Ronald E. Chance

Modifications in the B10 and B26–30 regions of the B chain of human insulin alter affinity for the human IGF-I receptor more than for the insulin receptor

Summary Inversion of the natural sequence of the B chain of human insulin (HI) from ProB28LysB29 to LysB28ProB29 generates an insulin analogue with reduced tendency to self-associate. Since this substitution increases the homology of insulin to insulin-like growth factor-I (IGF-I), we have examined the affinity of a series of insulin analogues with the general modified structure XaaB28ProB29 HI for binding to both human placental insulin and IGF-I receptors. The XaaB28ProB29 HI series is approximately equipotent to HI in binding to the insulin receptor with the exception of when Xaa = Phe, Trp, Leu, Ile, and Gly (40–60 % relative to HI). Substitution with basic residues in the B28 position increased the relative affinity to the IGF-I receptor approximately 1.5−2-fold (ArgB28ProB29 > OrnB28ProB29 = LysB28ProB29). Substitution with acidic residues reduced relative affinity for the IGF-I receptor approximately 2-fold (CyaB28ProB29 = GluB28ProB29 > AspB28ProB29). Combination of AspB10 substitution in conjunction with a modification in the B28–29 position (e.g. AspB10LysB28ProB29 HI) showed an additional 2-fold selective increase in affinity for the IGF-I receptor, suggesting that these two effects are additive. Addition of Arg residues at B31–32, on the backbone of either HI or AspB10 HI, increased affinity for the IGF-I receptor 10 and 28 fold, respectively, compared to HI, confirming the significance of enhanced positive charge at the C-terminal end of the insulin B-chain in increasing selectivity for the IGF-I receptor. This relative increase in IGF-I receptor affinity correlated largely, but not completely, with enhanced growth promoting activity in human mammary epithelial cells. In the case of LysB28ProB29 HI, growth activity correlated with dissociation kinetics from the insulin receptor which were shown to be identical with those of human insulin. [Diabetologia (1997) 40: S 54–S 61]

Chemical, Physical, and Biologic Properties of Biosynthetic Human Insulin

Human insulin derived via recombinant DNA technology was tested extensively by a complex battery of analytic procedures. This product, which is designated biosynthetic human insulin, was found to be chemically, physically, and immunologically equivalent to pancreatic human insulin and biologically equivalent to both pancreatic human insulin and purified pork insulin.

Hybrid insulin cocrystals for controlled release delivery

The ability to tailor the release profile of a drug by manipulating its formulation matrix offers important therapeutic advantages. We show here that human insulin can be cocrystallized at preselected ratios with the fully active lipophilically modified insulin derivative octanoyl-Nε-LysB29–human insulin (C8-HI). The cocrystal is analogous to the NPH (neutral protamine Hagedorn) crystalline complex formed with human insulin, which is commonly used as the long-acting insulin component of diabetes therapy. The in vitro and in vivo release rates of the cocrystal can be controlled by adjusting the relative proportions of the two insulin components. We identified a cocrystal composition comprising 75% C8-HI and 25% human insulin that exhibits near-ideal basal pharmacodynamics in somatostatin-treated beagle dogs. The dependence of release rate on cocrystal ratio provides a robust mechanism for modulating insulin pharmacodynamics. These findings show that a crystalline protein matrix may accommodate a chemical modification that alters the dissolution rate of the crystal in a therapeutically useful way, yet that is structurally innocuous enough to preserve the pharmaceutical integrity of the original microcrystalline entity and the pharmacological activity of the parent molecule.

Preparation of an Insulin with Improved Pharmacokinetics Relative to Human Insulin through Consideration of Structural Homology with Insulin-Like Growth Factor I

The Diabetes Control and Complications Trial has emphasized the need for improved control of blood glucose as a means to diminish long-term complications of diabetes. LysPro-insulin is an analog of human insulin whose design was modeled on structural homology with insulin-like growth factor I. An analysis of the structural conformation of insulin suggested that an inversion of amino acids B28 and B29 in the C-terminus of the B chain could yield an insulin analog with a faster onset of biological action. This insulin analog has proved to be virtually identical to human insulin in action, with one important exception. LysPro-insulin has demonstrated an improved time course of action in control of a mealtime glucose elevation. This offers the opportunity for improved convenience and safety for patients with insulin-dependent diabetes mellitus.

