Insulin crystallization

This is a crystalline product of insulin and an alkaline protein where the protein/insulin ratio is called the isophane ratio. This product gives a delayed and uniform insulin action with a reduction in the number of insulin doses necessary per day. Such a preparation may be made as follows 1.6 g of zinc-insulin crystals containing 0.4% of zinc are dissolved in 400 ml of water, with the aid of 25 ml of 0.1 N hydrochloric acid. To this are added aqueous solutions of 3 ml of tricresol, 7.6 g of sodium chloride, and sufficient sodium phosphate buffer that the final concentration is As molar and the pH is 6.9. 

Adams MJ, Blundell TL, Dodson EJ, Dodson GG, Vijayan M, Baker EN et al. Structure of rhombohedral 2 zinc insulin crystals. Nature 1969 224 491-5. 

More subtle effects of preservatives on injectable formulations are possible. Formulation of insulin is an illustrative case study. Insulin is usually formulated as a multiple-dose vial, since individual dosage varies among patients. Preservation of zinc insulin with phenol causes physical instability of the suspension, whereas methyl-paraben does not. However, the presence of phenol is required for obtaining protamine insulin crystals [9]. 

Addition of zinc in order to promote Zn-insulin crystal growth (which take longer to disassociate and, hence, longer to leak into the bloodstream from the injection depot site). 

Insulin suspensions. When the hormone is injected as a suspension of insulin-containing particles, its dissolution and release in subcutaneous tissue are retarded (rapid, intermediate, and slow insulins). Suitable particles can be obtained by precipitation of apolar, poorly water-soluble complexes consisting of anionic insulin and cationic partners, e.g the polycationic protein protamine or the compound aminoqui-nuride (Surfen). In the presence of zinc and acetate ions, insulin crystallizes crystal size determines the rate of dissolution. Intermediate insulin preparations (NPH or isophane, lente or zinc insulin) act for 18 to 26 h, slow preparations (protamine zinc insulin, ultralente or extended zinc insulin) for up to 36 h. 

Another zinc-utilizing enzyme is carbonate/dehydratase C (Kannan et al., 1972). Here, the zinc is firmly bound by three histidyl side chains and a water molecule or a hydroxyl ion (Fig. 27). The coordination is that of a distorted tetrahedron. Metals such as Cu(II), Co(Il), and Mn(ll) bind at the same site as zinc. Hg(II) also binds near, but not precisely at, this site (Kannan et al., 1972). Horse liver alcohol dehydrogenase (Schneider et al., 1983) contains two zinc sites, one catalytic and one noncatalytic. X-Ray studies showed that the catalytic Zn(II), bound tetrahedrally to two cysteines, one histidine, and water (or hydroxyl), can be replaced by Co(II) and that the tetrahedral geometry is maintained. This is also true with Ni(Il). Insulin also binds zinc (Adams etai, 1969 Bordas etal., 1983) and forms rhombohedral 2Zn insulin crystals. The coordination of the zinc consists of three symmetry-related histidines (from BIO) and three symmetry-related water molecules. These give an octahedral complex... 

Gursky, O. et al. (1994) Stereospecific dihaloalkane binding in a Ph-sensitive cavity in cubic insulin crystals. Proc. 

Schlichtkrull J. Insulin Crystals. Copenhagen Ejnar Munksgaard Publisher, 1958. 

Figure 9-25. Atomic arrangement in the 2Zn-insulin crystal. The smaller projection drawing shows the molecular packing in the insulin hexameters. Courtesy of Dorothy Hodgkin [37],...

Baker T, Dodson E, Dodson G, Hodgkin D, Hubbard R (1985) The water structure in 2Zn insulin crystals. In Moras D, Drenth J, Strandberg B, Suck D, Wilson K (eds) Crystallography in molecular biology. Plenum Press, New York, pp 179-192... 

Pertinent to this discussion of spontaneously seeded batch operations is an investigation by Schlichtkrull (S8) in which the seeded and unseeded cases are compared. In these experiments with insulin crystals,... 

Fig. 21. Cumulative distribution of insulin crystals. After Schlichtkrull (S8).

O. Gursky, J. Badger, Y. Li, and D. L. D. Caspar. Conformational changes in cubic insulin crystals. Biophys... 

Caspar et al. (1988) described an elegant analysis of the diffuse X-ray scattering from insulin crystals. They found two types of coupled motion one with a characteristic length of about 6 A and amplitude of about 0.4 A, the other with a characteristic length of about 20 A and smaller amplitude. The latter motion represents the jiggle of neighboring molecules of the lattice. The former represents the coupled fluidlike fluctuations within a protein molecule. The short-range motions appear to be similar to those detected by Mossbauer spectroscopy. 

Fig. 10 Surfaces of human insulin crystals showing that steps arise by different mechanisms depending on supersaturation. (A) At low supersaturation, steps are created by screw dislocations, D and (b) At high supersaturations, cjs > 10, steps occur at the edges of islands as a consequence of 2-D nucleation. (From Ref. l)...

The long-acting insulins in use today are mainly protamine insulin and zinc insulins. Protamine insulins are the salt-like compounds formed between the acid (insulin) polypeptide and the polypeptide protamine, which consists primarily of arginine. They are used in the form of neutral suspensions of protamine insulin crystals (isophane insulin). 

Schlichtkrull, J. (1958). Insulin Crystals. Ejnar Munksgaard Publisher, Copenhagen. Schmid-Antomarchi, H., De Weille, J., Fosser, M. and Lazdunski, M. (1987). J. Biol. Chem. 262. 15840-15844. 

Schlichtkrull, J. (1956) Insulin crystals 1. The minimum mole-fraction of metal in insulin crystals prepared with Zn, ... 

HUMULIN R REGULAR U-500 (concentrated insulin human injection, USP) [/s call expressed biosynthetic human insulin] Eli Lilly 20 mL vials of zinc-insulin crystals dissolved in a clear fluid 500 Units/mL 100 units/ mL (U-100) 0.24 mg sodium citrate 1.04 mg citric acid monohydrate 9.6 mg mannitol 0.8 mg polysorbate 80 16mg/mL glycerin, 2.5mg/mL m-cresol as a preservative 0.085 mg/mL (0.017 mg/ 100 units) zinc-oxide (sodium hydroxide and/or hydrochloric acid may be added during manufacture to adjust the pH)... 

Zinc Insulin Crystals. Crystalline preparation of the active antidiabetic principle of the internal secretion of Langerhans islands of the pancreas. The crystals contain a small amount of zinc (not less than 0.45% nor more than 0.9%), which is chemically combined with the active principle. Each milligram nf the crystals is equivalent to not less than 22 units of insulin. The product is marketed in the form of cryst zinc-insulin injection. Study of the binding of zinc by insulin Tanford, Epstein, J. Am. Chem. Soc. 76, 2170 (1954) Cunningham, et al ibid. 77, 5703 (1955). Proposed crystallographic structure Marcker, Graae, Acm Chem. Sea rid. 16, 41 (1962). 

The drug is a sterile suspension of Zinc-insulin crystals and protamine sulphate in buffered water for injection, usually combined in such a fashion that the solidphase of the suspension essentially comprises of crystals composed of insulin, protamine and zinc. 

Insulin crystal type soluble, amorphous, crystalline, microcrystalline... 

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