Oligomerization of Pharmaceutically Relevant Insulin Analogues for Varying Concentration and Salinity Revealed by Small-Angle X-ray Scattering.

Two different insulin analogues, insulin degludec and lithocholyl insulin, were studied by small-angle X-ray scattering with respect to their self-assembly and interactions in solution at different concentrations of insulin and salt, NaCl. Very different behavior was observed for the two. Insulin degludec, linked to a hexadecanedioic acid, consistently formed di-hexamers, without any further oligomeric growth upon screening of electrostatic repulsions, indicating a stable di-hexamer unit without further oligomerization, as expected in the presence of phenol. The other insulin analogue, linked to the sterol lithocholic acid, formed n-hexamers with n ranging from 1 to 15, increasing with NaCl concentration and insulin concentration, indicating attractive forces in competition with the electrostatic repulsion and solution entropy. At the highest concentration of insulin and NaCl, a liquid crystal phase was observed, which has not previously been identified, featuring a quadratic structure organized into layers, which might hold interesting properties for pharmaceutical applications.

Expression, purification, and characterization of the Degludec precursor DesB30.

Diabetes is a chronic metabolic disease, for which recombinant human insulin is the most effective and mainstream treatment. DesB30 is an insulin analogue in which the B chain lacks amino acid 30 (Thr) compared with human insulin. This analogue can be used to produce the long-acting insulin Degludec or Detemir. In this study, a spacer peptide was added before the sequence of DesB30 and the C-peptide was replaced with AAK, a short connecting peptide. The target gene was cloned under the control of AOX1 and expressed as a secretory protein in Pichia pastoris. A high-yield recombination strain was selected by screening for resistance to G418. The basal salts medium was optimized and a lower salt concentration medium was found to show the best effects. Both media were used to compare the yield of high-density fermentation. The maximum yield reached 4.51 g/L in 1/2 basal salt medium, which is the highest reported yield to date. The insulin precursor, which is single-stranded, was purified by weak cation exchange chromatograph and preparative reversed-phase high-performance liquid chromatography (RP-HPLC), from which 73.39% of product was recovered at over 95% purity. The double-stranded protein (DesB30) was obtained by digesting the insulin precursor with trypsin. Using preparative RP-HPLC, the product was obtained with over 95% purity. Finally, the structure of DesB30 was confirmed.

LAPS Insulin115: A novel ultra-long-acting basal insulin with a unique action profile.

AIMS: To conduct a comprehensive pre-clinical study of the novel ultra-long acting insulin analogue LAPS Insulin115. METHODS: Pharmacokinetic/pharmacodynamic studies comparing LAPS Insulin115 with other basal insulins were conducted in genetically diabetic (db/db) mice. Insulin signalling in the major target organs was analysed using Western blot after single subcutaneous injection in wild-type male Wistar rats. Using in vitro assays we analysed transendothelial transport, insulin receptor (IR) interaction, and the mitogenic and metabolic properties of LAPS Insulin115. Furthermore, IR downregulation after long-term exposure to high concentrations of LAPS Insulin115 was analysed using an in vitro desensitization/resensitization model. RESULTS: The novel Fc-conjugated insulin derivative LAPS Insulin115 showed an extensively prolonged pharmacokinetic and pharmacodynamic profile in rodents. Despite its size of 59 kDa, LAPS Insulin115 passes the vascular endothelial barrier and induces insulin signalling in all major target tissues in rats. In vitro, LAPS Insulin115 showed a very slow onset of action because of its reduced IR affinity; however, after long-term stimulation it was equipotent in respect to its metabolic potency and showed no increased mitogenic action when compared with regular insulin. Remarkably, under conditions of chronic exposure, LAPS Insulin115 does not induce irreversible desensitization of target cells, which is probably attributable to much less prominent IR downregulation. CONCLUSION: Thus, LAPS Insulin115 exhibits a unique in vivo and in vitro profile and thereby represents an excellent candidate for a once-weekly insulin analogue.

Investigation of the Physico-Chemical Properties that Enable Co-Formulation of Basal Insulin Degludec with Fast-Acting Insulin Aspart.

