Open-label study to assess the safety and pharmacodynamics of five oral insulin formulations in healthy subjects.

AIM: Orally delivered insulin is predicted to bear therapeutic advantages in diabetes management when compared to injectable insulin, because of its ability to mimic the natural route of endogenous insulin secreted by the pancreas into the portal vein and directly to the liver. Oramed Pharmaceuticals is developing an oral insulin product which consists of unmodified recombinant human insulin combined with adjuvants that protect it from enzymatic degradation in the gastrointestinal tract and promote its absorption from the gut. The aim was to determine the optimal adjuvants to insulin ratio which can provide for the best pharmacodynamic profile, while maintaining the safety of the product. METHODS: Eight healthy, male volunteers participated in this open-label study which included five independent visits. During each visit, subjects were administered one of the five encapsulated oral insulin formulations which contained equal amounts of insulin but varying proportions of adjuvants. Parameters measured included safety, C(max) and T(max) for insulin and C(min), T(min) and area under the curve (AUC) for glucose and c-peptide. Comparisons were made between formulations and between post-treatment time periods within each visit. RESULTS: All five oral insulin formulations were well tolerated and no serious adverse events were reported. All formulations resulted in a significant response in the response period (60-300 min) in comparison to baseline (0-60 min); this was captured both in the c-peptide response and the glucose response (all five formulations p < 0.05). However, none of the formulations turned out significantly different in response over the other. Formulation 5 showed the most profound reduction in c-peptide when AUC(0-60) (baseline) was compared to AUC(60-300) (p < 0.007). CONCLUSIONS: All five oral insulin formulations resulted in glucose and c-peptide reductions, where formulation 5 demonstrated the most pronounced effect on c-peptide concentration reduction. This formulation was deemed the lead formulation to be advanced to future clinical studies. This study also reinforces the notion that oral insulin can maintain its biological activity after delivery, suggesting a potential role for this product in management of diabetes.

Plasminogen activator activity in differentiating leukemia cells.

Plasminogen activator (PA) activity of human promyelocytic leukemia cell line HL-60 was assayed by following the conversion of plasminogen to plasmin and the plasmin-mediated hydrolysis of 14C-labeled globin. When HL-60 cells were induced to differentiate into macrophages by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), cell-associated PA activity and secretion of PA into the conditioned medium increased profoundly. PA activity increased earlier and as a result of lower concentrations of TPA than the ability of the cells to adhere. Exposure to 10(-6)M dexamethasone did not prevent TPA-induced adherence and produced a slight inhibition of cellular PA activity. These findings imply that TPA-induced differentiation of HL-60 cells to macrophage-like cells is associated with induction of PA activity.

Ageing and the mismatch repair system.

Age-related accumulation of mutations has been extensively documented, and it has been proposed as one of the prominent causes of malignancies in old age. The present review is focused on the particular case of DNA mismatch repair system (MMR), that has drawn increased attention for its possible relevance to malignancy. We also report on our own observations on an age-associated genomic instability that develops with age in the MMR system. Our study was performed on DNA samples that were prepared from peripheral blood cells, obtained at a 10-year interval from young and old human subjects. The two DNA samples from each individual were examined comparatively. The older individuals showed a significantly higher rate of microsatellite instability (MSI) in several loci examined, whereas no difference was found between the paired samples of any of the young subjects. We suggest that this increase in MSI with age may indicate an overall genomic instability in the elderly.

Low density lipoprotein particle size and risk factors of insulin resistance syndrome.

The present study aimed to examine the association between low density lipoprotein (LDL) particle size and glucose and insulin variables and with other risk factors that have been related to insulin resistance syndrome. LDL particle size was determined in two groups of subjects who participated in the first examination of the Jerusalem Diabetes Study and who were invited to be re-examined after 8-10 years. The first group were non-diabetic subjects who were found to have at the first examination high insulin levels (above the sex and age specific 90th percentile of the 2 h post-glucose load insulin distribution). The second group was a random sample of individuals who had normal insulin and glucose levels at baseline. Sex-, Age- and body mass index (BMI) mean adjusted LDL-cholesterol (C), triglyceride (TG) and high density lipoprotein cholesterol (HDL-C) levels were significantly different among the LDL subclass groups. Fasting glucose levels and hemoglobin A(1c) did not differ statistically by LDL subclasses. Fasting and 2-h post load insulin levels were significantly higher in persons with LDL subclasses III and IV (small LDL), intermediate in those with LDL subclass II, and lowest in those with LDL subclass I (large LDL). Insulin resistance had an effect on the association between lipids, lipoproteins and LDL particle size. Multivariate analyses indicated that LDL-C, HDL-C and TG were independently associated with LDL particle size variability. The addition of 'insulin resistance' or insulin and glucose levels had no independent effects on LDL particle size. In conclusion, an association of LDL particle size with the cluster of risk factors that characterize the insulin resistance syndrome has been demonstrated. The association of 'insulin resistance' and LDL particle diameter, however, is not mediated directly through the level of insulinemia but via alterations in lipid metabolism.