Research, Development, Production, and Safety of Biosynthetic Human Insulin

This paper provides some historical aspects on the research and development of Humulin® (rDNA origin), the first human health-care product derived from rDNA technology more than a decade ago. Also referred to as biosynthetic human insulin, Humulin® is currently produced via the human proinsulin route, using an Escherichia coli fermentation process. The authenticity, high purity, and safety of BHI has been investigated and verified by a complex battery of analytical and physicochemical methods. The daily treatment of more than two million diabetic patients worldwide with this rDNA human insulin not only demonstrates the value of rDNA technology in providing an important medical product, it is assurance that diabetic patients will have unlimited supplies of this vital hormone as well as potential analogue refinements.

Purification and analysis of the major components of chum salmon protamine contained in insulin formulations using high-performance liquid chromatography.

A simple high-performance liquid chromatographic method has been developed for the rapid purification and analysis of protamine components contained in insulin formulations. Only a single step is needed to separate peptides whose compositions, sizes, and unusual isoelectric points (pI 13.8) are nearly identical. The method involves their isocratic separation on a reversed-phase column using a pH 2 phosphate buffer and a low acetonitrile content as an eluant. The purified chum salmon components were analyzed by amino acid analysis, solid-phase amino acid sequencing, carboxypeptidase B digests, insulin complexation analysis, and a mass spectrophotometric procedure which gives an accurate mass of the intact peptides. This HPLC purification technique may also be applicable to protamines and other highly basic peptides isolated from other sources.

Immunogenicity of Insulin

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

Single chain insulin with high bioactivity

The instant invention provides polypeptide compounds of the formula b-BP-a having significant insulin activity, nucleotide sequences encoding said polypeptides, vectors comprising said nucleotide sequences, and cell lines transformed with said vectors useful for treating diabetes.

Self-Association Properties of Monomeric Insulin Analogs Under Formulation Conditions

AbstractPurpose. The purpose of the current study was to investigate the effects of two important excipients, zinc and m-cresol, on the self-association properties of a series of monomeric insulin analogs. In this way, the effects on formulation behavior of individual amino acid substitutions in the C-terminal region of the insulin B-chain could be compared. Methods. The self-association of ten insulin analogs was monitored by equilibrium and velocity analytical ultracentrifugation under three different conditions: (i) in neutral buffer alone; (ii) in neutral buffer containing zinc ion; and (iii) in neutral buffer containing both zinc ion and phenolic preservative (a typical condition for insulin formulations). The self-association properties of these analogs were compared to those of human insulin and the rapid-acting insulin analog LysB28ProB29-human insulin. Results. The analogs in the current study exhibited a wide range of association properties when examined in neutral buffer alone or in neutral buffer containing zinc ion. However, all of these analogs had association properties similar to human insulin in the presence of both zinc and m-cresol. Under these formulation conditions each analog had an apparent sedimentation coefficient of s* = 2.9−3.1 S, which corresponds to the insulin hexamer. Conclusions. Analogs with changes in the B27−B29 region of human insulin form soluble hexamers in the presence of both zinc and m-cresol, and m-cresol binding overrides the otherwise destabilizing effects of these mutations on self assembly.

Insulin and IGF-I Analogs: Novel Approaches to Improved Insulin Pharmacokinetics

Current insulin formulations do not mimic the normal glucose-induced release of insulin by the pancreas in a physiological manner.1 One limitation is the delayed absorption of hexameric insulin from the subcutaneous site of injection, such that soluble insulins (currently the most rapid acting formulations) are too slow and have too long a duration of action.2 Another limitation is that longer acting insulin formulations, such as human ultralente, exhibit too short a duration of action, show a pronounced peak in activity and are suspensions, resulting in variability in administration.3 The use of recombinant DNA technology and peptide chemistry have allowed the generation of insulin analogs with a wide variety of amino acid substitutions, which in turn halve been useful in mapping regions of the insulin nucleus that are associated with Zn2+ binding, dimer formation and insulin receptor interaction. This report will review the physical, biological and clinical characterization of several insulin analogs that have been designed to improve absorption characteristics and pharmacodynamics. Because of the structural homology between insulin and insulin-like growth factor-I (IGF-I), we have investigated specific IGF-like modifications in the insulin sequence to determine if these will transfer to pharmacokinetic differences in insulin absorption and clearance.