edrophonium, pyridostigmine, combination therapy comprising a cholinergic agonist or a demecarium, phospholine, metrifonate, Zanapezil, nitric oxide donor and a diabetes drug exhibits significantly ambenonium and combinations thereof. less adverse side effects to conventional combination thera In an embodiment of the invention, the phosphodiesterase pies. The inventors have discovered that combination therapy inhibitor is selected from a group consisting of anagrelide, 40 comprising a cholinergic agonist or a nitric oxide donor and a tadalafil, dipyridamole, dyphylline, Vardenafil, ciloStaZol. diabetes drug is associated with fewer and less severe inci milrinone, theophylline, Sildenafil, caffeine and combina dences of hypoglycemia, edema, weight gain, and liver dam tions thereof. age. Furthermore, the inventors discovered that lower dos ages of the diabetes drug, as compared to conventional BRIEF DESCRIPTION OF THE FIGURES 45 combination therapies, are required to provide glycemic con trol FIG. 1 illustrates the chemical structure for various cholin In particular, the inventors have discovered that the co ergic agonists. administration of a cholinergic agonist and a gluthathione FIG. 2 illustrates the chemical structure for various are increasing agent results in Synergistic improvements in insu oline-analogue cholinergic agonists. 50 lin sensitivity. Glutathione (GSH) is a ubiquitous tripeptide FIG. 3 illustrates the chemical structure for various spri that is involved in a variety of vital cellular processes includ opiperidines and spiroquinuclidines having cholinergic activ ing detoxification and quenching free radicals. There are a ity. number of agents that are available to increase glutathione FIG. 4 illustrates the chemical structure for various rigid including glutathione itself (or its esters), C-lipoic acid and analogues of acetylcholine having cholinergic activity. 55 the cysteine precursor and rate-limiting agent of GSH bio FIG. 5 illustrates the chemical structure for various synthesis, N-acetylcysteine or oxathiazolidine-4-carboxylic oxotremorine and pilocarpine cholinergic agonists. acid. The inventors have discovered that treatment with the FIG. 6 is a graph comparing the effect of bethanechol and cholinergicagonist, Such as bethanechol, in combination with N-acetylcysteine combination therapy on meal induced insu either N-acetylcysteine or C-lipoic acid or oxathiazolidine-4- lin sensitization in control diet and Sucrose diet rats. 60 carboxylic acid results in Synergistic improvements in insulin FIG. 7 illustrates the mechanistic pathway of the parasym sensitivity as compared to treatment with either a cholinergic pathetic signalling in the liver. agonist or a glutathione increasing agent alone. In addition, the combination therapy was associated with decreased inci DETAILED DESCRIPTION dence of gastrointestinal side effects (i.e. nausea, upset stom 65 ach, Vomiting) which are generally associated with treatment While the present invention is not limited to a particular with either bethanechol, N-acetylcysteine or O-lipoic acid model or mechanism of action, it appears the parasympathetic alone. US 8,673,964 B2 7 8 The present inventors have discovered that activation of the yguanidine, a hydroxyurea, a nitrosylated derivative thereof, parasympathetic pathway through administration of the cho a pharmaceutical Salt thereof, and a mixture thereof. linergic agonist bethanechol, in combination with a glu Any suitable nitric oxide donor or a pharmaceutical accept tathione replenishing agent such as N-acetylcysteine, is espe able salt thereof may be used may be used to practice the cially useful for the treatment of several secondary invention. Examples of suitable nitric oxide donors, include complications of obesity and diabetes, and particularly but are not limited to: an organic nitrate, an organic nitrite, a inflammation (Hsu et al., 2004). Systemic inflammation is metal-NO complex, a N-nitrosamine, a N-hydroxy nitro highly correlated with morbid obesity and diabetes, and may samine, a nitrosothiol, a C-nitro compound, a diazetine diox be responsible for many of the associated pathologies of these ide, a furoxan, a benzofuroxan, a oXatriazole-5-imine, a 10 Sydonoimine, an oximine, a hydroxylamine, a N-hydrox diseases. Indeed, even transient episodes of hyperglycemia, yguanidine, or a hydroxyurea. Such as might occur post-prandially can cause hepatic oxida As used herein, the term "diabetes drug” refers to any tive stress and release of inflammatory cytokines Such as composition known in the art to be useful in the treatment or TNF-a and IL-1 (Ling et al., 2004, Hsu et al., 2004). The prevention of insulin resistance and diabetes. Examples of inventors have determined that bethanechol in combination 15 diabetes drugs which may be used to practice the invention, with N-acetylcysteine is effective in providing relief of these include but are not limited to: complications, elevating hepatic GSH and improving the (a) an antioxidant such as vitamin E. Vitamin C, an isofla HISS response in patients. vone, Zinc, Selenium, ebselen, a carotenoid; The present inventors are the first to identify a vital rela (b) an insulin or insulin analogue Such as regular insulin, tionship between parasympathetic response and GSH status lente insulin, semilente insulin, ultralente insulin, NPH in controlling insulin response. or insulin lispro (Humalog(R). The present invention provides novel pharmaceutical com (c) an O-adrenergic receptor antagonist Such as prazosin, positions comprising (a) a modulator of hepatic parasympa doxazocin, phenoxybenzamine, teraZosin, phentola thetic tone, (b) at least one diabetes drug, and (c) a pharma mine, rauwolscine, yohimine, tolaZoline, tamsulosin, or ceutically acceptable carrier. As used herein a “modulator of 25 teraZosin; hepatic parasympathetic tone' is any compound which posi (d) a B-adrenergic receptor antagonist Such as acebutolol. tively affects the hepatic parasympathetic function. The atenolol, betaxolol, bisoprolol, carteolol, esmolol, meto modulator of hepatic parasympathetic tone may modulate prolol, nadolol, penbutolol, pindolol, propanolol, any of the components involved in the hepatic parasympa timolol, dobutamine hydrochloride, alprenolol, bunolol, thetic pathway (see FIG.7). The modulators may be generally 30 bupranolol, carazolol, epanolol, moloprolol, Oxpre be split into two classes: (1) those compounds which modu nolol, pamatolol, talinolol, tiprenolol, tolamolol, or late cholinergic receptors, for example cholinergic agonists toliprolol; and acetylcholinesterase antagonists, and (2) those com (e) a non-selective adrenergic receptor antagonist Such as pounds which modulate signaling components downstream carvedilol or labetolol; of cholinergic receptors, for example NOS stimulators, NO 35 (f) a first generation Sulphonylurea Such as tolaZamide, donors, and phosphodiesterase antagonists. tolbutamide, chlorpropamide, or acetohexamide. In a preferred embodiment of the invention, a novel phar (g) a second generation Sulphonylurea Such as glyburide, maceutical composition comprises: (a) a nitric oxide donor, glipizide, and glimepiride; (b) at least diabetes drug, and (c) a pharmaceutical acceptably (h) a biguanide agent Such as is metformin; carrier. 