Enteral administration of insulin in the rat.

1 The effect of the surfactant, Cetomacrogol 1000, on the absorption of insulin across the rectal mucosa has been studied. 2 Rectal administration of microenemata containing Cetomacrogal 1000 and insulin causes a rise in the plasma concentration of insulin and a consequent fall in the blood glucose concentration in diabetic and non-diabetic rats. 3 The hypoglycaemic response is dependent on both the concentration of surfactant and the dose of insulin administered. 4 The results suggest that the transport of insulin across the rectal mucosa is facilitated by Cetomacrogal 1000.

Absorption of protein via the intestinal wall. A quantitative model.

Intact, biological active insulin and pancreatic RNase can be absorbed from the intestinal lumen into the blood circulation. The absorption is dependent on the addition of bile acid (sodium cholate) and proteinase inhibitor. The quantitative absorption of insulin and pancreatic RNase has been demonstrated in an in situ model. The amount of insulin absorbed after 30 min from the ileum to the mesenteric vein was 0.025% of the initial amount. Sodium cholate (10 mg/ml) and 3000 KIU/ml aprotinin enhanced this absorption by 30 times. The amount of pancreatic RNase which was absorbed from the ileum to the blood was 0.002% of the initial amount during 30 min. Sodium cholate (10 mg/ml) and 3000 KIU/ml aprotinin increased the absorption by a factor of 200. No damage occurred to the intestine during the experimental procedures. The sieving characteristics of the intestinal wall were not altered by the presence of sodium cholate and proteinase inhibitor in the intestinal lumen. These results suggest that sodium cholate and proteinase inhibitors can facilitate the absorption of intact, biologically active proteins across the intestinal wall.

Bile salts facilitate the absorption of heparin from the intestine.

Heparin was absorbed through the rectal mucosa of rodents and primates only when administered in solutions containing sodium cholate or sodium deoxycholate (DOC). The absorption of heparin was monitored by following the increase in plasma radioactivity after administration of [35S]heparin, and by measurement of its biological effects: plasma lipase activity and prolongation of partial thromboplastin time (PTT). Following administration of the same concn of heparin in solutions lacking bile salts, there was almost no radioactivity in the blood, no prolongation of PTT and no release of plasma lipase activity. The PTT effect was found to be a less sensitive test of heparin absorption than the plasma lipase activity.

Oral administration of insulin in solid form to nondiabetic and diabetic dogs.

It was previously demonstrated that a biologically active insulin could cross the mucosal membrane in the gut by using surface active substances. In this report we describe studies in which insulin administered orally, in a solid formulation, was effectively absorbed in the canine model. The insulin was mixed with cholate and soybean trypsin inhibitor. It was delivered orally, as enterocoated microtablets, to nondiabetic and diabetic (pancreatectomized) dogs in a fasting state. The time interval between the administration of the drug and the beginning of a decrease in the plasma glucose levels was 60-140 min. This decrease reached a minimum level of 20-40 % of the initial values and lasted for more than 90 min following administration of the drug. In this model a pronounced increment in plasma insulin levels was shown prior to the drop of plasma glucose concentrations. It is concluded that with this novel oral insulin formulation a beneficial biological effect can be achieved in the treatment of diabetes.

Protease inhibitor activity in rat skeletal muscle.

The cytosol fraction obtained by homogenization of rat gastrocnemius muscle inhibited the activities of rat alkaline myofibrillar protease, bovine trypsin and bovine chymotrypsin. The inhibition of the three proteolytic enzymes by muscle cytosol changed differentially during ageing, fasting and following administration of glucocorticoid hormone. The inhibition exerted by the cytosol on the three proteases was also differentially affected by precipitation with trichloracetic acid, heat, dialysis and molecular sieve chromatography. It is suggested that the intracellular protease inhibitor(s) are involved in the regulation of muscle protein degradation.

Changes in acid proteolytic activity during differentiation of murine erythroleukemic cells.

