Monocomponent Insulin Research Articles

Human Monocomponent Insulinに関するブタ精製インスリンを対照とした二重盲検試験成績 (第2編)

Human Monocomponent Insulinに関するブタ精製インスリンを対照とした二重盲検試験成績 (第2編)

Monocomponent Insulin治療でInsulin Lipoatrophyを生じた1症例

Monocomponent Insulin治療でInsulin Lipoatrophyを生じた1症例

Is diabetes a disease for general practice?

Is diabetes a disease for general practice?

Insulin therapy-realising the potential.

Since its discovery in 1922, insulin has been the basis for treating diabetes both in patients with type I diabetes and those with type II diabetes when oral agents fail. For many years, commercial insulin production used animal sources and extracted insulin from bovine and porcine pancreas. While effective clinically, these insulin preparations also contained small amounts of impurities from other pancreatic components and were highly immunogenic. A significant development in insulin chemistry and the production process was the ability to produce pure animal insulin, allowing the introduction of a range of monocomponent insulins. From here, it was a logical step to the production of human insulin, initially by enzymatic modification of porcine insulin and then more generally by bacterial or yeast biosynthesis.

12-Month Chronic Toxicity Study of LY275585 (Human Insulin Analog) Administered Subcutaneously to Fischer 344 Rats

The monocomponent insulin analog LY275585 was administered to Fischer 344 rats in daily subcutaneous doses of 0, 20, or 200 U/kg for 12 months. Each treatment group consisted of 30 males and 30 females, and the vehicle control group consisted of 35 of each sex. Changes related to the pharmacologic activity of LY275585 included an increase in body weight, body weight gain, food consumption, and efficacy of food utilization in male and female rats given 200 U/kg. Pharmacologically related clinical chemistry effects occurring at 200 U/kg were an increase in mean serum glucose (24 h postdose) of males and females, a slight decrease in mean serum triglyceride of females, and decreased mean serum cholesterol of males and females. Fewer effects occurred in low-dose rats (20 U/kg) and included an increase of efficiency of food utilization only in female rats, a slight decrease in mean serum triglyceride in females, and decreased mean serum cholesterol in low-dose males and females. Three male and two female rats administered 200 U/kg died during the treatment period. Death of one male was attributed to hypoglycemia. Cause of death of four other high-dose rats was either not apparent, or the result of a variety of intercurrent disease processes unrelated to treatment. Survival in the low-dose (20 U/kg) and control groups was similar; one rat in each group died prior to treatment termination. The only compound-related lesion associated with treatment was a non-dose-related enhancement of the degree and incidence of inflammation at the site of subcutaneous injection. There were no compound-related changes in background lesions including neoplasms, or in hematologic or urinalysis values. In a comparison study, pharmacokinetic profiles of LY275585 and serum glucose values were determined in groups of 30 male and female rats administered single doses of 20 or 200 U/kg. Plasma levels of LY275585 peaked after 30 min, were dose related, and were similar in male and female rats. The hypoglycemia produced in rats receiving a single dose of 20 U/kg was maximal, suggesting saturation of the insulin receptor sites at this dose. The dose of 200 U/kg of LY275585 was, therefore, a supramaximal pharmacological dose when compared to an average daily human dose of 0.5 U/kg/ d. It is concluded that no toxicologically significant changes, other than moderate injection site inflammation, occurred in Fischer 344 rats treated with LY275585 at doses of 20 and 200 U/kg for 12 months.

Suppression of diabetes in nonobese diabetic mice by oral administration of porcine insulin.

Suppression of diabetes in nonobese diabetic mice by oral administration of porcine insulin.

Metabolic Derangements in Excessive Insulin and Sulfonylurea Therapy

Mild streptozotocin diabetic rats, characterized by normal or slightly elevated fasting blood glucose levels and glucose intolerance, treated with excessive doses of monocomponent pork insulin (0.5 U/day) (I) or glybenclamide (0.6 mg/day) (S) were compared to controls (C) and streptozotocin-diabetic rats without treatment (D). Intravenous glucose tolerance tests (0.75 g/kg) were performed in all animals and repeated after overtreatment. Insulin binding and insulin-induced D-(U-14C)-glucose transport and oxidation were also determined in isolated epididymal adipocytes. Diabetic rats showed a failure in the initial phase of insulin release and glucose intolerance as compared with (C). In overtreated rats glucose tolerance worsened (p < 0.05) after therapy. Maximal insulin binding by isolated adipocytes at tracer insulin concentration was unchanged after excessive insulin or sulfonylurea therapy. Besides, glucose transport and oxidation in the cells of overtreated rats were greater than in D and even greater than in C. These apparently divergent results, i.e. deterioration of glucose tolerance with increased insulin action in adipocytes suggest that overtreatment induces a state of resistance to hormone action in other target tissue(s) than the adipose one, possibly muscle.