PURPOSE: To study the self-association states of insulin degludec and insulin aspart alone and combined in pharmaceutical formulation and under conditions simulating the subcutaneous depot. METHODS: Formulations were made of 0.6 mM degludec at 3 and 5 Zn/6 insulin monomers, and 0.6 mM aspart (2 Zn/6 insulin monomers). Self-association was assessed using size-exclusion chromatography (SEC) monitored by UV and orthogonal reverse-phase chromatography. RESULTS: Simulating pharmaceutical formulation, degludec eluted as dihexamers, whereas aspart eluted as hexamers and monomers. Combining degludec at low zinc with aspart increased dihexamer content, indicating hybrid hexamer formation. At high zinc concentration, however, there was no evidence of this. Simulating the subcutaneous depot by removing preservative, degludec eluted as multihexamers and aspart as monomers. Aspart was incorporated into the multihexamer structures when combined with degludec at low zinc, but there was no such interaction with high-zinc degludec. SEC using progressively diluted concentrations of phenol and m-cresol showed that dissociation of aspart into monomers occurs before the formation of degludec multihexamers. CONCLUSION: Insulins degludec and aspart can be combined without forming hybrid hexamers, but this combinability is dependent on zinc and preservative concentration, and requires that degludec is fully dihexameric before addition of aspart.

Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris.

Glargine is an analog of Insulin currently being produced by recombinant DNA technology using two different hosts namely Escherichia coli and Pichia pastoris. Production from E. coli involves the steps of extraction of inclusion bodies by cell lysis, refolding, proteolytic cleavage and purification. In P. pastoris, a single-chain precursor with appropriate disulfide bonding is secreted to the medium. Downstream processing currently involves use of trypsin which converts the precursor into two-chain final product. The use of trypsin in the process generates additional impurities due to presence of Lys and Arg residues in the Glargine molecule. In this study, we describe an alternate approach involving over-expression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (Arg-Arg) at the end of B-chain of Glargine. KEX2 gene over-expression in Pichia was accomplished by using promoters of varying strengths to ensure production of greater levels of fully functional two-chain Glargine product, confirmed by HPLC and mass analysis. In conclusion, this new production process involving Kex2 protease over-expression improves the downstream process efficiency, reduces the levels of impurities generated and decreases the use of raw materials.

Extensive study of human insulin immunoassays: promises and pitfalls for insulin analogue detection and quantification.

BACKGROUND: Over the last few decades, new synthetic insulin analogues have been developed. Their measurement is of prime importance in the investigation of hypoglycaemia, but their quantification is hampered by variable cross-reactivity with many insulin assays. For clinical analysis, it has now become essential to know the potential cross-reactivity of analogues of interest. METHODS: In this work, we performed an extensive study of insulin analogue cross-reactivity using numerous human insulin immunoassays. We investigated the cross-reactivity of five analogues (lispro, aspart, glulisine, glargine, detemir) and two glargine metabolites (M1 and M2) with 16 commercial human insulin immunoassays as a function of concentration. RESULTS: The cross-reactivity values for insulin analogues or glargine metabolites ranged from 0% to 264%. Four assays were more specific to human insulin, resulting in negligible cross-reactivity with the analogues. However, none of the 16 assays was completely free of cross-reactivity with analogues or metabolites. The results show that analogue cross-reactivity, which varies to a large degree, is far from negligible, and should not be overlooked in clinical investigations. CONCLUSIONS: This study has established the cross-reactivity of five insulin analogues and two glargine metabolites using 16 immunoassays to facilitate the choice of the immunoassay(s) and to provide sensitive and specific analyses in clinical routine or investigation.

Concentrations of insulin glargine and its metabolites during long-term insulin therapy in type 2 diabetic patients and comparison of effects of insulin glargine, its metabolites, IGF-I, and human insulin on insulin and igf-I receptor signaling.

We investigated 1) the ability of purified glargine (GLA), metabolites 1 (M1) and 2 (M2), IGF-I, and NPH insulin to activate the insulin receptor (IR)-A and IR-B and IGF-I receptor (IGF-IR) in vitro; 2) plasma concentrations of GLA, M1, and M2 during long-term insulin therapy in type 2 diabetic patients; and 3) IR-A and IR-B activation in vitro induced by serum from patients treated with GLA or NPH insulin. A total of 104 patients (age 56.3 ± 0.8 years, BMI 31.4 ± 0.5 kg/m(2), and A1C 9.1 ± 0.1% [mean ± SE]) were randomized to GLA or NPH insulin therapy for 36 weeks. Plasma concentrations of GLA, M1, and M2 were determined by liquid chromatography-tandem mass spectrometry assay. IR-A, IR-B, and IGF-IR autophosphorylation was induced by purified hormones or serum by kinase receptor activation assays. In vitro, M1 induced comparable IR-A, IR-B, and IGF-IR autophosphorylation (activation) as NPH insulin. After 36 weeks, M1 increased from undetectable (<0.2 ng/mL) to 1.5 ng/mL (0.9-2.1), while GLA and M2 remained undetectable. GLA dose correlated with M1 (r = 0.84; P < 0.001). Serum from patients treated with GLA or NPH insulin induced similar IR-A and IR-B activation. These data suggest that M1 rather than GLA mediates GLA effects and that compared with NPH insulin, GLA does not increase IGF-IR signaling during long-term insulin therapy in type 2 diabetes.