40 (i) a benzoic acid derivative such as replaglinide; As used herein, any pharmaceutical compound or compo (i) a C-glucosidase inhibitor Such as acarbose and miglitol; sition is considered “pharmaceutically acceptable' If: (a) at (k) a thiazolidinedione such as rosiglitaZone, pioglitaZone, the dose and method of administration to the patient, it is not or troglitaZone; acutely toxic, and does not result in chronic toxicity dispro (1) a phosphodiesterase inhibitor Such as anagrelide, tadal portionate to the therapeutic benefit derived from treatment, 45 fil, dipyridamole, dyphylline, Vardenafil, cilostaZol, mil and (b) the dose and method of administration to the patient rinone, theophylline, or caffeine; reduces insulin resistance in the patient. (m) a cholineresterase antagonist Such as donepezil, As used herein, the phrase, “modulator of hepatic parasym tacrine, edrophonium, demecarium, pyridostigmine, pathetic tone' means a compound selected from the group Zanapezil, phospholine, metrifonate, neostigmine, or consisting of a cholinergic agonist, an NO donor, an NOS 50 galathamine; and stimulators, bethanechol, acetylcholine, donepezil, tacrine, (n) a glutathione increasing compound Such as N-acetyl edrophonium, demecarium, pyridostigmine, Zanapezil, phos cysteine, a cysteine ester, L-2-oxothiazolidine-4-car pholine, metrifonate, neostigmine, phenserine and boxolate (OTC), gamma glutamylcysteine and its ethyl galathamine, a cholinergic agonist, methacholine, BIBN 99. ester, gly tathtione ethyl ester, glutathione isopropyl DIBD, SCH-57790, SCH-217443, SCH-72788, arecoline, an 55 ester, lipoic acid, cysteine, methionine, or S-adenosyl arecoline analogue, Xanomeline, alvameline, milameline, RU methionine. 47213, sabcomeline, PD-151832, CDD-0034-C, CDD-0102, (o) GLP and glucagon like peptide analogues, such as a Spriopiperidine, a spiroquinuclidine, muscarine, cis-diox exanitide, DAC:GLP-1 (CJC-1131), Liraglutide, ZP10, olane, RS86, AF-30, ocvimeline, AF150(S), AF267B, SDZ BIM51077, LY315902, LY307161 (SR). 210-086, YM-796, a rigid analogue of acetylcholine, acclid 60 In one embodiment of the invention, the pharmaceutical ine, tasaclidine, oxotremorine, an oxotremorine analogue, composition comprises 3-morpholinosyndnonimine (SIN-1) pilocarpine, a pilocarpine analogue, thiopilocarpine, and a as the nitric oxide donor and a Sulphonylurea as the diabetes nitrosylated derivative thereof, an organic nitrate, an organic drug. In a preferred embodiment of the invention, the sulpho nitrite, a metal-NO complex, a N-nitrosamine, a N-hydroxy nylurea is glipizide. nitrosamine, a nitrosothiol, a C-nitro compound, a diazetine 65 In another embodiment of the invention, the pharmaceuti dioxide, a furoxan, a benzofuroxan, a oXatriazole-6-imine, a cal composition comprises 3-morpholinosyndnonimine Sydonoimine, an oximine, a hydroxylamine, a N-hydrox (SIN-1) as the nitric oxide donor and a C-glucosidase inhibi US 8,673,964 B2 9 10 tor as the diabetes drug. In a preferred embodiment of the In another embodiment of the invention, the pharmaceuti invention, the C-glucosidase inhibitor is acarbose. cal composition comprises bethanechol as the cholinergic In a further embodiment of the invention, the pharmaceu agonist and a C-glucosidase inhibitor as the diabetes drug. In tical composition comprises 3-morpholinosyndnonimine a preferred embodiment of the invention, the C-glucosidase (SIN-1) as the nitric oxide donor and a biguanide agent as the 5 inhibitor is acarbose. diabetes drug. In a preferred embodiment of the invention, the In a further embodiment of the invention, the pharmaceu biguanide inhibitor is metformin. tical composition comprises bethanechol as the cholinergic In a still further embodiment of the invention, the pharma agonist and a biguanide agent as the diabetes drug. In a ceutical composition comprises 3-morpholinosyndnonimine preferred embodiment of the invention, the biguanide inhibi (SIN-1) as the nitric oxide donor and a thiazolidinediones as 10 the diabetes drug. In a preferred embodiment of the invention, tor is metformin. the thiazolidinedione is pioglitaZone. In a still further embodiment of the invention, the pharma The pharmaceutical composition comprises 3-morpholi ceutical composition comprises bethanechol as the cholin nosyndnonimine (SIN-1) as the nitric oxide donorand a bigu ergic agonistandathiazolidinedlones as the diabetes drug. In anide agent as the diabetes drug. In a preferred embodiment 15 a preferred embodiment of the invention, the thiazolidinedi of the invention, the biguanide inhibitor is metformin. ones is pioglitaZone. In yet a further embodiment of the invention, the pharma In yet a further embodiment of the invention, the pharma ceutical composition comprises 3-morpholinosyndnonimine ceutical composition comprises bethanechol as the cholin (SIN-1) as the nitric oxide donor and a benzoic acid derivative ergic agonist and a benzoic acid derivative as the diabetes as the diabetes drug. In a preferred embodiment of the inven- 20 drug. In a preferred embodiment of the invention, the benzoic tion, the benzoic acid derivative is replaglinide. acid derivative is replaglinide. In yet a further embodiment of the invention, the pharma In another embodiment of the invention, the pharmaceuti ceutical composition comprises 3-morpholinosyndnonimine cal composition comprises bethanechol as the cholinergic (SIN-1) as the nitric oxide donor and a glutathione replen agonist and a GLP-1 analogue as the diabetes drug. In a isher as the diabetes drug. In a preferred embodiment of the 25 preferred embodiment of the invention, the GLP-1 is invention, the benzoic acid derivative is N-Acetyl Cysteine. exanatide. In another embodiment of the invention, the pharmaceuti In another embodiment of the invention, the pharmaceuti cal composition comprises: (a) a cholinergic agonist, (b) at cal composition comprises bethanechol as the cholinergic least one diabetes drug, and (c) a pharmaceutically acceptable agonistandan C.-adrenergic receptor antagonist as the diabe carrier is provided. 30 tes drug. In a preferred embodiment of the invention, the Any Suitable cholinergic agonist may be employed to prac C.-adrenergic receptor antagonist is prazosin. tice the invention. Examples of suitable cholinergic agonists, In another embodiment of the invention, the pharmaceuti include, but are not limited to: acetylcholine, methacholine, cal composition comprises bethanechol as the cholinergic bethanechol, BIBN 99 (FIG. 1), DIBD (FIG. 1), SCH-57790 agonistandan B-adrenergic receptor antagonist as the diabe (FIG. 1), SCH-217443 (FIG. 1), SCH-72788 (FIG. 1), 35 tes drug. In a preferred embodiment of the invention, the arecoline (FIG. 2), an arecoline analogue (FIG. 2), Xanome B-adrenergic receptor antagonist is atenolol. line (FIG. 2), alvameline (FIG. 2), milameline (FIG. 2), RU In another embodiment of the invention, the pharmaceuti 47213 (FIG. 2), sabcomeline (FIG. 2), PD-151832 (FIG. 2), cal composition comprises bethanechol as the cholinergic CDD-0034-C(FIG.2), CDD-0102 (FIG.2), a spiropiperidine agonist and a non-selective adrenergic receptor antagonist as (FIG. 3), a spiroquinuclidine (FIG. 3), muscarine (FIG. 3), 40 the diabetes drug. In a preferred embodiment of the invention, cis-dioxolane (FIG. 3), RS86 (FIG. 3), AF-30 (FIG. 3), the non-selective adrenergic receptor antagonist is carvedilol. ocvimeline (FIG. 3), AF150(S) (FIG. 3), AF267B (FIG. 3), In a still further embodiment of the invention, the pharma SDZ 210-086 (FIG. 3), YM-796 (FIG. 3), a rigid analogue of ceutical composition comprises a cholinergic agonist, a nitric acetylcholine (FIG. 4), acclidine (FIG. 4), tasaclidine (FIG. oxide donor and a diabetes drug such as glipizide, acarbose, 4), oxotremorine (FIG. 5), an oxotremorine analogue (FIG. 45 metformin, pioglitaZone, or repaglinide. 