Jens Jorgen Veilgaard Brange

Monomeric Insulins and Their Experimental and Clinical Implications

Due to the inherent pharmacokinetic properties of available insulins, normoglycemia is rarely, if ever, achieved in insulin-dependent diabetic patients without compromising their quality of life. Subcutaneous insulin absorption is influenced by many factors, among which the associated state of insulin (hexameric) in pharmaceutical formulation may be of importance. This review describes the development of a series of human insulin analogues with reduced tendency to selfassociation that, because of more rapid absorption, are better suited to meal-related therapy. DNA technology has made it possible to prepare insulins that remain dimeric or even monomeric at high concentration by introducing one or a few amino acid substitutions into human insulin. These analogues were characterized and used for elucidating the mechanisms involved in subcutaneous absorption and were investigated in preliminary clinical studies. Their relative receptor binding and in vitro potency (free-fat cell assay), ranging from 0.05 to 600% relative to human insulin, were strongly correlated (r = 0.97). In vivo, most of the analogues exhibited ∼100% activity, explainable by a dominating receptor-mediated clearance. This was confirmed by clamp studies in which correlation between receptor binding and clearance was observed. Thus, an analogue with reduced binding and clearance gives higher circulating concentrations, counterbalancing the reduced potency at the cellular level. Absorption studies in pigs revealed a strong inverse correlation (r = 0.96) between the rate of subcutaneous absorption and the mean association state of the insulin analogues. These studies also demonstrated that monomeric insulins were absorbed three times faster than human insulin. In healthy subjects, rates of disappearance from subcutis were two to three times faster for dimeric and monomeric analogues than for human insulin. Concomitantly, a more rapid rise in plasma insulin concentration and an earlier hypoglycemic response with the analogues were observed. The monomeric insulin had no lag phase and followed a monoexponential course throughout the absorption process. In contrast, two phases in rate of absorption were identified for the dimer and three for the normal hexameric human insulin. The initial lag phase and the subsequent accelerated absorption of soluble insulin can now be explained by the associated state of native insulin in pharmaceutical formulation and its progressive dissociation into smaller units during the absorption process. In the light of these results, the effects of insulin concentration, injected volume, temperature, and massage on the absorption process are now also understood. When given to diabetic patients immediately before a standard meal, the monomeric analogue lowered postprandial glucose excursions by ∼50% when compared with human insulin given at the same time. Subsequently, it was shown that three monomeric to dimeric analogues injected separately just before a meal gave glycemic control at least comparable to that of human insulin administered 30 min earlier. Lower plasma glucose concentrations (∼50%) were observed with the analogues from 1.5 h postprandially. Thus, monomeric analogues are faster in onset of action, can be given with the meal without losing glycemic control, and have the potential to minimize late hypoglycemia. Therefore, the development of these novel insulins represents a major step in the evolution of insulin preparations to subserve meal-related insulin requirements.

Insulin analogs with improved pharmacokinetic profiles

The aim of insulin replacement therapy is to normalize blood glucose in order to reduce the complications of diabetes. The pharmacokinetics of the traditional insulin preparations, however, do not match the profiles of physiological insulin secretion. The introduction of the rDNA technology 20 years ago opened new ways to create insulin analogs with altered properties. Fast-acting analogs are based on the idea that an insulin with less tendency to self-association than human insulin would be more readily absorbed into the systemic circulation. Protracted-acting analogs have been created to mimic the slow, steady rate of insulin secretion in the fasting state. The present paper provides a historical review of the efforts to change the physicochemical and pharmacological properties of insulin in order to improve insulin therapy. The available clinical studies of the new insulins are surveyed and show, together with modeling results, that new strategies for optimal basal-bolus treatment are required for utilization of the new fast-acting analogs.

