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0.00 0.02 0.04 0.06 Water content (g g_1dry weight)

Figure 11.13 The increasing rate of decomposition of a lyophilised rbST formulation with increasing water content following incubation in sealed vials at 47°C.

Reproduced from reference 29.

0.00 0.02 0.04 0.06 Water content (g g_1dry weight)

Figure 11.13 The increasing rate of decomposition of a lyophilised rbST formulation with increasing water content following incubation in sealed vials at 47°C.

Reproduced from reference 29.

(PLGA) is one of the commonest polymers used in microsphere form to deliver, inter alia, growth-hormone-releasing factor, a somato-statin analogue, ciclosporin, and LHRH antagonists.27

11.3.4 Routes of delivery

Table 11.7 summarises the invasive and non-invasive routes of delivery for peptides and proteins, involving direct injection of solutions, depot systems and a variety of oral, nasal, topical and other formulations.

11.4 A therapeutic protein and a peptide

11.4.1 Insulin

It is appropriate that the protein therapeutic substance with the longest pedigree is discussed here. There are three main types of insulin preparations:

• Those with a short duration of action which have a relatively rapid onset (soluble insulin, insulin lispro and insulin aspart)

• Those with an intermediate action (isophane insulin and insulin zinc suspension)

• Those with a slow action, slower in onset and lasting for long periods (crystalline insulin zinc suspension)

Some aspects of insulin were dealt with in section 9.4.4. Table 11.8 lists some of the insulin formulations designed to produce different durations of onset and action. Insulin is generally self-administered sub-cutaneously with injection pens, needle-free devices or pumps.

Precipitation of insulin and other proteins

Precipitation of insulin in pumps due to the formation of amorphous particles, crystals or fibrils of insulin can lead to changes in release pattern or to blockage which prevents insulin release. 'Amorphous' or 'crystalline' precipitates can be caused by the leaching of divalent metal contaminants or lowering of pH (due to CO2 diffusion or leaching of acidic substances), but can be prevented. More difficult to solve is the tendency of insulin to form fibrils as illustrated in Fig. 11.14.

It appears that the interactions leading to fibril formation result from the monomeric form, and from change in monomer conformation and hydrophilic attraction of the parallel ^-sheet forms. Fibril formation is also encouraged by contact of the insulin solution with hydrophobic surfaces. Contact with gamma-irradiated PVC leads to instability, apparently induced by chemical changes in the insulin.

Table 11.8 Effect of insulin formulation on its pharmacokinetics after subcutaneous injection

Producta

Formulation

Pharmacokineticsb

Humulin R Zinc-insulin crystalline suspension

Novolin R (acid regular)

Humulin N Isophane suspension protamine, zinc

Novolin N crystalline insulin (buffer water for injection)

Humulin 70/30c 70% isophane suspension Novolin 70/30c 30% zinc crystalline

Humulin U Extended zinc-insulin suspension - all crystalline

Humulin L 70% zinc-insulin crystalline suspension

Novolin L 30% amorphous insulin (cloudy suspension)

Humulin BR Zinc crystalline insulin dissolved in sodium diphosphate buffer

Rapid onset, short duration

Intermediate-acting, slower onset, longer duration than regular insulin

Intermediate-acting, faster onset, longer duration

Slow-acting, slow onset, longer, less-intense duration than R or N forms

Intermediate-acting, slower onset, longer duration

Start, 2.5 h; peak, 7-15 h; end, 22 h Rapid onset, short duration; use in pumps only a Humulin products (Eli Lilly & Company) contain recombinant human insulin derived from Escherichia coli; Novolin products (Novo Nordisk) are recombinant human insulin derived from Saccharomyces cerevisiae; both companies also sell other forms of recombinant human insulin and may have additional forms (formulations or new drugs) in clinical trials.

b The pharmacokinetics of each formulation may vary greatly among different individuals; the onset of therapeutic levels of insulin is referred to as the start of the effect, the maximum serum level of insulin is denoted the peak, and the time at which the insulin levels are below therapeutic levels is listed as the end of the therapeutic time course.

c Solution consists of 70% N form and 30% R form for both products.

Figure 11.14 Transmission electron micrographs of insulin fibrils formed (a) at 37°C, (b) at 80°C (x100 000).

Reproduced from J. Brange, Galenics of Insulin, Springer, Berlin, 1987.

Figure 11.14 Transmission electron micrographs of insulin fibrils formed (a) at 37°C, (b) at 80°C (x100 000).

Reproduced from J. Brange, Galenics of Insulin, Springer, Berlin, 1987.

Propylene glycol, glycerol, nonionic and ionic surfactants and calcium ions have been used in formulations to achieve greater stability, reducing fibril formation, but the most successful strategy is the addition of calcium ions or zinc, which appear to protect the hexameric form of the insulin (see section 9.4.4).

Recombinant human insulin: insulin lispro and insulin aspart

Chemical modifications to an endogenous protein, however minor, can lead to significant differences in properties and activity. Species differences can also be great. Salmon calcitonin is ten times more potent than human calcitonin, for example. Recombinant human protein analogues may be subtly different, as in the case of insulin lispro (Lilly) in which the sequence of proline and lysine at positions 28 and 29 in the B protein chain has been reversed (Fig. 11.15). This sole difference leads to hexamers which more rapidly dissociate to monomers on injection, giving a faster onset of action than human insulin in which the B28 is proline and B29 is lysine (such as the recombinant product Humulin S, which is injected up to an hour before meals).

As we have discussed, peptides become less soluble the closer they are to their isoelectric point. The first attempt to use this principle to prolong insulin action (circa 1988) was unsuccessful owing to injection site reactions and variable bioavailability.

Insulin glargine (Lantus, Aventis) has two arginines added to the B30 position (Fig. 11.15). Its isoelectric point changes from a pH of 5.4 to 6.7, making it a soluble (and thus clear) solution in the acid medium of pH 4 in which it is supplied. When it is injected sub-cutaneously, however, glargine precipitates at the physiological pH of 7.4, thereby delaying its absorption and prolonging its action. Substituting glycine for asparagine at the A21 position and adding zinc stabilises the hexamer and delays absorption further.

Glargine has a similar insulin receptor affinity as NPH (Neutral Protamine Hagedorn, an intermediate-acting insulin that initially achieves slower action through the addition of protamine to short-acting insulin) so that once absorbed in the circulation, glargine is

A chain

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