Hyperglycaemia is associated with impaired pulsatile insulin secretion: effect of basal insulin therapy.

AIM: Postprandial insulin pulsatility is impaired in patients with type 2 diabetes, but the effects of exogenous insulin therapy on pulsatile insulin secretion are not known. We addressed, whether pulsatile insulin secretion is related to glycaemic control, whether basal insulin supplementation increases postprandial insulin secretion, and if so, is this accomplished by a specific improvement in pulsatile insulin secretion? METHODS: Fourteen patients with type 2 diabetes underwent a mixed meal test before and after an 8-week treatment period with insulin glargine. Glucose, insulin and C-peptide levels were measured, and insulin pulsatility was determined by deconvolution analysis. RESULTS: Insulin treatment lowered fasting glycaemia from 179.6 ± 7.5 mg/dl to 117.6 ± 6.5 mg/dl (p < 0.001). Postprandial insulin and C-peptide levels increased significantly after the treatment period (p < 0.0001). The total calculated insulin secretion rate increased with insulin treatment (p = 0.0039), with non-significant increases in both pulsatile and non-pulsatile insulin secretion. Insulin pulse frequency was unchanged by the intervention. There was an inverse relationship between fasting and postprandial glycaemia and insulin pulse mass (r(2) = 0.51 and 0.56, respectively), whereas non-pulsatile insulin secretion was unrelated to either fasting or postprandial glucose concentrations (r(2) = 0.0073 and 0.031). CONCLUSIONS: Hyperglycaemia in type 2 diabetes is associated with a reduction in postprandial insulin secretion, specifically through a reduction in insulin pulsatility. Reducing chronic hyperglycaemia by basal insulin therapy enhances endogenous ß-cell function in the postprandial state. These data support the use of basal insulin regimens in the pharmacotherapy of overtly hyperglycaemic patients with type 2 diabetes.

Ligand-controlled assembly of hexamers, dihexamers, and linear multihexamer structures by the engineered acylated insulin degludec.

Insulin degludec, an engineered acylated insulin, was recently reported to form a soluble depot after subcutaneous injection with a subsequent slow release of insulin and an ultralong glucose-lowering effect in excess of 40 h in humans. We describe the structure, ligand binding properties, and self-assemblies of insulin degludec using orthogonal structural methods. The protein fold adopted by insulin degludec is very similar to that of human insulin. Hexamers in the R(6) state similar to those of human insulin are observed for insulin degludec in the presence of zinc and resorcinol. However, under conditions comparable to the pharmaceutical formulation comprising zinc and phenol, insulin degludec forms finite dihexamers that are composed of hexamers in the T(3)R(3) state that interact to form an R(3)T(3)-T(3)R(3) structure. When the phenolic ligand is depleted and the solvent condition thereby mimics that of the injection site, the quaternary structure changes from dihexamers to a supramolecular structure composed of linear arrays of hundreds of hexamers in the T(6) state and an average molar mass, M(0), of 59.7 × 10(3) kg/mol. This novel concept of self-assemblies of insulin controlled by zinc and phenol provides the basis for the slow action profile of insulin degludec. To the best of our knowledge, this report for the first time describes a tight linkage between quaternary insulin structures of hexamers, dihexamers, and multihexamers and their allosteric state and its origin in the inherent propensity of the insulin hexamer for allosteric half-site reactivity.

Plasma exposure to insulin glargine and its metabolites M1 and M2 after subcutaneous injection of therapeutic and supratherapeutic doses of glargine in subjects with type 1 diabetes.