Chemical Stability of Insulin. 1. Hydrolytic Degradation During Storage of Pharmaceutical Preparations

Hydrolysis of insulin has been studied during storage of various preparations at different temperatures. Insulin deteriorates rapidly in acid solutions due to extensive deamidation at residue AsnA21. In neutral formulations deamidation takes place at residue AsnB3 at a substantially reduced rate under formation of a mixture of isoAsp and Asp derivatives. The rate of hydrolysis at B3 is independent of the strength of the preparation, and in most cases the species of insulin, but varies with storage temperature and formulation. Total transformation at B3 is considerably reduced when insulin is in the crystalline as compared to the amorphous or soluble state, indicating that formation of the rate-limiting cyclic imide decreases when the flexibility of the tertiary structure is reduced. Neutral solutions containing phenol showed reduced deamidation probably because of a stabilizing effect of phenol on the tertiary structure (α-helix formation) around the deamidating residue, resulting in a reduced probability for formation of the intermediate imide. The ratio of isoAsp/Asp derivative was independent of time and temperature, suggesting a pathway involving only intermediate imide formation, without any direct side-chain hydrolysis. However, increasing formation of Asp relative to isoAsp derivative was observed with decreasing flexibility of the insulin three-dimensional structure in the formulation. In certain crystalline suspensions a cleavage of the peptide bond A8–A9 was observed. Formation of this split product is species dependent: bovine > porcine > human insulin. The hydrolytic cleavage of the peptide backbone takes place only in preparations containing rhombohedral crystals in addition to free zinc ions.

Studies of the Structure of Insulin Fibrils by Fourier Transform Infrared (FTIR) Spectroscopy and Electron Microscopy

Fibril formation (aggregation) of insulin was investigated in acid media by visual inspection, transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopy. Insulin fibrillated faster in hydrochloric acid than in acetic acid at elevated temperatures, whereas the fibrillation tendencies were reversed at ambient temperatures. Electron micrographs showed that bovine insulin fibrils consisted of long fibers with a diameter of 5 to 10 nm and lengths of several microns. The fibrils appeared either as helical filaments (in hydrochloric acid) or arranged laterally in bundles (in acetic acid, NaCl). Freeze-thawing cycles broke the fibrils into shorter segments. FTIR spectroscopy showed that the native secondary structure of insulin was identical in hydrochloric acid and acetic acid, whereas the secondary structure of fibrils formed in hydrochloric acid was different from that formed in acetic acid. Fibrils of bovine insulin prepared by heating or agitating an acid solution of insulin showed an increased content of beta-sheet (mostly intermolecular) and a decrease in the intensity of the alpha-helix band. In hydrochloric acid, the frequencies of the beta-sheet bands depended on whether the fibrillation was induced by heating or agitation. This difference was not seen in acetic acid. Freeze-thawing cycles of the fibrils in hydrochloric acid caused an increase in the intensity of the band at 1635 cm(-1) concomitant with reduction of the band at 1622 cm(-1). The results showed that the structure of insulin fibrils is highly dependent on the composition of the acid media and on the treatment.

Chemical stability of insulin. 2. Formation of higher molecular weight transformation products during storage of pharmaceutical preparations.

Formation of covalent, higher molecular weight transformation (HMWT) products during storage of insulin preparations at 4–45°C was studied by size exclusion chromatography. The main products are covalent insulin dimers (CID), but in protamine-containing preparations the concurrent formation of covalent insulin-protamine (CIP) products takes place. At temperatures ≥25°C parallel or consecutive formation of covalent oligo- and polymers can also be observed. Rate of HMWT is only slightly influenced by species of insulin but varies with composition and formulation, and for isophane (NPH) preparations, also with the strength of preparation. Temperature has a pronounced effect on CID, CIP, and, especially, covalent oligo- and polymer formation. The CIDs are apparently formed between molecules within the hexameric unit common for all types of preparations and rate of formation is generally faster in glycerol-containing preparations. Compared with insulin hydrolysis reactions (see the preceding paper), HMWT is one order of magnitude slower, except for NPH preparations.

Subcutaneous Insulin Absorption Explained by Insulin's Physicochemical Properties: Evidence From Absorption Studies of Soluble Human Insulin and Insulin Analogues in Humans