Treatment of allergy to heterologous monocomponent insulin with human semisynthetic insulin. Long-term study.

Eight type II (non-insulin-dependent) diabetic subjects (7 women, 1 man, aged 42-61 yr), initially treated with oral hypoglycemic agents and intermittently treated with conventional insulins, were identified as developing allergic reactions to porcine and mixed-species monocomponent insulin. Allergy was systemic (urticaria and nonthrombocytopenic purpura) and local delayed in two subjects and local immediate or biphasic in six subjects. Lipoatrophy was present in two subjects. After treatment with human semisynthetic insulin (Monotard HM and Actrapid HM), systemic allergy disappeared. Local allergy disappeared in five subjects and was reduced in three subjects. No lipoatrophy occurred in new injection areas. The clinical results were accompanied by a significant decrease in serum insulin-specific IgE after 6, 12, 18, 24, 30, and 36 mo. Insulin-specific IgG showed an evident decrease in five of eight patients, but the difference in mean values was not significant after 6, 18, 24, 30, and 36 mo. With one exception, intradermal skin tests were positive to human, bovine, and porcine insulin before and after human insulin treatment.

Comparison of human and porcine insulin therapies in children with newly diagnosed diabetes mellitus

A multicenter, longitudinal study of children below the age of 16 years with newly diagnosed Type 1 (insulin-dependent) diabetes treated either with porcine monocomponent insulin (n = 26) or semisynthetic human monocomponent insulin (n = 26) was performed during the first 24 months after onset of diabetes. The two groups were carefully matched for age, duration of disease symptoms, initial metabolic values, islet cell antibodies and HLA-DR antigens. During the 24-month observation period there was no significant difference between the two groups in respect to the clinical course, insulin dosage, HbA1 and residual B-cell activity. No child in either group had a real remission without necessitating insulin therapy. The prevalence of insulin antibodies increased slowly and was 62% in the group treated by human insulin and 52% in the porcine insulin-treated group after 24 months. The titres were generally low and there was no statistical difference between the two groups in respect to insulin antibody formation.

Clinical and Immunologic Studies of Semisynthetic Human Monocomponent Insulin (U-100) in Diabetic Children

Clinical and Immunologic Studies of Semisynthetic Human Monocomponent Insulin (U-100) in Diabetic Children

The human cellular immune response to insulin: a study in unexposed control subjects and type I diabetic patients on acute and chronic treatment.

Using a cellular cytotoxicity assay, we have investigated the antigenicity of pharmaceutical insulins and of highly purified insulin constituents (unformulated monocomponent (MC) insulin, insulin B-component and C-peptide) in control, insulin naive, subjects and in three groups of type I diabetic patients. These were: Group 1, newly diagnosed patients receiving either porcine or bovine MC insulins for less than one week; Group 2, established patients receiving porcine or bovine MC insulin for 2-6 years; Group 3, established patients receiving conventional bovine insulin for 2-6 years, and tested within two weeks of switching to either porcine or bovine MC insulins. In control subjects, there was a higher incidence of cytotoxic reactions with beef (72%) than with porcine (33%) pharmaceutical preparations (p less than 0.01) but there was a similar incidence of reactions with the purified beef and pork constituents. All patients, except those Group 3 patients receiving bovine MC insulins, had a significantly increased incidence of aggregate reactions to the spectrum of antigens. In porcine treated patients, there was an increase in the incidence of reactions to pharmaceutical preparations in Group 1 (p less than 0.05) and Group 3 (p less than 0.001), but this was absent from each of the bovine treated groups. All patient groups showed significant increases in the incidence of reactions against insulin constituents of their therapeutic analog. In Groups 1 and 2, but not 3, there was significant analog cross reactivity. We deduce that the reactivity seen in control subjects is principally directed against unidentified formulation constituents of bovine pharmaceutical preparations. Patients on both acute and chronic therapy with insulin show T-cell sensitization to MC insulin and its components. Chronic therapy with conventional bovine insulin induces tolerogenesis, but this is reversed on exposure to a fresh insulin analog.

Treatment of diabetic pregnant women with monocomponent insulins.