5), pilocarpine (FIG. 5), a pilocarpine analogue (FIG. 5), or In a second aspect, the invention provides a pharmaceutical thiopilocarpine (FIG. 5). A nitrosylated form of any these composition comprising (a) an acetylcholinesterase antago compounds can also be employed. nist, (b) a glutathione increasing compound and (c) a phar Preferably, the cholinergic agonist is a muscarnic receptor maceutically acceptable carrier. Examples of acetylcholinest agonist and more preferably the cholinergic agonist is acetyl- 50 erase inhibitors which may be used to prepare the choline and most preferably, the cholinergic agonist is pharmaceutical composition include, but are not limited to: bethanechol. phenserine, donepezil, galanthamine, rivastigme, tacrine, The diabetes drug used with the cholinergic agonist can be physostigmine, neostigmine, edrophonium, pyridostigmine, any of the diabetes drugs discussed above. demecarium, phospholine, metrifonate, Zanapezil, In one embodiment of the invention, pharmaceutical com- 55 ambenonium and combinations thereof. The gluthathione position comprises bethanechol as the cholinergic agonist increasing compound may be any of the compounds and a glutathione increasing compound as the diabetes drug. described above. In an preferred embodiment, the In preferred embodiment, the pharmaceutical composition gluthathione increasing compound is N-acetylcysteine. comprises bethanechol and N-acetylcystelne as the diabetes In a third aspect, the invention provides a pharmaceutical drug. In another preferred embodiment, the pharmaceutical 60 composition comprising (a) a phosphodiesterase antagonist, composition comprises bethanechol and C-lipoic acid as the (b) a glutathione increasing compound and (c) a pharmaceu diabetes drug. tically acceptable carrier. Examples of acetylcholinesterase In another embodiment of the invention, the pharmaceuti inhibitors which may be used to prepare the pharmaceutical cal composition comprises bethanechol as the cholinergic composition include, but are not limited to: anagrelide, tad agonist and a Sulphonylurea as the diabetes drug. In a pre- 65 alafil, dipyridamole, dyphylline, Vardenafil. cilostaZol, mil ferred embodiment of the invention, the sulphonylurea is rinone, theophylline, Sildenafil, caffeine and combinations glipizide. thereof. The gluthathione increasing compound may be any US 8,673,964 B2 11 12 of the compounds described above. In an preferred embodi skin permeability, as well as non-chemical modes such as the ment, the gluthathione increasing compound is N-acetylcys use of iontophoresis, electroporation or ultrasound. teine. The pharmaceutical compositions of the present invention The pharmaceutical compositions of the present invention may also include various other components which provide may be manufactured in a manner that is itself known, e.g., by additional therapeutic benefit, act to affect the therapeutic means of conventional mixing, dissolving, granulating, dra action of the pharmaceutical composition, or act towards gee-making, levigating, emulsifying, encapsulating, entrap preventing any potential side effects which may be posed as a ping or lyophilizing processes. result of administration of the pharmaceutical composition. Pharmaceutical compositions for use in accordance with Exemplary pharmaceutically acceptable components or the present invention thus may be formulated in conventional 10 adjuncts which are employed in relevant circumstances manner using one or more physiologically acceptable carriers include antioxidants, free radical scavenging agents, pep comprising excipients and auxiliaries which facilitate pro tides, growth factors, antibiotics, bacteriostatic agents, cessing of the active compounds into preparations which can immunosuppressives, anticoagulants, buffering agents, anti be used pharmaceutically. Proper formulation is dependent inflammatory agents, anti-pyretics, time release binders, upon the route of administration chosen. 15 anaesthetics, steroids, vitamins, and minerals. Administration of compositions of the invention may be The pharmaceutical compositions according to the inven made by a variety of Suitable routes including oral, topical tion can be used to treat or prevent insulin resistance and (including transdermal, buccal or Sublingual), nasal, inhala diabetes. The pharmaceutical compositions can also be used tion, and parenteral (including intraperitoneal, Subcutaneous, to treat or prevent other disorders related to insulin resistance intravenous, intradermal or intramuscular injection) with oral Such as impaired glucose intolerance, hyperglycemia, hyper or parenteral being generally preferred. It also will be appre lipideamia, hyperinsulinaemia, impaired glucose metabo ciated that the preferred method of administration and dosage lism, obesity, diabetic retinopathy, diabetic nephropathy, amount may vary with, for example, the condition and age of glomerulosclerosis, syndrome X, hypertension, heart dis the recipient. ease, cardiovascular disease, stroke, endothelial dysfunction, For injection, the agents of the invention may be formu 25 congestive heart failure, angina, chronic renal failure, acute lated in aqueous solutions, preferably in physiologically renal failure and peripheral artery disease. compatible buffers such as Hanks solution, Ringer's solu The precise dose for any of the pharmaceutical composi tion, or physiological saline buffer. tions of the present invention will depend on a number of For oral administration, the compounds can be formulated factors which will be apparent to those skilled in the art and in readily by combining the active compounds with pharmaceu 30 light of the disclosure herein. In particular these factors tically acceptable carriers well known in the art. Such carriers include: the identity of the compounds to be administered, the enable the compounds of the invention to be formulated as formulation, the route of administration employed, the tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, patient’s gender, age, and weight, and the severity of the Suspensions and the like, for oral ingestion by a patient to be condition being treated. Methods for determining dosage and treated. Pharmaceutical preparations for oral use can be 35 toxicity are well known in the art with studies generally obtained by Solid excipient, optionally grinding a resulting beginning in animals and then in humans if no significant mixture, and processing the mixture of granules, after adding animal toxicity is observed. The appropriateness of the dos suitable auxiliaries, if desired, to obtain tablets or dragee age can be assessed by monitoring insulin resistance using the cores. Suitable excipients are, in particular, fillers such as RIST protocol as set out in Lautt et al., 1998. Where the dose Sugars including lactose, Sucrose, mannitol, or Sorbitol, or 40 provided does not cause insulin resistance to decline to nor cellulose preparations such as; maize starch, wheat starch, mal or tolerable levels, following at least three days of treat rice starch, potato starch, gelatin, gum tragacanth, methyl ment, the dose can be increased. The patient should be moni cellulose, hydroxypropylmethyl-cellulose, Sodium car tored for signs of adverse drug reactions and toxicity, boxymethylcellulose, and/or polyvinylpyrrolidone. If especially with regard to liver and cardiovascular function. desired, disintegrating agents may be added. Such as the 45 Forpharmaceutical compositions comprising a nitric oxide cross-linked polyvinylpyrrolidone, agar, or alginic acid or a donor, the daily dosage of the nitric oxide donor will between salt thereof Such as sodium alginate. 