Objective To study the influence of molecular aggregation on rates of subcutaneous insulin absorption and to attempt to elucidate the mechanism of absorption of conventional soluble human insulin in humans. Research Design and Methods Seven healthy male volunteers aged 22-43 yr and not receiving any drugs comprised the study. This study consisted of a single-blind randomized comparison of equimolar dosages of 125I-labeled forms of soluble hexameric 2 Zn2+ human insulin and human insulin analogues with differing association states at pharmaceutical concentrations (AspB10, dimeric; AspB28, mixture of monomers and dimers; AspB9, GluB27, monomeric). After an overnight fast and a basal period of 1 h, 0.6 nmol/kg of either 125I-labeled human soluble insulin (Actrapid HM U-100) or 125I-labeled analogue was injected subcutaneously on 4 separate days 1 wk apart. Absorption was assessed by measurement of residual radioactivity at the injection site by external γ-counting. Results The mean ± SE initial fractional disappearance rates for the four preparations were 20.7 ± 1.9 (hexameric soluble human insulin), 44.4 ± 2.5 (dimeric analogue AspB10), 50.6 ± 3.9 (analogue AspB28), and 67.4 ± 7.4%/h (monomeric analogue AspB9, GluB27). Absorption of the dimeric analogue was significantly faster than that of hexameric human insulin (P < 0.001); absorption of monomeric insulin analogue AspB9, GluB27 was significantly faster than that of dimeric analogue AspB10 (P < 0.01). There was an inverse linear correlation between association state and the initial fractional disappearance rates (r = −0.98, P < 0.02). Analysis of the disappearance data on a log linear scale showed that only the monomeric analogue had a monoexponential course throughout. Two phases in the rates of absorption were identified for the dimer and three for hexameric human insulin. The fractional disappearance rates (%/h) calculated by log linear regression analysis were monomer 73.3 ± 6.8; dimer 44.4 ± 2.5 from 0 to 2 h and 68.9 ± 3.5 from 2.5 h onward; and hexameric insulin 20.7 ± 1.9 from 0 to 2 h, 45.6 ± 5.0 from 2.5 to 5 h, and 70.6 ± 6.3 from 5 h onward. Conclusions Association state is a major determinant of rates of absorption of insulin and insulin analogues. The lag phase and the subsequent increasing rate of subcutaneous soluble insulin absorption can be explained by the associated state of native insulin in pharmaceutical formulation and its progressive dissociation into smaller units during the absorption process.

Comparison of Subcutaneous Soluble Human Insulin and Insulin Analogues (AspB9, GluB27; AspB10; AspB28) on Meal-Related Plasma Glucose Excursions in Type I Diabetic Subjects

Objective To compare postprandial glucose excursions and plasma free insulin-analogue levels after subcutaneous injection of three novel human insulin analogues (AspB10; AspB9, GluB27; and AspB28) with those after injection of soluble human insulin (Actrapid HM U-100). To compare postprandial glucose excursions and plasma free insulin-analogue levels after subcutaneous injection of three novel human insulin analogues (AspB10; AspB9, GluB27; and AspB28) with those after injection of soluble human insulin (Actrapid HM U-100). Research Design and Methods Six male subjects with insulin-dependent diabetes, at least 1 wk apart and after an overnight fast and basal insulin infusion, received 72 nmol (∼ 12 U)s.c. of soluble human insulin 30 min before, or 72 nmol of each of the three analogues immediately before, a standard 500-kcal meal. Results Mean basal glucoses were similar on the 4 study days. Compared to human insulin (6.3 ± 0.8 mM), mean ± SE peak incremental glucose rises were similar after analogues AspB10 (5.4 ± 0.8 mM) and AspB9, GluB27 (5.4 ± 0.7 mM) and significantly lower after analogue AspB28 (3.6 ± 1.2 mM, P < 0.02). Relative to soluble human insulin (100% ± SE21), incremental areas under the glucose curve between 0 and 240 min were 79% ± 34 (AspB10, NS), 70% ± 29 (AspB9, GluB27, NS), and 43% ± 23 (AspB28, P < 0.02). Basal plasma free insulin levels were similar on the 4 study days. Plasma free insulin-analogue levels rose rapidly to peak 30 min after injection at 308 ± 44 pM (AspB10); 1231 ± 190 pM (AspB9, GluB27) and 414 ± 42 pM (AspB28) and were significantly higher than corresponding (i.e., 30 min postmeal) plasma free insulin levels of 157 ± 15 pM (P < 0.02 in each case). Conclusions Plasma profiles of the insulin analogues were more physiological than that of human insulin after subcutaneous injection. All three analogues given immediately before the meal are at least as effective as soluble human insulin given 30 min earlier. These analogues are promising potential candidates for short-acting insulins of the future.

Iontophoresis of monomeric insulin analogues in vitro: effects of insulin charge and skin pretreatment.

