• Identical glycodynamic profile of an intranasally delivered insulin formulation (Nasulin) in comparison to subcutaneous Aspar injection in non-diabetic rats
• Bi-phasic Transmucosal Insulin Release Mechanism: Delivered Intranasally (Nasulin) in the Control of Pre-meal and Post-meal Glucose Levels in Non-diabetic Rats
• Treatment of Severe Diabetic Ketoacidosis in Streptozocin-induced Diabetic Rats with a Low Dose Oral Insulin (Musulin)
• The Effects of Low Dose Oral Insulin (Musulin) in the Control of Hyperglycemia in Streptozocin-induced Diabetic Rats
• A long-acting transmucosal insulin formuation delivered per-orally (Musulin) as tested in non-diabetic rats
• Control of pre-meal and post-meal glucose levels with per-orally delivered insulin (Musulin) in non-diabetic rats

Frank K Leung, M.D.¹, Jiang Li M.D.¹, Emily C Leung, B.A.¹ and Chris Seet B.A.¹.
¹Lake bluff, Illinois .

This study is to evaluate the glucodynamic profile of an intranasally delivered insulin formulation ( IN ) in comparison to subcutaneous Aspar insulin injection ( AS ) and placebo control ( Control ). Procedures and materials: IN non-diabetic rats : IN group 1 ( n=6 ) ; IN group 2 ( n=4); IN grop 3 ( n=4); AS non-diabetic rats ( n=4 ); Control non-diabetic rats ( n=4 ). Average weight: 276 gm; Average baseline glucose: 98 mg/dl after fasting for 12 hours; Dose of insulin given: 0.2 unit/gram in the IN or AS groups. Average dosing volume: IN group 1: 0.1 ml of 500 u/ml formulation. IN group 2: 0.05 ml of 1000 u/ml formulation. IN group 3: 0.025 ml of 2000 u/ml formulations. Concurrent study was performed. Blood glucose was measured from the tail at time 0, 15, 30, 60, 120, 180 minutes. Each nostril of the IN rat received 50% of the dosing volume. Data and results: Glucose level was normalised as 100% at time zero. Control group: 100%, 109%, 106%, 101%, 97%, 90%. AS group: 100%, 81%, 64% 46%, 42%, 12%. IN group 1: 100%, 108%, 82%, 52%, 33%, 9%. IN group 2: 100%, 94%, 72%, 56%, 36%, 17%. IN group 3: 100%, 87%, 65%, 52%, 29%, 17%. Statistical analysis: SAS 8.01. AUC_T: IN group 1 is 7815; AS is 7452 (P=0.4545). Slopes: IN group 1 is -0.312 error of slope 0.077; AS is -0.4703 error of slope is 0.082 (P=0.1626), Control is -0.1095 error of slope 0.045 (P<0.0001). Tmax: IN group 1,2,3 and AS are all at 15 min.The slopes and AUC_T of IN group 2, and IN group 3 matched exactly the AS group results. Onset of significant hypoglycemia compared to Control group occurred within 15 minutes, except IN group 1 has a delay onset at 30 minutes. At 180 minutes, profound hypoglycemia occurred in all the groups of IN, AS. Conculsion: Intranasally delivered insulin in three separate highly concentrated insulin solutions, formulated with Coremed s drug delivery system, have near identical hypoglycemic effects and glucodynamic profile as Aspar subcutaneous injection, and is effective in lowering blood glucose over a wide range of insulin concentrations.

Frank K Leung, M.D. Jiang Li M.D. Emily Leung B.A. and Chris Seet B.A.

