Human insulin preparations

Table 11.3 Native and engineered human insulin preparations that have gained approval for general medical use. Reproduced in updated form with permission from Walsh, G. 2005. Therapeutic insulins and their large-scale manufacture. Applied Microbiology and Biotechnology, 67, 151-159...

Many successful protein products, including antibodies, have been marketed over the years for the treatment of a number of diseases. One of the oldest examples of a protein product is insulin, still one of the most successful drugs after 70-80 years of its discovery. Early insulin preparations, derived from natural sources, are being replaced by recombinant human insulin preparations and new formulations are being marketed that provide a more gradual and continuous release profile and maximise glucose control in diabetic patients. " ... 

Hypogiycemic reactions Hypoglycemia when using this concentrated insulin can be prolonged and severe. As with other human insulin preparations, hypoglycemia reactions may be associated with the administration of concentrated insulin. However, deep secondary hypoglycemic reactions may develop 18 to 24 hours after the original injection of concentrated insulin. 

Human insulin from E coli is available for clinical use as Humulin (Lilly) and dispensed as either regular, NPH, lente, or ultralente Humulin. Human insulin prepared biosynthetically in yeast is marketed by Novo Nordisk as human insulin injection in regular, lente, and NPH forms Novolin R, Monotard Human Insulin (Novolin L), and Novolin N. The same company also produces a human insulin marketed as Velosulin (regular) that contains a phosphate buffer. This reduces aggregation of regular insulin molecules when used in infusion pumps. However, because of the tendency of phosphate to precipitate zinc ions, Velosulin should not be mixed with any of the lente insulins. 

Injection of older insulin preparations sometimes led to atrophy of subcutaneous fatty tissue at the site of injection. This type of immune complication is almost never seen since the development of human insulin preparations of neutral pH. Injection of these newer preparations directly into the atrophic area often results in restoration of normal contours. Hypertrophy of subcutaneous fatty tissue remains a problem, even with the purified insulins, if injected repeatedly at the same site. However, this may be corrected by avoidance of that specific injection site or with liposuction. 

NPH Isophane Human Insulin Suspension. NPH isophane insulin, also called Humulin N, Insulatard NPH Human, or Novolin N is an intermediate-acting form of human insulin produced by recombinant DNA techniques. Mixtures Humulin 70/30 and Novolin 70/30 contain 70% NPH isophane and 30% regular, whereas Humulin 50/50 contains 50% NPH isophane and 50% regular. It is adrninistered subcutaneously and should not be given intravenously. Absorption is delayed because the insulin is conjugated with protamine in a complex of reduced isoelectric solubiUty. Therapeutically, this preparation is probably comparable to purified porcine NPH insulin. However, human NPH insulin may have a slightly shorter duration of action than comparable purified porcine products. 

Care must be taken when giving insulin to use the correct insulin. Names and packaging are similar and can easily be confused. The nurse carefully reads all drug labels before preparing any insulin preparation. For example, Humalog (insulin lispro) and Humulin R (regular human insulin) are easily confused because of die similar names. 

Fahrner, R.L., Lester, P.M., Blank, G.S., and Reifsnyder, D. H., Non-flammable preparative reversed-phase liquid chromatography of recombinant human insulin-like growth factor-I, ]. Chromatogr. A, 830, 127, 1999. 

Although a high degree of homology is evident between insulins from various species, the same is not true for proinsulins, as the C peptide sequence can vary considerably. This has therapeutic implications, as the presence of proinsulin in animal-derived insulin preparations can potentially elicit an immune response in humans. 

An alternative method (developed in the Eli Lilly research laboratories), entails inserting a nucleotide sequence coding for human proinsulin into recombinant E. coli. This is followed by purification of the expressed proinsulin and subsequent proteolytic excision of the C peptide in vitro. This approach has become more popular, largely due to the requirement for a single fermentation and subsequent purification scheme. Such preparations have been termed human insulin prb ... 

The toxicologist should be prepared to do nothing if the material is well-known, its properties are understood, and there is adequate characterization of the nature of the preparation supplied for example, human insulin or growth hormone produced by genetic engineering should not be submitted to prolonged safety tests in animals, provided that the molecular forms present are sufficiently well understood. 

Insulin, a hormone produced by the pancreas, is essential for the metabolism of glucose, proteins, and fats. Insulins are classified on the basis of the duration of action as rapid-, intermediate-, or long-acting and on the basis of source or species, such as human or animal (beef, pork, and mixtures of beef and pork). Table 10.1 summarizes insulin preparations currendy available in the United States. 

It is interesting to note that serine peptidases can, under special conditions in vitro, catalyze the reverse reaction, namely the formation of a peptide bond (Fig. 3.4). The overall mechanism of peptide-bond synthesis by peptidases is represented by the reverse sequence f-a in Fig. 3.3. The nucleophilic amino group of an amino acid residue competes with H20 and reacts with the acyl-enzyme intermediate to form a new peptide bond (Steps d-c in Fig. 3.3). This mechanism is not relevant to the in vivo biosynthesis of proteins but has proved useful for preparative peptide synthesis in vitro [17]. An interesting application of the peptidase-catalyzed peptide synthesis is the enzymatic conversion of porcine insulin to human insulin [18][19]. 

When Emma received her doctor s diagnosis, only one effective treatment for diabetes was available daily injection of extracts taken from the pancreases of a cow or pig. These extracts contained animal insulin that was enough like human insulin to metabolize glucose, a task that a diabetic s own body is unable to carry out. Preparing these extracts, however, was very time-consuming and expensive, and there were never enough cow and pig pancreases available to... 

Insulin aspart If insulin aspart is mixed with NPH human insulin, draw insulin aspart into the syringe first. Do not mix insulin aspart with crystalline zinc insulin preparations. When used in external subcutaneous infusion pumps for insulin, do not mix with any other insulins or diluent. 

As previously described, porcine insulin differs from human insulin by only one amino acid (the B30 residue). In the early 1970s, a method was developed by which porcine insulin could be converted into a preparation of identical amino acid sequence to that of human insulin (Figure 8.4). This method utilizes a combination of enzymatic and chemical treatment of the porcine product. 

An octapeptide, corresponding in sequence to the C-terminal octapeptide found in the human insulin B-chain, is prepared by chemical synthesis. This octapeptide is then coupled to the shortened insulin using a well-established chemical method, thus yielding a semi-synthetic human insulin product ( human insulin emp Box 8.2). 

Typically, 7.0 g of human insulin can be prepared from 10 g of purified porcine insulin. The process allows large-scale production of human insulin simply and inexpensively. The complete de novo synthesis of human insulin, while technically possible, is economically unattractive. 

Insulin allergy occurs in as many as 3% of patients receiving pork or beef insulin but smaller proportion in those using human insulin. However, the immuno-genicity of insulin is determined more by the purity of its preparations and since the use of monocomponent insulin, insulin allergy has become extremely rare. 

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