The aim of this study was to investigate the influence of association state and net charge of human insulin analogues on the rate of iontophoretic transport across hairless mouse skin, and the effect of different skin pretreatments on said transport. No insulin flux was observed with anodal delivery probably because of degradation at the Ag/AgCl anode. The flux during cathodal iontophoresis through intact skin was insignificant for human hexameric insulin, and only low and variable fluxes were observed for monomeric insulins. Using stripped skin on the other hand, the fluxes of monomeric insulins with two extra negative charges were 50-100 times higher than that of hexameric human insulin. Introducing three additional charges led to a further 2-3-fold increase in flux. Wiping the skin gently with absolute alcohol prior to iontophoresis resulted in a 1000-fold increase in transdermal transport of insulin relative to that across untreated skin, i.e. to almost the same level as stripping the skin. The alcohol pretreatment reduced the electrical resistance of the skin, presumably by lipid extraction. In conclusion, monomeric insulin analogues with at least two extra negative charges can be iontophoretically delivered across hairless mouse skin, whereas insignificant flux is observed with human, hexameric insulin. Wiping the skin with absolute alcohol prior to iontophoresis gave substantially improved transdermal transport of monomeric insulins resulting in clinically relevant delivery rates for basal treatment.

Absorption kinetics and action profiles of subcutaneously administered insulin analogues (AspB9GluB27, AspB10, AspB28) in healthy subjects.

Objective The subcutaneous absorption and resulting changes in plasma insulin or analogue, glucose, C-peptide, and blood intermediary metabolite concentrations after subcutaneous bolus injection of three soluble human insulin analogues (AspB9GluB27, monomeric; AspB28, mixture of monomers and dimers; and AspB10, dimeric) and soluble human insulin were evaluated. Research Design and Methods Fasting healthy male volunteers (n = 7) were studied on five occasions 1 wk apart randomly receiving 0.6 nmol·kg−1 s.c. 125I-labeled AspB10 or soluble human insulin (Novolin R, Novo, Copenhagen); 1st study and 0.6 nmol·kg−1 s.c. 125I-labeled AspB28, AspB9GluB27 or soluble human insulin (2nd study). Residual radioactivity at the injection site was measured over 8 h with frequent venous sampling for plasma immunoreactive insulin or analogue, glucose, C-peptide, and blood intermediary metabolite concentrations. Results The three analogues were absorbed 2–3 times faster than human insulin. The mean ± SE time to 50% residual radioactivity was 94 ± 6 min for AspB10 compared with 184 ± 10 min for human insulin (P < 0.001), 83 ± 8 min for AspB28 (P < 0.005), and 63 ± 9 min for AspB9GluB27 (P < 0.001) compared with 182 ± 21 min for human insulin. delta Peak plasma insulin analogue levels were significantly higher after each analogue than after human insulin (P < 0.005). With all three analogues, the mean hypoglycemic nadir occurred earlier at 61–65 min postinjection compared with 201–210 min for the reference human insulins (P < 0.005). The magnitude of the hypoglycemic nadir was greater after AspB9GluB27 (P < 0.05) and AspB28 (P < 0.001) compared with human insulin. There was a significantly faster onset and offset of responses in C-peptide and intermediary metabolite levels after the analogues than after human insulin (P < 0.05). Conclusions The rapid absorption and biological actions of these analogues offer potential therapeutic advantages over the current short-acting neutral soluble insulins.

Insulin Formulation and Delivery

Ever since its introduction in 1922, insulin has provided a major stimulus for scientific research in numerous and diverse fields, including protein chemistry, structure, synthesis, and biosynthesis, polymer biochemistry, metabolism, endocrinology, cellular biology, immunogenicity, radioimmunoassay, receptor-ligand interactions, molecular genetics, and recombinant DNA technology. More importantly, in the context of this book, insulin has for many years served as a model compound for research into protein drug formulation and delivery. The epoch-making discovery by Banting and Best (1922) prolonged the life expectancy for all insulin-dependent diabetic patients from two years to several decades. However, despite the major advances that have occurred relating to production, purification, and pharmaceutical formulation, insulin-replacement therapy is far from ideal (Zinman, 1989; Home et al., 1989). The optimal method of insulin delivery must be safe, should provide insulin to diabetic patients in a way that will correct the metabolic abnormalities of diabetes mellitus, and must be psychologically and socially acceptable. Metabolic control should be maintained the closest possible to normal as this gives the best hope of preventing, delaying, arresting, or even reversing progression of long-term complications in diabetic patients. Sophisticated and sometimes complicated systems, such as continuous infusion pumps, have been developed to enable optimal regulation of