0.01 mg/kg and 100 mg/kg depending on the particular nitric For administration by inhalation or aerosol, the composi oxide donor used. Where the nitric oxide donor is SIN-1, the tions can be prepared according to techniques well known in daily dose will between 0.01 mg/kg and 40 mg/kg and pref the art of pharmaceutical formulation. The compositions can 50 erably 0.15 mg/kg. The daily dosage of the diabetes drug will be prepared as Solutions in Saline, using benzyl alcohol or depend on the particular drug used. Where the drug is gliz other Suitable preservatives and absorption promoters to pide, the daily dosage will between 0.1 mg/kg and 10 mg/kg, enhance bioavailability, fluorocarbons or other solubilizing and more preferably between 1 mg/kg and 5 mg/kg. Where or dispersing agents known in the art. The compositions of the the diabetes drug is acarbose, the daily dosage will be present invention are conveniently delivered in the form of an 55 between 1 and 100 mg/kg, and preferably 10 mg/kg and 40 aerosol spray presentation from pressurized packs or a nebu mg/kg. Where the diabetes drug is metformin, the daily dos lizer, with the use of a suitable propellant, e.g., dichlorodif age will be between 10 and 1000 mg/kg, and preferably 50 luoromethane, trichlorofluoromethane, dichlorotetrafluoro and 200 mg/kg. Where the diabetes drug is pioglitazone, the ethane, carbon dioxide or other Suitable gas. In the case of a daily dosage will be between 0.1 and 10 mg/kg, and prefer pressurized aerosol the dosage unit may be determined by 60 ably between 0.5 mg/kg and 5 mg/kg. Where the diabetes providing a valve to deliver a metered amount. Capsules and drug is repaglinide, the daily dosage will be between 0.1 and cartridges of e.g. gelatin for use in an inhaler or insufflator 10 mg/kg, and preferably between 0.5 mg/kg and 5 mg/kg. may beformulated containing a powder mix of the compound Forpharmaceutical compositions comprising a cholinergic and a suitable powder base Such as lactose or starch. agonist, the daily dosage of the cholinergic agonist will Transdermal delivery systems may be passive transdermal 65 depend on the particular cholinergic agonist used. Where the delivery systems, or may include the use of skin penetration cholinergic agonist is bethanechol, a single dosage will be enhancing agents, or “permeation enhancers, to increase between 0.01 mg and 100 mg and preferably between 0.1 mg US 8,673,964 B2 13 14 and 10 mg. Where the cholinergic agonist is bethanechol, a through a cannula in the jugular vein, Supplemented with a single dosage will be between 0.01 mg and 10 mg and pref 0.65 mg (0.01 ml) bolus injection when required. The rats are erably between 0.1 and 10 mg. The amount of the diabetes placed on a temperature-controlled Surgical table (Harvard drug will be the similar to the amounts discussed above. Apparatus, Kent, England) and rectal temperature is moni Where the diabetes drug is N-acetylcysteine, a single dosage tored and held at 37-37.5° C. Spontaneous respiration is will be between 100 mg and 5 g, and preferably between 500 allowed through a tracheal tube. mg and 1 g The pharmaceutical composition may be admin An arterial-venous shunt is established, as previous istered to have it peak when blood glucose is high, Such as described (Lautt WWetal. CanJ Physiol Pharmacol. 1998: after a meal. So as to allow glucose uptake at that time. The 76 (12): 1080-6), for monitoring mean arterial blood pressure combination of drugs may be formulated into the same pill 10 (MAP), blood glucose leveland for drug delivery. Briefly, two containing the cholinergic agonist and/or the nitric oxide catheters (polyethylene tubing PE60), one inserted into the donor and the diabetes drug. Alternatively, a kit may be used right femoral artery and the other into the right femoral vein, comprising of multiple pills with the appropriate dose of are connected to the two openings of a three way vascular diabetic drug and/or the cholinergic agonist and nitric oxide circuit consisting of a T tube connected with silicon tubing. donor, such as, but not limited to, a “blister pack', including 15 The third opening of the circuit is connected to a pressure instructions or directions printed on or associated with the transducer for the recording of the shunt pressure which, packaging. when the silicon tubing toward the venous side of the circuit The pharmaceutical compositions of the present invention is closed by clamping, represented the systemic arterial blood can be targeted to the liver of the patient thereby eliminating pressure. Blood samples are taken from the arterial side of the deleterious systemic effects. The pharmaceutical composi shunt for the glucose measurement. Flowing blood within the tions can be conjugated to bile salts or albumin for preferen shunt assures the real time measurement of the blood glucose tial delivery to the liver. Alternatively, the pharmaceutical concentration, which is essential for the euglycemic clamp compositions can be encapsulated within liposomes which test as mentioned below. An infusion line is inserted into the are preferentially targeted to the liver. The pharmaceutical venous side of the loop for drug delivery. Another infusion compositions of the present invention can be administered 25 line connected to the jugular vein is established for glucose either in active form or as precursor which is metabolized by infusion. Animals are heparinized (100 IUkg-1) to prevent to the active form by enzymes in the liver. Where the phar clotting in the vascular loop. maceutical composition is targeted to the liver, the dosage In addition to the standard RIST Surgical preparation, a may be reduced. laparotomy with portal venous puncture (21 gx/4 inch) and Although the present invention has been described with 30 an IG puncture (22 gauge, 1 inchiv catheter inserted into the reference to illustrative embodiments, it is to be understood stomach and glued into place) are performed. that the invention is not limited to these precise embodiments, A control RIST is performed as previously described and that various changes and modifications may be effected (Lautt WW et al. Can J. Physiol Pharmacol. 1998: 76 (12): therein by one skilled in the art. All Such changes and modi 1080-6). Briefly, following completion of surgery, animals fications are intention to be encompassed in the appended 35 are allowed a 30-min stabilization period. The baseline glu claims. cose levels are then determined by samples taken at 5-min intervals and continued until three successive stable determi EXAMPLES nations were made. The mean of these three data points is regarded as the baseline for the RIST. To perform the RIST, Example 1 40 human insulin (50 ml J-kg-1 in 0.5 ml saline) is infused into the femoral vein at the rate of 0.1 milmin-1 for 5-min. After Effect of Bethanechol and N-Acetylcysteine on 1-min of insulin infusion, the first test glucose sample is Insulin Sensitivity in Rats with Insulin Resistance determined and a variable glucose infusion (10%) is initiated. Produced by High Sucrose Feeding Blood samples are taken every 2-minand the glucose infusion 45 rate is adjusted accordingly to maintain euglycemia. The Animal Groups—Male Sprague Dawley rats having a RIST index is the amount of glucose (mgkg-1) infused, to starting weight of >220.0 g (Charles River, St. Constant, maintain euglycemia, over the test period that terminated Quebec, Canada) were fed a normal rat chow diet and regular when no further glucose infusion was required (approxi tap water, with and without the exposure to a 35% liquid mately 30 min). At the end of a RIST, the animal is at its Sucrose solution for a 2 week period. Six groups of animals 50 pretest glycemic level. were established under the following treatments: (1) control Depending on the test group, the bethanechol or an equiva diet, saline treatment (n=6); (2) control diet, bethanechol (0.5 lent volume of saline is administered ipv (0.5 ml bolus, rate of ug/kg ipv) and N-acetyl-L-cysteine (200 mg/kg) treatment 0.05 ml/min plus 0.03 ml for catheter dead-space volume). (n=8); (3) control diet, bethanechol (0.5ug/kg ipv) (n=5); (4) Depending on the test group, the N-acetylcysteine or an control diet, N-acetyl-L-cysteine (200 mg/kg) (n=4); (5) 55 equivalent volume of saline is then administered iv (1.0 ml Sucrose diet, saline treatment (n=4); (6) Sucrose diet, bolus, rate of 0.1 ml/min). The animals were allowed to rest bethanechol (0.5 g/kg ipv) and N-acetyl-L-cysteine (200 for 60 minutes from the initiation of the drug administration. mg/kg) treatment (n=5); (7) sucrose diet, bethanechol (0.5 The animals were administered a mixed liquid meal (10 ug/kg ipv) (n-6); and (8) Sucrose diet, N-acetyl-L-cysteine ml/kg) by intragastric infusion (1.0 ml/min, add 0.1 ml test (200 mg/kg) (n=3). 