OBJECTIVE: In vivo, after subcutaneous injection, insulin glargine (21(A)-Gly-31(B)-Arg-32(B)-Arg-human insulin) is enzymatically processed into 21(A)-Gly-human insulin (metabolite 1 [M1]). 21(A)-Gly-des-30(B)-Thr-human insulin (metabolite 2 [M2]) is also found. In vitro, glargine exhibits slightly higher affinity, whereas M1 and M2 exhibit lower affinity for IGF-1 receptor, as well as mitogenic properties, versus human insulin. The aim of the study was to quantitate plasma concentrations of glargine, M1, and M2 after subcutaneous injection of glargine in male type 1 diabetic subjects. RESEARCH DESIGN AND METHODS: Glargine, M1, and M2 were determined in blood samples obtained from 12, 11, and 11 type 1 diabetic subjects who received single subcutaneous doses of 0.3, 0.6, or 1.2 units · kg(-1) glargine in a euglycemic clamp study. Glargine, M1, and M2 were extracted using immunoaffinity columns and quantified by a specific liquid chromatography-tandem mass spectrometry assay. Lower limit of quantification was 0.2 ng · mL(-1) (33 pmol · L(-1)) per analyte. RESULTS: Plasma M1 concentration increased with increasing dose; geometric mean (percent coefficient of variation) M1-area under the curve between time of dosing and 30 h after dosing (AUC(0-30h)) was 1,261 (66), 2,867 (35), and 4,693 (22) pmol · h · L(-1) at doses of 0.3, 0.6, and 1.2 units · kg(-1), respectively, and correlated with metabolic effect assessed as pharmacodynamics-AUC(0-30h) of the glucose infusion rate following glargine administration (r = 0.74; P < 0.01). Glargine and M2 were detectable in only one-third of subjects and at a few time points. CONCLUSIONS: After subcutaneous injection of glargine in male subjects with type 1 diabetes, exposure to glargine is marginal, if any, even at supratherapeutic doses. Glargine is rapidly and nearly completely processed to M1 (21(A)-Gly-human insulin), which mediates the metabolic effect of injected glargine.

Effect of insulin analogues on insulin/IGF1 hybrid receptors: increased activation by glargine but not by its metabolites M1 and M2.

BACKGROUND: In diabetic patients, the pharmacokinetics of injected human insulin does not permit optimal control of glycemia. Fast and slow acting insulin analogues have been developed, but they may have adverse properties, such as increased mitogenic or anti-apoptotic signaling. Insulin/IGF1 hybrid receptors (IR/IGF1R), present in most tissues, have been proposed to transmit biological effects close to those of IGF1R. However, the study of hybrid receptors is difficult because of the presence of IR and IGF1R homodimers. Our objective was to perform the first study on the pharmacological properties of the five marketed insulin analogues towards IR/IGF1R hybrids. METHODOLOGY: To study the effect of insulin analogues on IR/IGF1R hybrids, we used our previously developed Bioluminescence Resonance Energy Transfer (BRET) assay that permits specific analysis of the pharmacological properties of hybrid receptors. Moreover, we have developed a new, highly sensitive BRET-based assay to monitor phophatidylinositol-3 phosphate (PIP(3)) production in living cells. Using this assay, we performed a detailed pharmacological analysis of PIP(3) production induced by IGF1, insulin and insulin analogues in living breast cancer-derived MCF-7 and MDA-MB231 cells. RESULTS: Among the five insulin analogues tested, only glargine stimulated IR/IGF1R hybrids with an EC50 that was significantly lower than insulin and close to that of IGF1. Glargine more efficiently stimulated PIP(3) production in MCF-7 cells but not in MDA-MB231 cells as compared to insulin. In contrast, glargine metabolites M1 and M2 showed lower potency for hybrid receptors stimulation, PIP(3) production, Akt and Erk1/2 phosphorylation and DNA synthesis in MCF-7 cells, compared to insulin. CONCLUSION: Glargine, possibly acting through IR/IGF1R hybrids, displays higher potency, whereas its metabolites M1 and M2 display lower potency than insulin for the stimulation of proliferative/anti-apoptotic pathways in MCF-7 cells.

Controlled release of modified insulin glargine from novel biodegradable injectable gels.

