BZ043, a novel long-acting amylin analog, reduces gastric emptying, food intake, glycemia and insulin requirement in streptozotocin-induced diabetic rats.

Amylin analogs are important adjunctive drugs in the treatment of diabetes mellitus. However, a dual therapy with insulin involves inconvenient multiple injections. Here we describe a novel n-terminal PEGylated human amylin analog - BZ043 - and its potential to improve the control of glycemia using lower doses of insulin. The effect of BZ043 over the insulin-mediated control of fed-glycemia was investigated in rats with streptozotocin-induced diabetes treated with the basal analog glargine (GLAR). Fasted rats (3 h) received a single treatment of BZ043 (16, 64 or 128 nmol/kg), GLAR (1.5 IU or 6.0 IU) or BZ043 plus GLAR low dose (1.5 IU) in separate injections, and had free access to 5% glucose rich chow and water. BZ043 dose-proportionally prevented the meal-related increase of glycemia, and the co-treatment (64 or 128 nmol/kg) with GLAR restored normoglycemia without abrupt variations of glycemia. BZ043 showed a prolonged anti-hyperglycemic effect and, together with GLAR, promoted a long-lasting normoglycemia, in vivo. We conceive that combining BZ043 and GLAR in a fixed-ratio co-formulation might conveniently improve the control of diabetes mellitus.

Physico-chemical properties of co-formulated fast-acting insulin with pramlintide.

Since the discovery of amylin its use has been discouraged by the inadequacy of the protocol involving multiple injections in addition to insulin. We aimed here to develop a combined fixed-dose formulation of pramlintide with fast-acting insulin. We have investigated the compatibility of regular and fast-acting insulin analogues (Aspart, AspB28, and LisPro, LysB28ProB29) with the amylin analogue pramlintide by using electrospray ionization - ion mobility spectrometry-mass spectrometry (ESI-IMS-MS), kinetic aggregation assays monitored by thioflavin T, and transmission electron microscopy (TEM) in the evaluation of the aggregation product. Insulin interacts with pramlintide, forming heterodimers as probed by ESI-IMS-MS. While their interaction is likely to delay the amyloid aggregation of pramlintide in phosphate-buffered solution pH 7.0, they do not prevent aggregation at this condition. At acidic sodium acetate solution pH 5.0, combination of pramlintide and the fast-acting insulin analogues become stable against amyloid aggregation. The co-formulated product at high concentration of both pramlintide (600 µg/mL,150 µM) and LisPro insulin (50 IU/mL, 300 µM) showed also stability against amyloid aggregation. These data indicate the physico-chemical short-term stability of the co-formulated preparation of LisPro insulin with pramlintide, which could bring benefits for the combined therapy.

Monoconjugation of Human Amylin with Methylpolyethyleneglycol.

Amylin is a pancreatic hormone cosecreted with insulin that exerts unique roles in metabolism and glucose homeostasis. The therapeutic restoration of postprandial and basal amylin levels is highly desirable in diabetes mellitus. Protein conjugation with the biocompatible polymer polyethylene glycol (PEG) has been shown to extend the biological effects of biopharmaceuticals. We have designed a PEGylated human amylin by using the aminoreactive compound methoxylpolyethylene glycol succinimidyl carbonate (mPEGsc). The synthesis in organic solvent resulted in high yields of monoPEGylated human amylin, which showed large stability against aggregation, an 8 times increase in half-life in vivo compared to the non-conjugated amylin, and pharmacological activity as shown by modulation of cAMP production in MCF-7 cell line, decrease in glucagon and modulation of glycemia following subcutaneous administration in mice. Altogether these data reveal the potential use of PEGylated human amylin for the restoration of fasting amylin levels.

Amylin induces hypoglycemia in mice.

Amylin is a 37-aminoacid pancreatic protein that exerts control over several metabolic events such as glycemia and lacticemia. Amylin has long been shown to induce increases in arterial plasma glucose. We decided to investigate whether amylin plays additional roles in the glucose metabolism. We evaluated glucose homeostasis using whole blood from the tail tip of fasting, conscious, unrestrained normal and streptozotocyn-induced diabetic mice following subcutaneous administration of mouse amylin. Subcutaneous injection of 1 µg mouse amylin caused a transient decrease in whole blood glucose in both normal and diabetic mice in the absence of insulin. The blood glucose levels were lowest approximately 2 hours after amylin administration, after that they gradually recovered to the levels of the control group. The hypoglycemic effect followed a dose-dependent response ranging from 0.1 to 50 µg / mouse. These results reveal the ability for amylin in the direct control of glycemia at low doses in the absence of insulin.