60 meal to account for catheter dead-space Volume). Blood glu Methodology—All animals are 24 hour fasted. The ani cose samples were taken every 5 minutes for a minimum of 90 mals are prepared using the standard RIST Surgical prepara minutes to profile the glycemic response to the test meal. tion. The rats are anaesthetized with an intraperitoneal injec Once a stable glycemia has been achieved, a second RIST is tion of sodium pentobarbital (65 mg kg-1: Somnotol, MTC performed. A liver sample is taken for glutathione determi Pharmaceuticals, Ont). Maintained anesthesia is achieved 65 nation. throughout the experiment by a continuous infusion of pen Results—As shown in Table 1 and in FIG. 6, rats treated tobarbital sodium (0.5 mg ml-1 saline given at 50 ulmin-1) with bethanechol and N-acetylcystelne showed the greatest US 8,673,964 B2 15 16 potentiation in RIST as compared to the control rats. The combination therapy is more effective than either combination therapy was more effective than either bethanechol or pioglitaZone alone for restoring insulin sensi bethanechol or N-acetylcystelne alone for restoring insulin tivity in sucrose fed rats. sensitivity in Sucrose fed rats. Example 6 TABLE 1. Effect of Bethanechol (Bch) and N-acetylcysteine (NAC) combination therapy on meal induced insulin Effect of Bethanechol and Repaglinide on Insulin sensitization in Sucrose fed rats. All values refer to Sensitivity in Rats with Insulin Resistance Produced the percent potentation of RIST as compared to control. 10 by High Sucrose Feeding Control Diet Sucrose Diet The experiment is completed as described in Example 1 Bch BCH NAC Bcf BCH NAC with N-acetylcysteine being replaced with repaglinide. The Control NAC alone alone Control NAC alone alone combination therapy is more effective than either 15 52.17 28.221 9.06 33.5 1.84 62.99 45.59 3 bethanechol or repaglinide alone for restoring insulin sensi 30.48 30.13 15.19 27.39 -10 45.9 13.22 - 13.85 tivity in sucrose fed rats. 63.48 36.18 35.62 57.25 1.89 69.76 -1322 -2.63 66.99 45.13 33.87 1.89 -10.68 45.25 1422 51.291 51.07 60.33 53.66 7.71 Example 7 45.6 109.OS 22.36 140.19 56.98 Effect of Neostigmine and N-Acetylcysteine on Insulin Sensitivity in Rats with Insulin Resistance Produced by High Sucrose Feeding Example 2 25 The experiment is completed as described in Example 1 Effect of Bethanechol and Glipizide on Insulin with benthanecol being replaced with neostigmine. The com Sensitivity in Rats with Insulin Resistance Produced bination therapy is more effective than either neostigmine or by High Sucrose Feeding N-acetylcysteine alone for restoring insulin sensitivity in Sucrose fed rats. The experiment is completed as described in Example 1 30 with the N-acetylcysteine being replaced with glipizide. The Example 8 combination therapy is more effective than either bethanechol orglipizide alone for restoring insulin sensitivity in Sucrose fed rats. Effect of SIN-1 and Glipizide on Insulin Sensitivity 35 in Rats with Insulin Resistance Produced by High Example 3 Sucrose Feeding Effect of Bethanechtol and Acarbose on Insulin The experiment is completed as described in Example 1 Sensitivity in Rats with Insulin Resistance Produced with bethanechol being replaced with SIN-1 and N-acetyl by High Sucrose Feeding 40 cysteine being replaced with glipizide. The combination therapy is more effective than either SIN-1 or glipizide alone The experiment is completed as described in Example 1 for restoring insulin sensitivity in Sucrose fed rats. with the N-acetylcystelne being replaced with acarbose. The combination therapy is more effective than either bethanechol oracarbose alone for restoring insulin sensitivity 45 Example 9 in Sucrose fed rats. Effect of SIN-1 and Acarbose on Insulin Sensitivity Example 4 in Rats with Insulin Resistance Produced by High Sucrose Feeding Effect of Bethanechol and Metformin on Insulin 50 Sensitivity in Rats with Insulin Resistance Produced by High Sucrose Feeding The experiment is completed as described in Example 1 with bethanechol being replaced with SIN-1 and N-acetyl The experiment is completed as described in Example 1 cysteine being replaced with acarbose. The combination with N-acetylcysteine being replaced with metformin. The 55 therapy is more effective than either SIN-1 or acarbose alone combination therapy is more effective than either for restoring insulin sensitivity in Sucrose fed rats. bethanechol or metformin alone for restoring insulin sensi tivity in sucrose fed rats. Example 10

Example 5 60 Effect of SIN-1 and Metformin on Insulin Sensitivity Effect of Bethanechol and Pioglitazone on Insulin in Rats with Insulin Resistance Produced by High Sensitivity in Rats with Insulin Resistance Produced Sucrose Feeding by High Sucrose Feeding 65 The experiment is completed as described in Example 1 The experiment is completed as described in Example 1 with bethanechol being replaced with SIN-1 and N-acetyl with N-acetylcysteine being replaced with pioglitazone. The cysteine being replaced with metformin. The combination US 8,673,964 B2 17 18 therapy was more effective than either SIN-1 or metformin index shows a greater reversal of insulin resistance than when alone for restoring insulin sensitivity in Sucrose fed rats. each of bethanechol and SIN-1 was given alone. Example 15 Example 11 Effect of SIN-1, Bethanechol and 5 Mg/Kg of Effect of SIN-1 and Pioglitazone on Insulin Glipizide on Insulin Sensitivity in Rats with Insulin Sensitivity in Rats with Insulin Resistance Produced Resistance Produced by Hepatic Denervation by High Sucrose Feeding An animal is prepared as in Example 1. A control RIST is The experiment is completed as described in Example 1 10 done. The response is within normal range. A RIST is then with bethanechol being replaced with SIN-1 and N-acetyl done after Surgical denervation. Bethanechol is administered cysteine being replaced with pioglitaZone. The combination by infusion into the portal vein at a rate of 2.5 mg/kg/min therapy is more effective than either SIN-1 or pioglitazone beginning 10 minutes prior to the insulin administration and alone for restoring insulin sensitivity in Sucrose fed rats. continuing for the entire test period. The resulting RIST index 15 shows reversal of the insulin resistance. On a second animal, a control RIST is done. The response Example 12 is within the normal range. A RIST is then done after surgical denervation and the RIST index shows significant insulin Effect of SIN-1 and Repaglinide on Insulin resistance. SIN-1 is administered by infusion into the portal Sensitivity in Rats with Insulin Resistance Produced vein at a rate of 10.0 mg/kg/min for a period of 2 minutes. The by High Sucrose Feeding resulting RIST index shows reversal of the insulin resistance. On a third animal a control RIST is done. The response is The experiment is completed as described in Example 1 within the normal range. A RIST is then done after surgical with bethanechol being replaced with SIN-1 and N-acetyl denervation and the RIST index shows significant insulin cysteine being replaced with repaglinide. The combination resistance. First bethanechol is administered as described therapy is more effective than either SIN-1 or repaglinide 25 above. Five minutes prior to the end of the test period SIN-1 alone for restoring insulin sensitivity in Sucrose fed rats. is administered by infusion into the portal veinata rate of 10.0 mg/kg/min for a period of 2 minutes. Thirty seconds prior to Example 13 