The objective of this study was to investigate the duration of biological effects of modified insulin glargine released from a novel biodegradable injectable gel in type II diabetic Zucker diabetic fatty (ZDF) rats. Modified insulin glargine was purified from the marketed formulation by process of dialysis followed by freeze-drying, and the purity was confirmed by the single peak, corresponding to insulin glargine in the HPLC chromatogram. To determine and to compare the biological activity of purified insulin glargine with marketed formulation, it was suspended in isotonic saline solutions and administered subcutaneously to ZDF rats at a dose of 10 IU/kg of insulin and the blood glucose levels were measured. The blood glucose levels of ZDF rats after a subcutaneous injection of a suspension of purified insulin glargine decreased below 200 mg/dL within 2 h and remained at this level up to 6 h, then steadily raised above 400 mg/dL in 12 h. Insulin glargine particles were loaded into a novel biodegradable injectable gel formulation prepared from a blend of polylactic-co-glycolic acid (PLGA) and biocompatible plasticizers. Approximately 0.1 mL of insulin glargine-loaded gel prepared with PLGA was administered subcutaneously to the ZDF rats, and blood glucose levels were measured. The PLGA gel formulations prepared with insulin glargine particles had duration of action of 10 days following a single subcutaneous injection. The addition of zinc sulfate to the formulations prepared with purified insulin glargine particles further slowed down the drop in blood glucose concentrations.

Insulin and glucose profiles during continuous subcutaneous insulin infusion compared with injection of a long-acting insulin in Type 2 diabetes.

AIMS: To compare insulin and glucose profiles during basal continuous subcutaneous infusion of a rapid-acting insulin analogue and once daily subcutaneous injection of a long-acting insulin analogue in Type 2 diabetes. METHODS: Twenty-one patients with Type 2 diabetes treated with oral glucose-lowering agents were randomized in this two-period crossover study to an equivalent 24-h dose of continuous subcutaneous infusion of insulin aspart and subsequently once-daily bedtime subcutaneous injection of insulin glargine, or vice versa, for eight consecutive days. Plasma profiles of insulin and glucose were recorded. RESULTS: On the last day of each treatment period, the area under the curve (AUC) for glucose was 10% lower on the continuous subcutaneous infusion regimen compared with the insulin injection regimen (P = 0.002). This was accomplished by a flat exogenous insulin infusion profile compared with a peaking profile with injected insulin (AUC was 74% higher after injection compared with pre-injection levels (P = 0.001)). During the last 6 days in each treatment period, the intra-subject variability of exogenous fasting insulin levels in the mornings was 41% lower during insulin infusion compared with insulin injection (P = 0.012). The corresponding intra-subject variability for fasting glucose only showed a tendency to be lower during infusion as compared to the injection regimen (28%; P = 0.104). Thirteen symptomatic-only or minor hypoglycaemic episodes were recorded during the entire infusion period compared with three episodes during the injection period. CONCLUSIONS: Basal continuous subcutaneous infusion of a rapid-acting insulin analogue improved plasma insulin (more flat insulin profile with a lower variability) and glucose (lower AUC) profiles compared with once-daily subcutaneous injection of a long-acting insulin analogue in Type 2 diabetes.

Nasal insulin delivery in the chitosan solution: in vitro and in vivo studies.

The effects of chitosan concentrations, osmolarity, medium and absorption enhancers in the chitosan solution on nasal insulin delivery were studied in vitro and in vivo. The penetration of insulin through the mucosa of rabbit nasal septum was investigated by measuring the transmucosal flux in vitro, while the nasal absorption of insulin in vivo was assessed by the efficiency in lowering the blood glucose levels in normal rats. It was demonstrated that increasing concentrations of chitosan up to 1.5% (w/v) caused an increase in the permeability of insulin across the nasal mucosa. Insulin given intranasally in hypo- or hyperosmotic formulation showed a higher hypoglycemic effect than insulin delivered in isoosmotic formulation. Insulin formulation in chitosan solution prepared with deionized water brought to a higher relative pharmacological bioavailability (Fr) value than that prepared with 50 mM pH 7.4 phosphate buffer. A formulation containing both 1% chitosan and 0.1% ethylenediaminetetraacetic acid (EDTA), 5% polysorbate 80 (Tween 80) or 1.2% beta-cyclodextrin (beta-CD) did not lead to a higher Fr than insulin formulated with 1% chitosan alone. The formulation containing both 5% hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and 1% chitosan was more effective at reducing blood glucose levels than the formulation containing 5% HP-beta-CD or 1% chitosan alone. The studies indicated that chitosan concentrations, osmolarity, medium and absorption enhancers in chitosan solution have significant effect on the insulin nasal delivery. The results of in vitro experiments were in good agreement with that of in vivo studies.

Absorption kinetics and action profiles after sequential subcutaneous administration of human soluble and lente insulin through one needle.