Proteolytic activity was measured in murine erythroleukemic 745 cell line grown in culture, before and after the addition of agents which promote differentiation. The 36,000 X g soluble fraction of the cells degraded [14C]globin with maximal activity at pH 3.6, while the insoluble fraction failed to degrade [14C]globin within a pH range of 2.5-9.0. The acid protease activity in the soluble fraction of the undifferentiated murine erythroleukemic cells increased during the first 2 days in culture and remained constant during the following 4 days. We suggest that this activity resides in the lysosomes since it migrates together with the lysosomal marker alpha-mannosidase on colloidal silica gradients, shows maximum activity at acid pH and is sensitive towards inhibition by pepstatin. Induced differentiation of the cells by dimethyl sulfoxide, butyric acid or hexamethylene bisacetamide was concomitantly associated with a marked reduction in protease activity and the accumulation of hemoglobin within the cells. In contrast, in a non-inducible variant of 745 cell line DMSO failed to affect proteolysis. It is suggested that in murine erythroleukemic cells changes in acid protease activity are associated with the cellular triggered by chemical inducers.

Protease inhibitor activity in human skeletal muscle.

Myofibrillar protease activity and activity of inhibitors toward trypsin, chymotrypsin, elastase, and the myofibrillar protease were determined in human skeletal muscle. The protease activity was found to increase in patients with acute and chronic inflammation as well as in patients with metastatic and nonmetastatic tumors. The inhibitory activity directed against trypsin and chymotrypsin was not affected by acute inflammation, while the inhibition of elastase and the myofibrillar protease was increased. Chronic inflammation did not affect the ability of the muscle cytosol to inhibit trypsin and elastase, but increased the inhibition of chymotrypsin and the myofibrillar protease. Nonmetastatic tumors produced an increase in the activity of inhibitors toward trypsin, chymotrypsin, and elastase, while patients bearing metastatic tumors had a high level of cytosolic inhibitors of all the tested proteolytic activities. These results indicate that the myofibrillar protease and the cytosolic protease inhibitors in human skeletal muscle are differentially affected by catabolic conditions.

Induction of differentiation in human myeloid leukemic cells by proteolytic enzymes.

Exogenous serine proteases were found to induce differentiation in human myeloid leukemic cells from either in vitro established long-term cell lines or in primary cultures of cells derived directly from patients with acute myeloid leukemia. Exposure of the human promyelocytic cell line HL-60 to trypsin, chymotrypsin, or elastase induced the appearance, within 3-6 days, of neutrophilic granulocytes defined by their morphology, their ability to reduce nitroblue tetrazolium, and their efficient phagocytosis of latex particles. Upon further incubation monocyte-like cells appeared. While these cells developed into fully mature macrophages other types of cells disappeared and on day 12 the culture consisted of a pure macrophage population. The inducing effect could be observed when the enzyme was presented alone, whereas a synergistic effect was noted when the protease was added in the presence of subthreshold concentrations of chemicals known to induce differentiation in this cell line such as dimethylsulfoxide, retinoic acid, butyric acid, or hexamethylene bisacetamide. Optimal induction of differentiation by trypsin required a 48 hr continuous exposure to the enzyme. When the protease was removed earlier no appreciable differentiation was noticed. The protease-induced differentiation involved a direct interaction with the cells and was not due to a proteolytic cleavage of a serum component because it could be obtained in serum-free cultures. The enzymatic activity of the protease was needed for its effect on cell maturation: Addition of protease inhibitors such as soybean-trypsin inhibitor or trasylol completely blocked differentiation induced by the proteases but had no effect on differentiation induced by the other inducers. It is still to be determined whether a proteolytic process is a general molecular event in cell differentiation or induction by chemicals involves a mechanism different from that initiated by exogenous proteases.

Phorbol ester-induced adhesion of murine erythroleukemia cells: possible involvement of cellular proteases.

Phorbol ester tumor promoters produce a rapid increase in adhesiveness of murine erythroleukemia (MEL) cells. Following treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and other tumor promoters, these cells adhere to the surface of the culture dish or become agglutinated to each other. Structurally related compounds which are devoid of tumor promoting activity failed to induce agglutination of MEL cells. Pentamidine isethionate (PI) and tosylamide-phenylethyl-chloromethyl ketone, two known inhibitors of trypsin-like enzymes, prevent the phorbol esters-induced adherence and agglutination. A short exposure to TPA results in an increase in protease activity at the alkaline pH range. This TPA-induced proteolytic activity is inhibited by PI. Induction of erythroid differentiation by hexamethylene-bisacetamide is associated with a decrease in TPA-induced cell adhesion and TPA-induced proteolytic activity. Taken together, these results suggest the participation of an alkaline proteolytic activity in the membranal changes evoked by phorbol esters.

Inhibition of phorbol-ester-induced adhesion of differentiating human myeloid leukemic cells by pentamidine-isethionate.