Very little immunological research has been undertaken in pregnant diabetic women in relation to insulin therapy. We investigated the relations between treatment with insulins of varied immunogenic character and the presence of immune factors such as insulin antibodies, immune complexes and insulin antiinsulin complexes as well as some maternal and neonatal complications of diabetic pregnancy. 128 insulin treated diabetic pregnant women and 121 of their newborns were included in the study. The incidence of insulin antibodies, immune complexes and insulin antiinsulin complexes was lower in patients treated with highly purified insulins than in those treated with conventional insulins. The insulin antibody levels were significantly related to the occurrence of maternal and neonatal morbidity. The presence of insulin antiinsulin complexes in the cord blood of infants of diabetic mothers was related to the presence of these complexes in their mothers. Our results seem to indicate that the use of highly purified insulin could favour the outcome of diabetic pregnancy.

Immunogenicity of biosynthetic human insulin: Humoral immune response in diabetic patients beginning insulin treatment and in patients previously treated with other insulins

The immunogenicity of biosynthetic human insulin (BHI) was studied in diabetic patients who had never received insulin treatment (Study A) and in diabetic patients who had already been treated with monocomponent insulin (Study B). The results of both studies were compared to matched control groups receiving other forms of insulin treatment. Blood samples obtained were tested for anti-insulin antibodies and circulating immune complexes using two different methods. After six months of treatment, the values of anti-insulin antibodies in those patients in Study A who were treated with BHI were significantly lower than those observed in control patients treated with monocomponent (P less than 0.02) or conventional insulin (P less than 0.001). At the sixth month of Study A no significant difference in the percentage of circulating immune complex positivity was seen between the three groups. In Study B no significant difference in the values of insulin antibodies or immune complexes was observed between patients who were switched to BHI and those who continued monocomponent insulin. No side effects were observed. The data show that the immunogenicity of BHI is even lower than that of monocomponent insulin.

Effects of insulin on pressor responsiveness and baroreflex function in diabetes mellitus.

The effects of insulin on pressor responsiveness to alpha agonist (phenylephrine) and angiotensin II, and baroreflex function were studied in fifteen diabetic patients without autonomic neuropathy. The dose of phenylephrine required to increase systolic pressure by 25 mmHg (PD25) was significantly increased from 38 +/- 7 to 62 +/- 9 micrograms (p less than 0.05) after IV injection of 4 U of Actrapid monocomponent insulin. The dose of angiotensin II required to increase systolic pressure by 30 mmHg (AD30) was also increased from 0.29 +/- 0.07 to 0.48 +/- 0.10 micrograms (p less than 0.01). Following insulin administration, the dose-response curves for phenylephrine and angiotensin II were shifted to the right. The baroreflex sensitivity was not affected by insulin. In contrast, there was no significant change in PD25, AD30 or baroreflex sensitivity after the injection of saline. These results suggest that insulin attenuates the pressor responsiveness to alpha agonist and angiotensin II, which may be one of the significant mechanisms in insulin-induced vasodilation.

Purification of insulin-binding antibody by affinity chromatography using monocomponent insulin as ligand

Insulin-binding antibody (IBA) was purified by affinity chromatography using porcine monocomponent (MC) insulin as the ligand. The purity of the antibody was compared with that of the antibody extracted using porcine crystalline (Cr) insulin. Comparing the antibody solutions obtained with MC insulin (MC-lig-sol) or Cr insulin (Cr-lig-sol), the content of IBA in Cr-lig-sol was higher than in MC-lig-sol, but the content of proinsulin-binding antibody (PBA) in MC-lig-sol was very small and statistically lower than that in Cr-lig-sol (P less than 0.01). Adding native MC insulin to a competitive radioimmunoassay suppressed the IBA titer obtained with MC insulin more than that obtained with Cr insulin. By adding native proinsulin in a similar assay system, the PBA titer obtained with Cr insulin was suppressed more than that extracted with MC insulin. Scatchard analysis of the 2 solutions showed that the affinity constants of high affinity antibodies were almost identical, but that of low affinity antibody in MC-lig-sol was larger than in Cr-lig-sol. The binding capacity of low affinity antibody in Cr-lig-sol was 15 times as much as that in MC-lig-sol. Using MC insulin, instead of Cr insulin, as the ligand in affinity chromatography increased the purity of recovered IBA. chromatography increased the purity of recovered IBA.

Metabolic instability in type I diabetic patients. Studies on insulin absorption, hepatic production of metabolites and glucose counterregulation.