the end of the test period a bolus injection of 5 mg/kg of glipizide is given. A RIST is taken and the result is greater Effect of Zaprinast and N-Acetylcysteine on Insulin 30 insulin resistance than bethanechol or SIN-1 alone. Sensitivity in Rats with Insulin Resistance Produced Example 16 by High Sucrose Feeding Effect of SIN-1, Bethanechol and 2.5 Mg/Kg of The experiment is completed as described in Example 1 Glipizide on Insulin Sensitivity in Rats with Insulin with bethanechol being replaced with Zaprinast. The combi 35 Resistance Produced by Hepatic Denervation nation therapy is more effective than either Zaprinastorn-ace tylcysteine alone for restoring insulin resistance in Sucrose An animal is prepared as in Example 1. A control RIST is fed rats, done. The response is within normal range. A RIST is then done after Surgical denervation. Bethanechol is administered Example 14 40 by infusion into the portal vein at a rate of 2.5 mg/kg/min beginning 10 minutes prior to the insulin administration and Effect of SIN-1 and Bethanechol on Insulin continuing for the entire test period. The resulting RIST index Sensitivity in Rats with Insulin Resistance Produced shows reversal of the insulin resistance. by Hepatic Denervation On a second animal, a control RIST is done. The response 45 is within the normal range. A RIST is then done after surgical An animal is prepared as in Example 1. A control RIST is denervation and the RIST index shows significant insulin done. The response is within normal range. A RIST is then resistance. SIN-1 is administered by infusion into the portal done after Surgical denervation of the hepatic nerves reaching vein at a rate of 10.0 mg/kg/min for a period of 2 minutes. The the liver along the hepatic artery and the index shows insulin resulting RIST index shows reversal of the insulin resistance. resistance. Bethanechol is administered by infusion into the 50 On a third animal a control RIST is done. The response is portal vein at a rate of 2.5 mg/kg/min beginning 10 minutes within the normal range. A RIST is then done after surgical prior to the insulin administration and continuing for the denervation and the RIST index shows significant insulin entire test period. The resulting RIST index shows reversal of resistance. First bethanechol is administered as described the insulin resistance. above. Five minutes prior to the end of the test period SIN-1 On a second animal, a control RIST is done. The response 55 is administered by infusion into the portal veinata rate of 10.0 is within the normal range. A RIST is then done after surgical mg/kg/min for a period of 2 minutes. Thirty seconds prior to denervation and the RIST index shows significant insulin the end of the test period a bolus injection of 2.5 mg/kg of resistance. SIN-1 is administered by infusion into the portal glipizide is given. A RIST is taken and the result is greater vein at a rate of 10.0 mg/kg/min for a period of 2 minutes. The insulin resistance than bethanechol or SIN-1 alone. resulting RIST index shows reversal of the insulin resistance. 60 Example 17 On a third animal a control RIST is done. The response is within the normal range. A RIST is then done after surgical Effect of SIN-1, Bethanechol and 2.5 Mg/Kg of denervation and the RIST index shows significant insulin Glipizide on Insulin Sensitivity in Rats with Insulin resistance. First bethanechol is administered as described Resistance Produced by Hepatic Denervation above. Five minutes prior to the end of the test period SIN-1 65 is administered by infusion into the portal veinata rate of 10.0 An animal is prepared as in Example 1. A control RIST is mg/kg/min for a period of 2 minutes. The resulting RIST done. The response is within normal range. A RIST is then US 8,673,964 B2 19 20 done after Surgical denervation. Bethanechol is administered On a second animal, a control RIST is done. The response by infusion into the portal vein at a rate of 2.5 mg/kg/min is within the normal range. A RIST is then done after surgical beginning 10 minutes prior to the insulin administration and denervation and the RIST index shows significant insulin continuing for the entire test period. The resulting RIST index resistance. SIN-1 is administered by infusion into the portal shows reversal of the insulin resistance. vein at a rate of 10.0 mg/kg/min for a period of 2 minutes. The On a second animal, a control RIST is done. The response resulting RIST index shows reversal of the insulin resistance. is within the normal range. A RIST is then done after surgical On a third animal a control RIST is done. The response is denervation and the RIST index shows significant insulin within the normal range. A RIST is then done after surgical resistance. SIN-1 is administered by infusion into the portal denervation and the RIST index shows significant insulin vein at a rate of 10.0 mg/kg/min for a period of 2 minutes. The 10 resistance. First bethanechol is administered as described resulting RIST index shows reversal of the insulin resistance. above. Five minutes prior to the end of the test period SIN-1 On a third animal a control RIST is done. The response is is administered by infusion into the portal veinata rate of 10.0 within the normal range. A RIST is then done after surgical mg/kg/min for a period of 2 minutes. Thirty seconds prior to denervation and the RIST index shows significant insulin the end of the test period abolus injection of 200 mg/kg, 100 resistance. First bethanechol is administered as described 15 mg/kg or 50 mg/kg of metformin is given. ARIST is taken and above. Five minutes prior to the end of the test period SIN-1 the result is greater insulin resistance than bethanechol or is administered by infusion into the portal veinata rate of 10.0 SIN-1 alone. The lower doses of metforminareas effective as mg/kg/min for a period of 2 minutes. Thirty seconds prior to the higher doses. the end of the test period a bolus injection of 1.25 mg/kg of glipizide is given. A RIST is taken and the result is greater Example 20 insulin resistance than bethanechol or SIN-1 alone. Effect of SIN-1, Bethanechol and Pioglitazone on Example 18 Insulin Sensitivity in Rats with Insulin Resistance Produced by Hepatic Denervation Effect of SIN-1, Bethanechol and Acarbose on 25 Insulin Sensitivity in Rats with Insulin Resistance An animal is prepared as in Example 1. A control RIST is Produced by Hepatic Denervation done. The response is within normal range. A RIST is then done after Surgical denervation. Bethanechol is administered An animal is prepared as in Example 1. A control RIST is by infusion into the portal vein at a rate of 2.5 mg/kg/min done. The response is within normal range. A RIST is then 30 beginning 10 minutes prior to the insulin administration and done after Surgical denervation. Bethanechol is administered continuing for the entire test period. The resulting RIST index by infusion into the portal vein at a rate of 2.5 mg/kg/min shows reversal of the insulin resistance. beginning 10 minutes prior to the insulin administration and On a second animal, a control RIST is done. The response continuing for the entire test period. The resulting RIST index is within the normal range. A RIST is then done after surgical shows reversal of the insulin resistance. 