Medical brain notes 37

  Insulin INTRODUCTION   Insulin was discovered by Banting and Best in 1921 (Bliss, 1982). Soon afterward, manufacturing pro-cesses were developed to extract the insulin from porcine and bovine pancreata. From 1921 to 1980, efforts were directed at increasing the purity of the insulin and providing different formulations for altering time-action for improved glucose control (Brange, 1987a,b; Galloway, 1988). Purification was improved by optimizing extraction and processing conditions and by implementing chromatographic processes (size exclusion, ion exchange, and re-versed-phase) (Kroeff et al., 1989) to reduce the levels of both general protein impurities as well as insulin-related proteins such as proinsulin and insulin polymers. Formulation development focused on improving chemical stability by moving from acidic to neutral formulations and by modifying the time-action profile through the uses of various levels of zinc and protamine. The evolution of recombinant DNA (rDNA) ...

  CHEMICAL DESCRIPTION   Insulin, a 51-amino acid protein, is a hormone that is synthesized as a proinsulin precursor in the β-cell of the pancreas and is converted to insulin by enzymatic cleavage. The resulting insulin molecule is composed of two polypeptide chains that are connected by two interchain disulfide bonds (Fig. 1) (Baker et al., 1988). The A-chain is composed of 21 amino acids and the B-chain is composed of 30 amino acids. The interchain disulfide linkages occur between A 7 –B 7  and A 20 –B 19 , respectively. A third intrachain disulfide bond is located in the A-chain, between residues A 6  and A 11 .   In addition to human insulin and insulin analog products, which are predominately used today as the first-line therapies for the treatment of diabetes, bovine and porcine insulin preparations have also been made commercially available (Table 1; Fig. 1); however, all major manufacturers of insulin have discontinued production of these products marki...

  PHARMACOLOGY AND FORMULATIONS   Normal insulin secretion in the nondiabetic person falls into two categories: (i) insulin that is secreted in response to a meal and (ii) the background or basal insulin that is continually secreted between meals and during the nighttime hours. The pancreatic response to a meal typically results in peak serum insulin levels of 60–80  m U/mL whereas basal serum insulin levels fall within the 5–15  m U/mL range (Galloway and Chance, 1994). Because of these vastly different insulin demands, considerable effort has been ex-pended to develop insulin formulations that meet the pharmacokinetic and pharmacodynamic require-ments of each condition. More recently, insulin analogs and insulin analog formulations have been developed to improve pharmacokinetic and pharma-codynamic properties.

  Regular and Rapid-Acting Soluble Formulations   Initial soluble insulin formulations were prepared under acidic conditions and were chemically unstable. In these early formulations, considerable deamidation was identified at Asn A21  and significant potency loss was observed during prolonged storage under acidic conditions. Efforts to improve the chemical stability of these soluble formulations led to the development of neutral, zinc-stabilized solutions.   The insulin in these neutral, regular formula-tions is chemically stabilized by the addition of zinc ( ~ 0.4% relative to the insulin concentration) and phenolic preservatives. As mentioned above, the addition of zinc leads to the formation of discrete hexameric structures (containing 2 Zn atoms per hexamer) that can bind six molecules of phenolic preservatives, e.g., m-cresol (Fig. 2). The binding of these excipients increases the stability of insulin by inducing the formation of a specific hexameric conformati...

  Intermediate-Acting Insulin Formulations   There are two widely used types of intermediate-acting insulin preparations: Neutral Protamine Hagedorn (NPH) and Lente. Both formulations achieve extended time-action by necessitating the dissolution of a precipitated and/or crystalline form of insulin. This dissolution is presumed to be the rate-limiting step in the absorption of intermediate- and long-acting insulin. Consequently, the time-action of the formulation is prolonged by further delaying the dissociation of the hexamer into dimers and monomers.     NPH, named after its inventor H.C. Hagedorn (Hagedorn et al., 1936), is a neutral crystalline suspension that is prepared by the cocrystallization of insulin with protamine. Protamine consists of a closely related group of very basic proteins that are isolated from fish sperm. Protamine is heterogeneous in composition; however, four primary components have been identified and show a high degree of sequence homology ...

  Long-Acting Insulin Formulations   The normal human pancreas secretes approximately 1 unit of insulin (0.035 mg) per hour to maintain basal glycemic control. Adequate basal insulin levels are a critical component of diabetes therapy because they regulate hepatic glucose output, which is essential for energy production by the brain. Consequently, long-acting insulin formulation must provide a very different pharmacokinetic profile than “meal-time” insulin formulation.   There are three long-acting insulin preparations currently commercially available: Ultralente, which was developed in the 1950s and two insulin analogs, Lantus (insulin glargine) and Levemir (insulin detemir), which have been recently approved (Table 1; Fig. 1). Ultralente and Lantus derive theirprotracted time-action profiles from the slow and relatively constant dissolution of solid particles in the subcutaneous tissue. This slow dissolution precedes the dissociation of insulin into absorbable units, an...

  PHARMACEUTICAL CONCERNS   Chemical Stability of Insulin Formulations   Insulin has two primary routes of chemical degradation upon storage and use: hydrolytic transformation of amide to acid groups and formation of covalent dimers and higher order polymers. Primarily the pH, the storage temperature, and the components of the specific formulation influence the rate of formation of these degradation products. The purity of insulin formulations is typically assessed by high-perfor-mance liquid chromatography using reversed-phase and size exclusion separation modes (USP Monographs: Insulin, 2006). In acidic solution, the main degradation reaction is the transformation of asparagine (Asn) at the terminal 21 position of the A-chain to aspartic acid. This reaction is relatively facile at low pH, but is extremely slow at neutral pH (Brange et al., 1992b). This was the primary degrada-tion route in early soluble (acidic) insulin formula-tions. However, the development of neutral...