A surgical or skin wound is first and naturally filled by the blood of the patient. A blood clot, mainly formed of red cells within a fibrin and platelets network, is normally formed and seals the wound. These blood components, including red cells, then actively participate to the healing phase as reported many years ago by Moorhead et al. (1943). So, the tissue repair of a wound occurs within a blood environment and the synthesis of collagen, which is a basic mechanism of the tissue repair, is the result of a natural response of inflammatory cells and fibroblasts to blood components.
The platelets growth factors give the right signals to these cells, but fibroblasts in particular also need to migrate into and attach to scaffolding structures. The blood clot is the natural scaffold which fills the tissue defect or cavity of a wound and it is not surprising to consider a major blood protein like hemoglobin to become the basis of a physiological material to fill tissue defects or cavities where blood is absent or has been removed.
Collagen from animal origin was the first physiological filling or sealing material proposed mainly because it was insoluble and did not diffuse out of its implantation site. So it seems legitimate to propose an insoluble human blood component to replace animal collagen if and when possible.
Globin is the protein constituting hemoglobin which, itself, contains 4 peptide chains (2 alpha and 2 beta chains), each associated with one heme, Braunitzer et al. (1961), Hill et al. (1962). The heme consists of a tetrapyrole structure containing one positively charged iron atom. There are 4 hemes per molecule, responsible for the red color of hemoglobin. The concentration of hemoglobin in blood is very high, reaching a mean value of 140 mg/ml. Peripheral blood is the most accessible human tissue, which makes it particularly attractive as a source of autologous biomaterials.
Broad and potent bactericidal properties of hemoglobin or soluble derivatives have been known for a long time: Hobson et al. (1958), Liepke et al. (2003). We have shown that the insoluble globin offers these unique antimicrobial properties in specific conditions and opens the possibility to use a physiological biopolymer to prevent local infection in a wound or an implanted area.
The processes for preparing globin and removing the heme have been known for a very long time, Schulz (1898), Anson-Mirsky (1930). Unlike hemoglobin, which is completely soluble at physiological pH, globin is notably insoluble under the same conditions. The insoluble nature of globin under physiological conditions has made its pharmaceutical applications difficult or non obvious.
For this reason, most experiments have sought to prepare chemically modified globin derivatives to make them soluble at physiological pH and use them, for instance, as plasma substitutes. Solubilisation experiments at neutral pH were carried out by Strumia et al., (1951) and (1952), using a prolonged alkaline treatment which resulted in partial deamidation of the globin at the asparagine and glutamine residues, converted respectively to aspartic acid and glutamic acid, Vars (1952).
Other solubilisation experiments were carried out by Volckmann (1988), by succinylation. The solubility of the modified globin comes from the global increase of its negative charges and decrease of its isoelectric pH. All these efforts however, failed to develop any long term medical use of a chemically modified soluble globin.
The first and only medical application of the natural insoluble globin was developed by Burroughs-Wellcome. An injectable product combining a preparation of acid globin (soluble only at acid pH) with insulin has been developed and patented: Reiner (1939); Reiner et al. (1939).
Globin is neutralized and precipitates in situ, after intramuscular injection. It then contributes to a gradual release of the insulin from this complex: Rabinowitch et al. (1947); Berg et al. (1953). This association called “Globin Insulin” was marketed from 1940 until 1978 and injected every day in many diabetic patients for years, demonstrating the long term safety of injections of insoluble globin. Globin-Insulin (1944), a commercial booklet of this pharmaceutical product, describes the product, its efficacy and safety.