35 denervation and the RIST index shows significant insulin On a second animal, a control RIST is done. The response, resistance. SIN-1 is administered by infusion into the portal is within the normal range. A RIST is then done after surgical vein at a rate of 10.0 mg/kg/min for a period of 2 minutes. The denervation and the RIST index shows significant insulin resulting RIST index shows reversal of the insulin resistance. resistance. SIN-1 is administered by infusion into the portal On a third animal a control RIST is done. The response is vein at a rate of 10.0 mg/kg/min for a period of 2 minutes. The 40 within the normal range. A RIST is then done after surgical resulting RIST index shows reversal of the insulin resistance. denervation and the RIST index shows significant insulin On a third animal a control RIST is done. The response is resistance. First bethanechol is administered as described within the normal range. A RIST is then done after surgical above. Five minutes prior to the end of the test period SIN-1 denervation and the RIST index shows significant insulin is administered by infusion into the portal veinata rate of 10.0 resistance. First bethanechol is administered as described 45 mg/kg/min for a period of 2 minutes. Thirty seconds prior to above. Five minutes prior to the end of the test period SIN-1 the end of the test period a bolus injection of 3 mg/kg, 1.5 is administered by infusion into the portal veinata rate of 10.0 mg/kg and 0.75 mg/kg of pioglitaZone is given. A RIST is mg/kg/min for a period of 2 minutes. Thirty seconds prior to taken and the result is greater insulin resistance than the end of the test period a bolus injection of 40 mg/kg, 20 bethanechol or SIN-1 alone. The lower doses of pioglitazone mg/kg or 10 mg/kg of acarbose is given. A RIST is taken and 50 are as effective as the higher doses. the result is greater insulin resistance than bethanechol or SIN-1 alone. The lower doses of acarbose are as effective as Example 21 the higher doses. Effect of SIN-1, Bethanechol and Repaglinide on Example 19 55 Insulin Sensitivity in Rats with Insulin Resistance Produced by Hepatic Denervation Effect of SIN-1, Bethanechol and Metformin on Insulin Sensitivity in Rats with Insulin Resistance An animal is prepared as in Example 1. A control RIST is Produced by Hepatic Denervation done. The response is within normal range. A RIST is then 60 done after Surgical denervation. Bethanechol is administered An animal is prepared as in Example 1. A control RIST is by infusion into the portal vein at a rate of 2.5 mg/kg/min done. The response is within normal range. A RIST is then beginning 10 minutes prior to the insulin administration and done after Surgical denervation. Bethanechol is administered continuing for the entire test period. The resulting RIST index by infusion into the portal vein at a rate of 2.5 mg/kg/min shows reversal of the insulin resistance. beginning 10 minutes prior to the insulin administration and 65 On a second animal, a control RIST is done. The response continuing for the entire test period. The resulting RIST index is within the normal range. A RIST is then done after surgical shows reversal of the insulin resistance. denervation and the RIST index shows significant insulin US 8,673,964 B2 21 22 resistance. SIN-1 is administered by infusion into the portal fatty acids, TGs, and HDL/LDL cholesterol), reduction in vein at a rate of 10.0 mg/kg/min for a period of 2 minutes. The blood pressure, weight loss and effect on liver function. resulting RIST index shows reversal of the insulin resistance. Inclusion Criteria. The subjects for the study are male On a third animal a control RIST is done. The response is subjects between the ages of 40 and 60, able to give informed within the normal range. A RIST is then done after surgical consent, with mild to moderate type 2 diabetes (have HBA1c denervation and the RIST index shows significant insulin levels >8%, fasting glucose levels of >110 mb/dL), in other resistance. First bethanechol is administered as described wise good general health, with no other significant illnesses, above. Five minutes prior to the end of the test period SIN-1 blood pressure 160/90, with no known target organ damage. is administered by infusion into the portal veinata rate of 10.0 End organ damage includes the following: proliferative mg/kg/min for a period of 2 minutes. Thirty seconds prior to 10 retinopathy, serum creatinine greater than 2, ischemic heart the end of the test period a bolus injection of 3 mg/kg, 1.5 disease, congestive heart failure, peripheral vascular disease mg/kg and 0.75 mg/kg of repaglinide is given. A RIST is and peripheral neuropathy. taken and the result is greater insulin resistance than Exclusion Criteria—Exclusion criteria include the follow bethanechol or SIN-1 alone. The lower doses of repaglinide 15 ing: significant digestive abnormalities such as malabsorp are as effective as the higher doses. tion or chronic diarrhea, significant organ malfunction including (but not limited to) liver disease, pulmonary dis Example 22 ease, ischemic heart disease, heart failure, stroke, peripheral vascular disease, hypertension (BP greater than 160/90), and Effect of Bethanechol, NAC and Repaglinide on anemia (hematocrit less than 30); other serious or chronic Insulin Sensitivity in Rats with Insulin Resistance illness; history of serious or chronic illness; any significant Produced by Hepatic Denervation complications from diabetes such as kidney damage (renal insufficiency, serum creatinine greater than 2), eye damage An animal is prepared as in Example 1. A control RIST is (proliferative retinopathy), diabetic neuropathy, coronary done. The response is within normal range. A RIST is then 25 artery disease, or peripheral vascular disease; Smoking, alco done after Surgical denervation. Bethanechol is administered hol or drug abuse; insulin treatment; positive HIV or hepatitis by infusion into the portal vein at a rate of 2.5 mg/kg/min (B or C) screening tests (subjects will be notified of these test beginning 10 minutes prior to the insulin administration and results). continuing for the entire test period. The resulting RIST index Patients on antihypertensive medication are excluded even shows reversal of the insulin resistance. 30 if blood pressure is well controlled because antihypertensive On a second animal, a control RIST is done. The response medication may affect blood glucose during the test meal is within the normal range. A RIST is then done after surgical challenge, thus introducing a confounding variable. denervation and the RIST index shows significant insulin Study Design. During the 12 week study the patient visits resistance. NAC (200 mg/mL) is administered by infusion the clinic four times. At the first visit (prior to the start of the into the portal vein at a rate of 0.1 mL/min for a period of 2 35 trial) the patient’s baseline levels are measured by blood minutes. The resulting RIST index shows reversal of the draw. The tests taken are: fasting glucose, TGs, AST, ALT. insulin resistance. total bilirubin, alkaline phosphatase, HDL, LDL, total cho On a third animal a control RIST is done. The response is lesterol, free fatty acids, fasting insulin, C-peptide and within the normal range. A RIST is then done after surgical HbA1c. The patient then undergoes a Meal Tolerance Test denervation and the RIST index shows significant insulin 40 using a standard can of Vanilla Boost as the model meal (237 resistance. First bethanechol is administered as described ml with 10 g of protein, 4 g of fat and 41 carbohydrates). above. Five minutes prior to the end of the test period NAC is Blood samples are collected via an indwelling catheter and administered by infusion into the portal vein at a rate of 0.1 sent for analysis. The timeline is as follows: mL/min for a period of 2 minutes. Thirty seconds prior to the end of the test period abolus injection of 3 mg/kg, 1.5 mg/kg 45 and 0.75 mg/kg of repaglinide is given. A RIST is taken and T = -1:10 (fasted) glucose the result is greater insulin resistance than bethanechol or T = -1:OO glucose NAC alone. The lower doses of repaglinide areas effective as PATIENT WAITS ONE HOUR the higher dose. 50 T = O:OO glucose, Insulin, c-peptide, lactate, Free Fatty Acids (FFA), Glucagon, triglycerides (TGL) Example 23 PATIENT