In vitro activity of biosynthetic human diarginylinsulin

Abstract

In diarginylinsulin two arginine residues are located at the C-terminal end of the B-chain (ArgB31 and ArgB32). This accounts for a shift of the isoelectric point from pH 5.4 in native insulin to pH 7.0 in diarginylinsulin leading to pharmacodynamic characteristics of an intermediate acting insulin when administered s.c. as pH 4.0-5.0 solution. We have investigated insulin receptor binding and biological activity of biosynthetic human diarginylinsulin in human adipocytes and compared to native insulin and proinsulin. Association- and dissociation studies of insulin receptor binding revealed no differences for diarginylinsulin and native insulin. In competition studies under steady-state binding conditions, half-maximal displacement of tracer occurred at 352 +/- 33 pmol/l, 337 +/- 32 pmol/l and 3640 +/- 480 pmol/l for diarginylinsulin, insulin and proinsulin, respectively. The biologic potency of human diarginylinsulin was evaluated by the ability to stimulate D-glucose transport and by the assessment of the antilipolytic activity. Activation of D-glucose transport was half-maximal at 49.6 +/- 5.4 pmol/l (diarginylinsulin), 44.8 +/- 5.8 pmol/l (insulin) and at 476.7 +/- 134.3 pmol/l (proinsulin). Half-maximal inhibition of lipolysis occurred at 13.9 +/- 3.4 pmol/l, 15.4 +/- 2.9 pmol/l and 138.4 +/- 38.6 pmol/l, respectively. In conclusion, diarginylinsulin has almost identical insulin receptor binding characteristics and full biological activity in vitro compared to native insulin. This pharmacodynamically intermediate acting insulin preparation is therefore of potential therapeutical value.

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

“…This molecule possesses two additional Arg residues at the C-terminal end of the insulin B chain, and, in fact, is a naturally occurring intermediate in the biosynthetic processing of proinsulin to insulin. Although previous studies have indicated that DiArg insulin is approximately equipotent with native insulin in terms of insulin receptor binding and stimulation of glucose uptake into adipocytes [26], we found in the present study that DiArg insulin is approximately 1.5-times more potent than insulin in the placental insulin receptor binding assay (Table 2). However, it was found to be 10.2 times more potent than insulin at displacing [ Substitution of Asp for His at B10 increased IGF-I receptor affinity to an extent similar to that reported previously for binding to the insulin receptor [27], and this effect was additive when added onto a background of Xaa B28 Pro B29 insulin with Xaa = Asp, Lys or Val (Table 2).…”

Insulin glargine: the first clinically useful extended-acting insulin in half a century?

“…This is well recognised and easily explained by the observation that insulin in the circulation is very rapidly cleared through its receptors (with a fractional clearance corresponding to a T ½ of 4 -5 min), so that an insulin of high affinity will have a proportionately low plasma insulin concentration. This accounts for the relatively equal in vivo potency of insulins as diverse as B10-Asp (with three times the receptor affinity of human insulin) and proinsulin (ten times lower) [15].…”

A 16-week comparison of the novel insulin analog insulin glargine (HOE 901) and NPH human insulin used with insulin lispro in patients with type 1 diabetes.

“…Insulin analogs designed to more closely mimic endogenous basal insulin patterns have been produced through recombinant DNA technology (3)(4)(5)(6). A novel insulin analog, 21 A -Gly-30 B a-L-Arg-30 B b-L-Arg-human insulin (HOE 901 or insulin glargine), has been designed with a modified isoelectric point that results in precipitation at neutral tissue pH and thus delayed absorption from subcutaneous tissue (7)(8)(9)(10).…”