This study evaluates the insulin pharmacokinetics of a transmucosal insulin solution ( IN ), formulated with Coremed s drug delivery system, and its efficacy in controlling pre-meal and post-meal glucose levels in non-diabetic rats. Procedures and materials: Group 1 ( total n=4): insulin was sampled from the jugular vein at time 0, 5, 15, 30, 60, 120 minutes for insulin pK estimates. Average weight: 430 gm. IN dose: 0.2 u/gm. Group 2 ( n=4) : as placebo control. Group 3 ( n=4) : as experimental group receiving IN intranasally. Group 2 and 3: average weight: 350 gm, IN dose: 0.1 u/gm, Average baseline glucose: 105 mg/dl. IN insulin concentration: 2000 units/ml. Blood glucose was sampled from the tail at time 0, 15, 30, 45, 60, 120, 180, 240 minutes.IN was given as a single dose at time 0. Food ( 2ml of D50W) was fed orally at time 30 minutes. Results: 1) Group 1 insulin levels (miu/dl): 2, 962, 2310, 383, 1930, 1050. Repeated experiment of Group 1: 2, 870, 1070, 885, 1810, 643. 2) Group 2 (control) glucose: 100%, 109%, 109%, 127%, 146%, 167%, 140%, 119% 3) Group 3 (IN) glucose: 100%, 91%, 69%, 64%, 57%, 65%, 79%, 70%. 4) Difference between Group 2 and 3: 0%, 18%, 40%, 64%, 89%, 102%, 61%, 49%. Statistical analysis; SAS 8.01.Tukey s Studentized Range (HSD). Bi-phasic insulin release mechanism was confirmed with a repeated experiment. In both experiments, the first peak insulin release occurred at 15 minutes and the second peak at 60 minutes.Significant at 5%: IN at 15 to 240 mintues. AUC_mean: IN (66.542); control (121.594). The glucose level was maintained euglycemically at steady and leveled state from the time of food intake at 30 to 240 minutes, without evidence of hyperglycemia as compared to the control group.The maximal difference in glucose levels occurred at 120 minutes between the two groups. Conclusion: Bi-phasic insulin release mechanism is effective in lowering the pre-meal and post-meal glucose levels, and maintains a steady euglycemic state from the time of food intake to 240 minutes. IN achieved a 45.3% reduction of the Control ( AUC_mean ).

Frank K Leung, M.D., Jiang Li M.D. Emily C Leung, B.A. and Chris Seet B.A.

The major obstacles in developing an effective oral insulin have been lack of efficacy and huge dose requirements, making treatment cost ineffective. The doses reported in literature had been around 20-30 units/ kg for oral insulin and 1 unit/kg for inhaled insulin. This study evaluates the effects of a dose of 0.01 unit/gm weight (10 units/kg) of an orally delivered Musulin in severe diabetic ketoacidotic rats. Procedures and materials: All rats were induced diabetes with Streptozocin. Average weight: 214 gm. Musulin concentation: 250 units/ml. Average dosing volume: 0.01ml. Musulin group, n=4, Placebo control ( C), n=4 and Aspar insulin injection control ( ASP), n=4. At time 0, Musulin at 0.01 u/gm was given per-orally, C was given placebo and ASP as Novolog at 0.01u/gm was given s.c. injection. Glucose was sampled from the tails at time 0, 15, 30, 60, 120, 180, 240, 300, 360, 420, 480 minutes. Data and results: Musulin group: average baseline glucose: 589 mg/dl; positive for blood ketones; average total dose of Musulin: 2 units. C and ASP were comparable. Glucose was expressed as percent of baseline levels. Musulin group: 100%, 107%, 108%, 106%, 100%, 89%, 78%, 67%, 60%, 52%, 49%. ASP: 100%, 91%, 80%, 58%, 41%, 31%, 21%, 13%, 10%, 8%, 6%. C: 100%, 105%, 110%, 112%, 106%, 100%, 96%, 93%, 87%, 83%, 74%. Statistical analysis: SAS 8.01.Tukey s Studentized Range (HSD) Test . AUC_mean: Placebo (92.4); Musulin (75.5); ASP (26.5).Significance at 5%: Musulin at 240 -480 minutes. ASP 15-480 minutes.% reduction in AUC_mean: Musulin (18.3%); ASP (71.3%). Ration: 18.3/71.3=26 Conclusion: In the treatment of diabetic ketoacidosis with a 10 units/kg dose of insulin, Musulin has a signficant hypoglycemic effect with long-sustained action. There was an initial transient rise in glucose for two to three hours in both the IN and C groups.Musulin lowered its baseline glucose level by 49% at eighth hour, or 31% compared to control. Musulin achieved 26% of the ASP efficacy based on the ratio of AUC_means.

Frank K Leung, M.D. Jiang Li M.D. Emily C Leung, B.A. and Chris Seet B.A.