In order to investigate the causes underlying metabolic instability in type I diabetes mellitus, we studied 8 unstable (group 1) and 4 well-controlled (group 2) diabetic patients, matched for age and duration of diabetes. Subjects were connected overnight to an artificial pancreas and brought to normoglycemia. On the following morning, insulin administration was discontinued for 6 hours and both metabolic and hormonal studies were carried out during this period. After insulin withdrawal, group 1 showed a faster rise of blood glucose (peak: 324.63 +/- 24.93 vs 175.25 +/- 42.63 mg/dl, p less than 0.01), beta-OH-butyrate (peak: 2,273.25 +/- 415.78 vs 550.50 +/- 158.17 mumol/l, p less than 0.01), and glycerol (164.10 +/- 38.90 vs 28.25 +/- 10.6 mumol/l, p less than 0.01). C-peptide secretion increased in group 2 from 0.09 +/- 0.052 to 0.22 +/- 0.099 pmol/ml whereas it remained almost undetectable in group 1 (p less than 0.01, group 1 vs group 2). Growth hormone, cortisol and immunoreactive glucagon were not significantly different in the two groups at any time after insulin withdrawal. Free insulin, after repeated s.c. or i.m. injection of porcine monocomponent insulin (10 IU), was not different in the two groups. We concluded that type I diabetic patients showing severe metabolic instability produced more glucose, ketone bodies and glycerol after insulin withdrawal than control 'stable' patients. This difference could not be accounted for by an excessive secretion of counterregulatory hormones or by an erratic insulin absorption from the injection sites and may have been related to the degree of B-cell failure, as measured by the absence of C-peptide and/or to the degree of insulin resistance.

Peripheral circulatory effects of insulin in diabetes.

Peripheral circulatory effects of insulin were studied in the diabetic patients with and without autonomic neuropathy. Forearm blood flow, calf venous volume and calf venous distensibility were measured by strain gauge plethysmography. In the diabetic patients with autonomic neuropathy, mean blood pressure fell from 96 +/- 5 to 88 +/- 5 mmHg after an intravenous injection of 4 U of monocomponent insulin (p less than 0.001). Forearm vascular resistance decreased from 53.99 +/- 8.29 to 45.88 +/- 7.76 mmHg X ml-1 X 100ml-1 X min-1 after insulin (p less than 0.01). Insulin increased calf venous volume from 1.20 +/- 0.19 to 2.23 +/- 0.44 ml/100ml (p less than 0.05) and calf venous distensibility from 0.039 +/- 0.004 to 0.082 +/- 0.016 ml/mmHg (p less than 0.05). In contrast, in the diabetic patients without autonomic neuropathy, there were no significant changes in the mean blood pressure, forearm vascular resistance, calf venous volume and calf venous distensibility. Symptoms of hypoglycaemia did not occur in any patient. These results suggest that insulin has a vasodilator action on both resistance and capacitance vessels, which may be one of the main factors in insulin-induced hypotension.

Cellular cytotoxicity to membrane-associated insulin in an autologous system

A method is described for the measurement of T-cell cytotoxicity to purified preparations of insulin, their high molecular weight fractions and C-peptide derivatives. The technique assesses cellular cytotoxicity to membrane associated insulin in an autologous system. Peripheral blood lymphocytes from 19 type 1 diabetic patients were stimulated in vitro for 6 days with the pharmaceutical preparation(s) used therapeutically. At the end of this period, they were set up in a 4-h cytotoxicity assay against 51Cr labelled PHA blast transformed lymphocytes in the presence of therapeutic insulin, monocomponent insulin, B-component or C-peptide. The results presented here demonstrate cytotoxicity to insulin B-component in diabetic patients who have received pork insulin for 2-6 years. The possible reasons for this finding are discussed.

Differential binding of sulphated insulin to adipocytes and hepatocytes.

The polymerization and precipitation of highly purified insulins which causes major problems in portable infusion systems does not occur with sulphated insulin. To compare the biological behaviour of sulphated insulin with that of a neutral highly purified monocomponent insulin, insulin receptor studies were performed on human and rat adipocytes and rat hepatocytes. Sulphated insulin displayed a lower affinity for binding to both human and rat adipocytes compared with neutral insulin, approximately four times the concentration being required to achieve half-maximal displacement of monoiodoinsulin (p less than 0.05 and 0.01, respectively). A 20-fold higher concentration of sulphated insulin was required for half-maximal displacement from rat hepatocytes (p less than 0.025). However, sulphated insulin bound to liver membranes with an affinity more closely resembling that for adipocytes rather than hepatocytes. Differences in the intracellular processing of the negatively charged insulin could account for the observed lower affinity of binding to hepatocytes.

Monocomponentインスリンの持続的皮下注入療法 (CSII) に続発し, ヒトインスリンが著効を呈したLipoatrophyの1例

Monocomponentインスリンの持続的皮下注入療法 (CSII) に続発し, ヒトインスリンが著効を呈したLipoatrophyの1例