CONSUMES BOOST LIQUID MEAL Hyperglycemic Control with Bethanechol and T = O:10 glucose, Insulin, lactate, FFA N-Acetylcysteine in Drug Naive Pre-Diabetic Males T = O:20 glucose, Insulin, lactate, FFA T = 0:30 glucose, Insulin, lactate, FFA 55 T = O:45 glucose, Insulin, lactate, FFA Objective To determine the effectiveness of bethanechol T = 1:00 glucose, Insulin, c-peptide, glucagon, lactate, and n-acetyl-cysteine (NAC) combination therapy in control FFA, TGL ling postprandial hyperglycemia. T = 1:30 glucose, Insulin, lactate, FFA T = 2:00 glucose, Insulin, c-peptide, glucagon, lactate, Summary of Study Design. The study is an open label FFA, TGL study. The study subjects are prefdiabetic males 40-60 years, 60 T = 3:00 glucose, Insulin, c-peptide, glucagon, lactate, The trial involves at least 20 men and follows them through at FFA, TGL least 12 weeks of treatment. Patients take the two compounds T = 4:00 glucose, Insulin, c-peptide, glucagon, lactate, preferably one hour prior to their three major meals. The FFA, TGL outcomes to be investigated are reductions in HbA1c levels either by >0.9% or below 7.5%, decrease in fasting glucose to 65 The patients receives the drugs to take home. The drugs <110 mg/dL, improving post prandial control of hyperglyce (bethanechol and N-acetylcysteine) are to be taken 1 hour mia/hyperinsulinemia, improvement of lipid profile (free prior to breakfast, lunch and dinner. US 8,673,964 B2 23 24 The second visit occurs 4 weeks after the start date. Phy Xie H & Lautt WW (1995). Induction of insulin resistance by sician checks patients blood pressure, weight and glucose. cholinergic blockade with atropine in the cat. Jauton phar Patients fill out a simple questionnaire to determine if any macol 15, 361-369. other major changes in their lifestyle have occurred. Any left The invention claimed is: over drug is returned to the physician and counted and new 5 1. A pharmaceutical composition comprising: (a) drug is given to last the next four weeks. bethanechol, (b) N-acetylcysteine, and (c) a pharmaceutically Visit three occurs after 8 weeks. At the safety visit the acceptable carrier, where the composition does not comprise physician again checks patients blood pressure, weight and a growth factor. glucose. Patients fill out a simple questionnaire to determine 2. The pharmaceutical composition according to claim 1, if any other major changes in their lifestyle have occurred. 10 wherein the bethanechol is nitrosylated. Old left over drug is returned to the physician and counted and 3. The pharmaceutical composition according to claim 1, new drug is released for the following four weeks. wherein the composition is a pharmaceutical salt thereof. During visit four which is at the 12 week mark the entire 4. The pharmaceutical composition according to claim 1, procedure of the first visit is repeated. Prior to the blood being further comprising a pharmaceutically acceptable liver tar withdrawn from the patient, he takes the combination therapy 15 geting Substance, wherein the liver targeting Substance is and then drinks the liquid test meal. The patient also the selected from the group consisting of albumin, a liposome, patient fills out the questionnaire. and a bile salt. Results—The combination therapy when taken prior to the 5. A method of treating or inhibiting type II diabetes, insu meal positively affects at least one the endpoints. lin resistance, or impaired glucose intolerance, comprising administering a therapeutically effective amount of the phar REFERENCES maceutical composition according to claim 1. 6. The pharmaceutical composition according to claim 1, Beyer J. Krause U, DobronzA, Fuchs B, Delver J R & Wagner further comprising insulin or an insulin analogue. R (1990). Assessment of Insulin Needs in Insulin-Depen 7. The pharmaceutical composition according to claim 6. dent Diabetics and Healthy Volunteers Under Fasting Con 25 wherein the insulin analogue is lente insulin, semilente insu ditions. Horm Metab Res Suppl 24, 71-77. lin, ultralente insulin, NPH or insulin lispro. Brownlee M. (2001). Biochemistry and molecular cell biol 8. The pharmaceutical composition according to claim 1, ogy of diabetic complications. Nature 414, 813-819. further comprising an antioxidant. Hsu C C, Yen H F, Yin MC, Tsai C M, Hsieh C. H. Five 9. The pharmaceutical composition according to claim 8. cysteine-containing compounds delay diabetic deteriora 30 tion in Balb/cA mice. J Nutr. 2004 December; 134 (12): wherein the antioxidant is selected from the group consisting 3245-9. of vitamin E, vitamin C, an isoflavone, Zinc, Selenium, ebselen, and a carotenoid. Latour MG & Chan CC (2002). A Rapid Insulin Sensitivity 10. The pharmaceutical composition according to claim 1, Test (RIST) in the Anesthetized Mice (Abstract). Diabetes further comprising a glucagon like peptide (GLP) or gluca 51 (Suppl. 2), A422. 35 Lautt W. W. (1999). The Hiss Story Overview: A Novel gon like peptide analogue. Hepatic Neurohumoral Regulation of Peripheral Insulin 11. The pharmaceutical composition according to claim Sensitivity in Health and Diabetes. CanJPhysiol Pharma 10, wherein the glucagon like peptide analogue is selected col 77, 553-562. from the group consisting of exanitide, DAC:GLP-1 (CJC Lautt WW (2003). New Paradigm for Insulin Resistance: The 40 1131), Liraglutide, ZP10, BIM51077, LY315902, and LY307161 (SR). Hiss Story. In: Atherosclerosis, Hypertension and Diabe 12. The pharmaceutical composition according to claim 1, tes. Eds: G. N. Pierce, M. Nagano, P. Zahradka, And N. S. further comprising alipoic acid. Dhalia. Kluwer Academic Publishers, Bost. Chapter 21, 13. The pharmaceutical composition according to claim 1, pp. 263-276. further comprising metformin. Lautt W. W. Macedo MP. Sadri P. Takayama S, Ramos F D & 45 Legare D J (2001). Hepatic Parasympathetic Nerve-De 14. The pharmaceutical composition according to claim 1, pendent Control OfPeripheral Insulin Sensitivity Is Deter further comprising a Sulphonylurea. mined By Feeding And Fasting: Dynamic Control Of Hiss 15. The pharmaceutical composition according to claim 14, wherein the Sulphonylurea is selected is from the group Dependent Insulin Action. Am J Physiol 281, G29-G36. consisting of tolaZamide, tolbutamide, chlorpropamide, Lautt, W.W., Wang, X. Sadri, P. Legare, D.J., And Macedo, 50 acetohexamide, glyburide, glipizide, and glimepiride. M. P. (1998). Rapid Insulin Sensitivity Test (RIST). Can.J. 16. The pharmaceutical composition according to claim 1, Physiol. Pharmacol. 76: 1080-1086. further comprising carvedilol or labetolol. Ling PR, Mueller C. Smith RJ, Bistrian BR. Hyperglycemia 17. The pharmaceutical composition according to claim 1, induced by glucose infusion causes hepatic oxidative stress further comprising prazosin, doxazocin, phenoxybenzamine, and systemic inflammation, but not STAT3 or MAP kinase 55 teraZosin, phentolamine, rauwolscine, yohimine, tolaZoline, activation in liver in rats. Metabolism. 2003 July; 52 (7): tamsulosin, carvedilol, or teraZosin. 868-74. 18. The pharmaceutical composition according to claim 1, Moore MC, Satake S. Baranowski B, Hsieh PS, Neal DW & further comprising acebutolol, atenolol, betaxolol, biso Cherrington Ad (2002). Effect of Hepatic Denervation on prolol, carteolol, esmolol, metoprolol, nadolol, penbutolol. Peripheral Insulin Sensitivity In Conscious Dogs. Am J 60 Physiol Endocrinol Metab 282, E286-E296. pindolol, propanolol, timolol, dobutamine hydrochloride, Porszasz. R. Legvari G. Nemeth J. Literati P. Szolcsanyi J & alprenolol, bunolol, bupranolol, carazolol, epanolol, molo Szilvassy Z (2002). The Sensory Nitrergic Nature of The prolol, oXprenolol, pamatolol, talinolol, tiprenolol, tolam Hepatic Insulin Sensitizing Substance Mechanism In Con olol, and toliprolol. scious Rabbits. Eur: J Pharmacol 443, 211-212.