Mary A. Root

New Forms of Insulin

This paper reviews some recent developments in insulin purification and formulation. Neutral Regular insulin (unbuffered Regular insulin with pH adjusted to neutrality, designated NRI) maintained nearly full potency when stored at room temperature for twelve months, while the potency of acid Regular (“commercial”) insulin (ARI) had dropped 20 per cent. When NRI and ARI were administered in combination with NPH or Ultralente insulin, comparable time activity responses were elicited. In fasted normal subjects subcutaneous doses of NRI and ARI gave similar blood glucose responses. Thirty-seven patients with allergy to commercially available insulin were treated with one or both of two highly purified insulins (single peak and single component pork insulin). Improvement occurred in 78 per cent. Single component insulin was usually, but not always, superior to single peak insulin when both were tested in the same patient. Insulin was administered orally with an absorption promoter, Brij 98, to twenty normal and thirteen diabetic fasted subjects in crossover studies. Under optimum conditions, definite responses were observed in about 50 per cent of subjects. Serum insulin measurements in the normals and diabetics revealed that increased concentrations were not necessarily associated with falls in blood glucose concentrations in spite of delivery of the insulin by the portal route. Because of the amounts of insulin required, the limitations of test conditions, and a high incidence of nausea, this project has been discontinued.

Immunogenicity of Insulin

Highly purified bovine and porcine insulins (single component insulin) did not produce antibodies in rabbits, and the highly purified porcine insulin was less immunogenic in diabetic patients than USP porcine insulin. In rabbits one porcine high molecular weight fraction (peak A) and two bovine high molecular weight fractions (peak A and peak B) separated from USP insulin by Sephadex chromatography were immunogenic, producing increases in the amount of 125I-labeled single component insulin bound by the serum. Both peaks (A and B) derived from bovine insulin produced significantly greater insulin binding in rabbits than did the same peaks derived from porcine insulin. In diabetic patients single component porcine insulin produced less binding of insulin to the serum than did USP insulin.

Clinical Pharmacologic Studies with Human Insulin (recombinant DNA)

Normal fasting subjects were used to study the pharmacokinetics of human insulin (recombinant DNA). Purified pork insulin (PPI) was used as a control agent. There was no difference in serum concentrations between neutral regular human insulin and PPI after intravenous administration. When given subcutaneously, peak concentrations are occasionally higher for human insulin than for PPI. The bioavailability indices for the two insulins are essentially the same. NPH human insulin produced a slightly higher serum concentration after 4 h than did NPH PPI. Studies with 70/30 NPH-regular mixtures suggest that the affinity of protamine for human insulin is less than that for PPI. The serum insulin concentrations after lente human insulin and PPI were not different. These studies, and a review of the published clinical pharmacologic literature, indicate that when present the differences between human insulin and PPI are minimal.

The plasma glucose response of normal fasting subjects to neutral regular and NPH biosynthetic human and purified pork insulins.

Using doses of 0.1 and 0.15 U/kg, the hypoglycemic activities of neutral regular and NPH biosynthetic human insulin (BHI) and purified pork insulin were compared in normal fasting subjects. Neutral regular insulin was administered by the intravenous and subcutaneous routes and NPH subcutaneously. Comparison of the plasma glucose curves disclosed no statistically significant differences between the maximum effects and the length of time to achieve the maximum effect. Moreover, a dose-response difference between 0.1 and 0.15 U/kg could not established. It is concluded that the hypoglycemic activities of neutral regular and NPH BHI and purified pork insulin are the same.