The effects of sequential administration through one needle of human soluble and human lente insulin on plasma insulin levels and action profiles, assessed with glucose clamping, were studied in six healthy volunteers. Insulin kinetics after administration of human soluble insulin (0.22 IU/kg) alone were compared with those after sequential administration of 1) human soluble insulin followed by human lente insulin and 2) human lente insulin followed by human soluble insulin. Total insulin dose in both sequences was 0.55 IU/kg, 40% of which was short-acting insulin. Plasma insulin levels were not significantly different at any time point between 0 and 240 min after soluble insulin compared with either combination. Although insulin levels were slightly but significantly lower at 30 and 105 min after lente followed by soluble insulin compared with soluble followed by lente insulin, these differences probably reflect chance occurrences. Glucose requirements were not significantly different after either of the three administrations. We therefore conclude that the unwanted retarding effect after mixing of human soluble insulin with human lente insulin in the syringe on the onset of action of the soluble insulin can be prevented by sequential subcutaneous injection of these insulins with two syringes through one needle.

Relationship between subcutaneous blood flow and absorption of lente type insulin.

To study the relationship between the absorption of intermediate acting insulin and the local subcutaneous blood flow (SBF) 8 diabetic patients were given subcutaneous injections of 125I labeled human lente type insulin and 133Xenon in the abdominal wall. External measurements of the tracer disappearance were performed and the insulin absorption rate and SBF were calculated. A curvilinear relationship between the insulin absorption rate and SBF was demonstrated with an initial almost linear relation but with decreasing impact of SBF when this was in the higher physiological range.

A comparison of the pharmacokinetics of human protamine sodium insulin with human isophane insulin following subcutaneous injection in normal subjects.

The pharmacokinetics of human protamine sodium insulin 0.6 mg% were compared with those of human isophane zinc insulin using the glucose clamp technique. The insulin preparations were administered subcutaneously at a dose of 0.5 U/kg in 7 normal subjects; a placebo injection served as control. Plasma insulin and C-peptide concentrations were then measured over 24 hours. After correction of the results to allow for endogenous insulin secretion the two insulin preparations gave similar values throughout the study except that protamine sodium insulin achieved an earlier peak in plasma insulin concentration (90 minutes) compared to isophane (120 minutes). When measured by an index of the dextrose infusion rate required to maintain euglycaemia the two insulin preparations gave similar values for the first 16 hours, maximum values being obtained during the fourth hour after administration. Using this index isophane zinc insulin gave higher values during the 17th, 19th and 21st hours following administration suggesting it may have a slightly more prolonged action than protamine sodium insulin.

[Relations between the site of insulin absorption and hypoglycemia in insulin-dependent diabetes during physical exertion]. / Rapporto tra sedi di assorbimento dell'insulina e ipoglicemia nel diabete insulino-dipendente durante l'esercizio fisico.

Hypoglycaemia often arises after physical exercise in insulin-dependent diabetics treated with fast and slow acting insulin. Some claim that this is closely linked to the injection site for fast insulin and related to altered mobilization of the hormone from its subcutaneous deposit. Others attribute it to hyperinsulinism. The hypoglycaemia occurring after physical exercise in subjects treated with slow insulin and that arising even hours after physical exercise are then considered.

The subcutaneous absorption of human and bovine ultralente insulin formulations.

The subcutaneous absorption of 125I-labelled semisynthetic human ultralente insulin was compared with that of 125I-labelled beef ultralente in eight insulin-dependent diabetic patients using a balanced cross-over design. In randomized order, 6 U of human ultralente was given in the thigh and in the abdominal wall while 24 U was given in the contralateral thigh and 6 U of bovine ultralente in the abdominal wall. On a second occasion the injection sites of human and bovine ultralente were reversed. Human ultralente was absorbed significantly faster than bovine ultralente. Thus, the 50% disappearance time of the radioactivity in the thigh was 15 +/- 3 h versus 44 +/- 9 h for the high dose, and 13 +/- 2 h versus 21 +/- 5 h for the small dose for human and bovine ultralente, respectively. Correspondingly, values for the abdomen (6U) were 9 +/- 1 h and 16 +/- 2 h. The difference between the absorption rates for 6 U and 24 U was statistically significant for bovine ultralente only. Human ultralente, but not bovine ultralente showed faster absorption from the abdomen than from the thigh. The absorption rate of human ultralente insulin, though faster than bovine ultralente insulin, makes it suitable as the basal insulin preparation for multiple insulin injection regimens.