The major obstacles in developing an effective oral insulin have been lack of efficacy and huge dose requirements, making treatment cost ineffective. The doses reported in literature had been 20-30 units/ kg for oral insulin and 1 unit/kg for inhaled insulin. This study evaluates the effects of a dose of 0.005 unit/gm weight ( 5 units/kg ) of an orally delivered Musulin in diabetic rats. Procedures and materials: All rats were induced diabetes with Streptozocin. Average weight: 295 gm. Musulin concentation: 25 units/ml. Musulin group: n=4, Placebo control ( C): n=4 and Aspar insulin injection control ( ASP): n=4. At time 0, Musulin at 0.005 u/gm was given per-orally, C was given placebo and ASP as Novolog at 0.005u/gm was given as s.c. injection. Glucose was sampled from the tails at time 0, 15, 30, 60, 120, 180, 240, 300, 360, 420, 480 minutes. Data and results: Musulin group: average baseline glucose: 446 mg/dl. Average total dose of Musulin and ASP: 1.4 units. C and ASP were comparable.Glucose was expressed as percent of baseline levels. Musulin group: 100%, 106%, 107%, 102%, 86%, 78%, 54%, 63%, 58%, 52%, 45%. ASP: 100%, 93%, 74%, 61%, 48%, 37%, 30%, 29%, 36%, 37%, 38%. C: 100%, 105%, 110%, 112%, 106%, 100%, 96%, 93%, 87%, 83%, 74%. There was an initial transient rise in glucose for two hours in both C and Musulin groups.Statistical analysis: SAS 8.01. Tukey s Studentized Rand (HSD) Test. AUC_mean: Placebo (92.39); Musulin (69.57); ASP (40.04). Signifcance at 5%: ASP at 15 - 480 minutes and Musulin at 120 - 480 minutes. The differences between Musulin and ASP glucose levels at 420 and 480 minutes were not significant.% of reduction AUC_mean by ASP was 56.7% and by Musulin 24.7%.Ratio: 24.7/56.7=44 Conclusion: At a low dose of 5 units/kg, Musulin has sustained effectiveness in lowering hyperglycemia in diabetic rats by 55% of it baseline level. At the seventh and eighth hours, there was no difference in the glucose levels between Musulin and Aspar injection groups. Musulin achieved 44% of the efficacy by Aspar injection, as measured by the ratio of their AUC_means.

Frank K Leung, M.D. Jiang Li M.D. and Emily C Leung, B.A.

This study is to evaluate a long-acting transmucosal insulin, formulated with Coremed s drug delivery system, delivered per-orally as tested in non-diabetic rats. Procedures and materials: Average weight: 310 gm. Placebo Control ( C): n=4. Musulin: n=4.Dose: 0.04 unit/gm. Average dosing volume:0.25 ml. Glucose is sampled from the tail at time (minutes): 0, 15, 30, 60, 120, 180, 240 300, 360, 420, 480. Musulin concentration: 500 units/ml.The study is concurrent. Results: Glucose is normalised as 100% of baseline value. C (%) : 100, 99, 102, 101, 92, 90, 85, 85, 81, 79, 75. Musulin (%): 100, 97, 90, 82, 74, 69, 60, 54, 53, 54, 55. Statistical Analysis: SAS 8.01.. P value at 5% significance: na, 0.47, 0.02, 0.016, 0.13, 0.035, 0.005, 0.007, 0.004, 0.004, 0.004. Significant onset of glucose-lowering effect occurred at 15 minutes and lasted till the end of the experiment at 480 minutes.After initial decline of glucose level from baseline 100% to 69% at 180 minutes. The Musulin group glucose levels were sustained between 50% to 60% at a steady state from 240 to 480 minutes. Conclusion: Musulin has a long-acting duration and sustained its glucose-lowering effects at a steady state during most of the eight hours study period.

Frank K Leung, M.D. Jiang Li M.D. Emily c Leung, B.A. and Chris Seet B.A.

This study is to evaluate the effects of food intake on the glucose responses in non-diabetic rats treated with Musulin, delivered per-orally. Procedures and materials: Study was concurrent. All are non-diabetic rats with average weight 300 gm. Placebo control (C ), n=4. Musulin, n=4. Musulin concentration: 2000 units/ml. Dose: 0.2 unit/gm. Food intake was given as one ml of D50W per-orally at 30 minutes of the experiment. Average dosing volume: 0.03ml given as one single administration at time 0. Blood glucose was sampled from the tails at time 0, 15, 30, 45, 60, 120, 240, 300, 360. Data and results: Glucose was normalized as percentage of the baseline glucose at time 0.% glucose of C: 100%, 109%, 127%, 145%, 167%, 140%, 119%, 95%, 89%.% glucose of Musulin group: 100%, 91%, 70%, 63%, 57%, 65%, 79%, 71%, 71%, 66%. Statistical analysis: SAS 8.01. Percent glucose-lowering effects of Musulin compared to C: 0%, 16.5%, 35.2%, 50%, 61%, 61%, 43.2%, 40.1%, 25.4%, 27%. Significant glucose-lowering effect occurred at 15 minutes. Tmax at 60 minutes. Glucose was lowered by 16.% compared to control, 35.2% just before food intake. 61% at time 60 to 120 minutes or 30 to 90 minutes after food intake. From time 60 to 260 minutes, glucose was maintained at steady level between 60-80% of the baseline glucose levels. Interpretation: Musulin has onset of action at 15 minutes at dose of 0.2 u/gm delivered per-orally. It exerts maximal effect between 30 to 90 minutes. Musulin lowered pre-meal and post-meal glucose significantly and maintained the glucose levels at a steady state over the period of the experiment.