Human myeloid leukemia cells can be induced to differentiate into macrophage-like cells by various phorbol esters, particularly 12-O-tetradecanoyl-phorbol-14-acetate (TPA). In this study, the effect of several known protease inhibitors on TPA-induced differentiation of human acute promyelocytic leukemia cells (line HL-60) was tested. Among the test compounds, only pentamidine-isethionate (PI), an inhibitor of trypsin-like enzymes, prevented one early marker of differentiation, e.g. cell adherence to plastic and glass surfaces. However, PI failed to affect other markers of differentiation and did not inhibit readherence of scraped and resuspended TPA-treated cells. Exposure to TPA resulted in a decrease in the cellular alkaline proteolytic activity and an increase in the acid proteolytic activity. PI further inhibited the residual activity of the alkaline protease in the 36,000 g pellet fraction of the TPA-treated cells, but did not reduce this activity in control cells. The present results indicate, on the basis of the differential effects of PI, that the emergence of differentiation markers in HL-60 cells following exposure to TPA is independent of the induction of adherence.

Univariate and bivariate admixture analyses of serum glucose and glycated hemoglobin distributions in a Jerusalem population sample.

Univariate and bivariate analyses of fasting glucose and glycated hemoglobin (HbA1c) levels and of glucose levels 2 hours after an oral glucose load test were performed in a random sample of the Jewish population of Jerusalem, aged 25-64 years. Using ln-transformed data, we found that a mixture of two distributions fits the glucose data significantly better than a single distribution in the age groups 25-44 and 45-64 years. The fasting glucose results indicate that 1.1% of subjects aged 25-44 and 3.7% of subjects aged 45-64 without known diabetes come from an upper distribution with mean values of 154 mg/dl and 224 mg/dl, respectively. Estimates from the analysis of glucose levels after a load test indicate that an additional 2.1% of younger subjects and 3.8% of older subjects belong to a minor distribution with a high mean glucose value. The frequency distribution of HbA1c is also bimodal in all age groups. Yet the bimodality indicates that only 0.1% and 2.3% of subjects in the two age groups, respectively, come from minor distributions with mean levels of 13.0-15.7%, compared with HbA1c values of 5.0% and 5.3% for the main distributions. Using glucose levels, we found that specificity rates are consistently greater than 99%, whereas sensitivity rates vary with age. The use of cutoff points suggested by the National Diabetes Data Group (140 mg/dl for fasting glucose level and 200 mg/dl for glucose level after an oral glucose load test) indicates a lower sensitivity rate in the younger subjects with a minimal improvement among older subjects. A mixture of bivariate log-normal distributions fitted to the fasting and 2-hour glucose levels in subjects aged 45-64 indicates a larger proportion (6.3%) belonging to the minor distribution compared with those obtained when a single variable is used. Yet this combined score shows a low specificity rate. No similar improvement in separating "normal" from "abnormal" subjects is achieved when a mixture of bivariate distributions is fitted to the glucose and HbA1c variables. Admixture of glucose and HbA1c distributions is demonstrated. Bivariate analysis of these distributions does not, however, provide better discrimination of putatively abnormal subjects than univariate analysis.

Biochemical and morpho-cytochemical evidence for the intestinal absorption of insulin in control and diabetic rats. Comparison between the effectiveness of duodenal and colon mucosa.

A combined biochemical and morpho-cytochemical investigation was carried out in order to assess insulin absorption by the duodenal and colon epithelium. Insulin was introduced in the lumen of the rat duodenum or colon in combination with sodium cholate and aprotinin. Blood analysis made at several time points has demonstrated a rapid increase in circulating levels of insulin followed by significant and consistent decreases in blood glucose. This indicates that biologically active insulin is absorbed by the intestinal mucosa and transferred to the circulation. Because of the initial high blood glucose levels, the lowering of the glycaemic values was more significant in diabetic animals. Also, levels of circulating insulin remained higher for longer time when the administration was performed in the colon. The integrity of the intestinal wall after insulin administration, evaluated morphologically, was retained. Application of protein A-gold immunocytochemistry has established the pathway for insulin absorption. In both duodenal and colon epithelial cells the labelling for insulin was detected in the endosomal compartment, in the Golgi apparatus and in association with the baso-lateral plasma membrane interdigitations. Some labelling was also present in the interstitial space and in capillary endothelial plasmalemmal vesicles. Insulin introduced in the lumen of the rat duodenum and colon appears thus to be rapidly internalized by the epithelial cells and transferred through a transcytotic pathway to the interstitial space from which it reaches the blood circulation. This exogenous insulin then induces significant decreases in plasma glucose levels which lasts for several hours. The results obtained support the possibility for the clinical development of an oral preparation of insulin.