A Comparison of Acid Regular and Neutral Regular Insulin: Responses of Normal Fasted Subjects to Varying Doses of Regular Insulin

Acid Regular insulin (ARI) and neutral Regular insulin (NRI) were compared in normalfasting subjects by means of a Latin-square experimental design. Both blood glucose and serum immunoreactive insulin were measured after the administration of 0.1 and 0.2 U. per kilogram intravenously, and 0.1, 0.2, and 0.3 U. per kilogram subcutaneously. No statistically significant differences were detected between ARI and NRI. Analysis of blood glucose and serum insulin data pooled from the ARI and NRI treatments disclosed resistance to increasing doses of insulin as the blood glucose fell below 50 to 60 mg. per 100 ml. Thus, relatively small increments in hypoglycemic activity occurred when the intravenous dose was doubled from 0.1 to 0.2 U. per kilogram or the subcutaneous dose increased from 0.2 to 0.3 U. per kilogram in spite of doubling the hypoglycemic effect when the subcutaneous dose was doubled from 0.1 to 0.2 U. per kilogram. In addition, for each 0.1 U. per kilogram of subcutaneouslyadministered insulin the maximum serum levels were approximately 15 to 30 μU. per milliliter andthe duration of effect was extended by one hour. Therefore, following 0.1 U. per kilogram the maximum effect was over by three hours, after 0.2 U. per kilogram by four hours, and 0.3 U. per kilogram by five hours. These results demonstrate the feasibility of studying the time action of insulin in normal subjects and document the conclusion that duration of effect is a function of the dose given.

Effect of chronic administration of glucagon to rats and rabbits.

Summary Daily administration of large doses of glucagon to rats and rabbits for 6 months did not produce any toxic reactions. The rate of weight gain of treated animals was the same as that of control animals. Diabetes was not produced and there was no evidence of chronic hyperglycemia. The liver glycogen concentration of the treated animals was significantly greater than that of the untreated animals.

Extended Production of Insulin by Isolated Rabbit Pancreatic Islets; Evidence for Biosynthesis of Insulin

Summary Methods for prolonged primary suspension cultures of rabbit pancreatic islets were established, and incorporation of carbon-14 labeled amino acids into insulin and other proteins was demonstrated.

Metabolic Reduction of 1-(p-Acetylbenzenesulfonyl)-3-cyclohexylurea (Acetohexamide) in Different Species

Summary Metabolism of acetohexamide 1 - (p-acetylbenzenesulfl) -β -cyclohexylurea was investigated in dogs, rabbits, rats, and man. Trace amounts of unchanged drug were excreted by dogs and man. All species excreted the reduced form 1-(p-α-hydroxyethylbenzenesulfonyl) −3 -cyclohexylurea. In a dog, a rabbit and 2 human subjects, 49 to 59% was recovered as metabolite in the urine, but only 18% in the pooled urine of 6 rats. Chromatograms of rat urine revealed no other metabolites. The authors are indebted to Messrs. D. O. Woolf and L. H. Howard for infrared and ultraviolet absorption data, and to Miss Ann VanCamp for x-ray diffraction patterns.

Effect of Glucagon on insulin Hupoglycemia

Summary Experiments in rabbits and mice have demonstrated that the presence of glucagon in insulin solutions in ratios of from 1:100 to 1:1 did not influence the degree of hypoglycemia produced by the insulin, and consequently did not alter the potency of the insulin preparation as determined by the mouse convulsion assay.

Dopamine agonist-induced hyperglycemia in rats: Structure-activity relationships and mechanisms of action

The concentration of blood glucose was measured in rats after administration of a number of drugs characterized as dopamine agonists. Compounds that cause release of dopamine, or agents that block the reuptake of dopamine, did not elevate blood glucose. Some direct dopamine receptor stimulants (lergotrile, bromocriptine, apomorphine) caused hyperglycemia, but other agonists (e.g. pergolide) did not. Further experiments with lergotrile, the most active hyperglycemic dopamine agonist, revealed that the blood glucose increase was accompanied by a marked elevation in liver glycogen, indicating a gluconeogenic effect of the compound. This hypothesis was supported by using inhibitors of gluconeogenesis (L-tryptophan or 3-mercaptopicolinic acid) to block lergotriles hyperglycemic action. Structure-activity relationships among close analogues of lergotrile suggest that the cyano moiety in the lergotrile molecule may be of importance in the hyperglycemic action of lergotrile. These results indicate that central dopamine stimulation per se does not cause hyperglycemia in rats.