Properties and Application of Reacombinant Human Lactoferrin to Enhance Healing of Diabetic Wounds

Introduction Lactoferrin (LF) is a member of the transferrin family of nonheme iron-binding proteins.[1] LF is found mainly in external secretions of mucosal epithelia, such as milk, saliva, tears, seminal fluid, sweat, and nasal mucus, as well as in bile, pancreatic fluid, and intestinal secretions in mammals. LF is also found in the secondary granules of neutrophils, which are the main source of LF in plasma as it is secreted upon neutrophil stimulation. As shown in Table 1, several physiological functions, such as broad-spectrum anti-infective, immunomodulatory, and anti-inflammatory activities, have been attributed to LF.[2,3] Pharmaceutical grade recombinant human lactoferrin (rhLF), produced in Aspergillus niger var. awamori, is a 78,000 Dalton glycoprotein and is identical in all material respects to its natural counterpart, native lactoferrin (LF).[4,5] rhLF is a 692-amino acid glycoprotein with a molecular weight of approximately 80 kDaltons. The protein has two lobes, each containing a binding site for ferric iron (Fe3+).[6] rhLF is structurally equivalent to native human LF, as demonstrated by a comparison of the three-dimensional structure, molecular weight, biological activity, and other physicochemical properties, and differs only in the nature of glycosylation.[7] Anti-Infective Functions LF is present in exocrine secretions that are commonly exposed to normal flora, such as milk, tears, nasal exudates, saliva, bronchial mucus, gastrointestinal fluids, cervico-vaginal mucus, and seminal fluid, and is thought to play a critical role in host primary defense against infections. rhLF prevents Escherichia coli translocation from the neonatal rat intestine after artificial infection protecting against high levels of illness and death.[8] LF exerts both bacteriostatic and bactericidal effects on bacteria by respectively binding iron needed for bacterial growth or by destabilizing the outer membranes of bacteria.[3] Intravenously administered LF has been shown to protect against E. coli-induced mortality with 70 percent of treated mice surviving a lethal dose of E. coli.[9,10] Further, protection by bovine LF against mortality induced by Toxoplasma gondii infections in mice has also been described,[11] and prophylactic administrations of intravenous (IV) and oral LF have been shown to protect against kidney infections and bacterial titers following IV administration of Staphylococcus aureus.[12] rhLF, when administered orally, is also capable of suppressing Helicobacter pyloris infection in mice.[13] LF and peptides derived from LF have been shown to inhibit the growth of yeast, filamentous fungi, and parasitic protozoa by binding to the lipid bilayer of biological membranes, forming pores, which is ultimately followed by cell lysis.[14–17] LF has been recognized as a potent inhibitor of various viruses via blockage of viral entry, including rotavirus,[18] human cytomegalovirus, and human immunodeficiency viruses,[19,20] herpes simplex virus,[21] and human hepatitis C virus.[22] Anti-Inflammatory Functions LF has several anti-inflammatory properties (Table 1). In a pilot clinical study, rhLF reduced gut inflammation and survival in patients with severe gut graft-vs-host disease (GVHD).[23] Th2-dominated responses are involved in the pathogenesis of GVDH and thus a reversal towards a Th1 environment appears to be protective.[24,25] rhLF’s anti-GVHD effect may be mediated by its ability to shift the immune responses from Th2 to Th1 by up-regulating IL-18 and IFN-gamma and by down-regulating IL-4, IL-5, and IL-10 (Figure 1).[26,27] LF is protective against bacterial lipopolysaccharide (LPS)-induced generalized systemic inflammation and mortality in several different animal models. Lee, et al., showed that oral bovine LF reduced the mortality from 74 percent to 17 percent in endotoxemic newborn piglets.[28] Kruzel, et al., also describe protection in mice against endotoxin-induced pathological and cytokine changes or mortality by intraperitoneal or IV administrations.[29,30] Sepsis is mediated by TNF-alpha, and studies with mice and human samples suggest that LF inhibits the in-vivo production of TNF-alpha from local sources, such as keratinocytes.[31–33] Human LF has also been shown to be protective in inflammation induced by allergic asthma. In a sheep model of asthma, LF administered by inhalation inhibited late-phase bronchoconstriction and delayed bronchial hyper-responsiveness following an antigen challenge.[34] As in the case of GVHD, the pathogenesis of asthma appears to involve a predominance of Th2 immune responses,[35] and therefore the ability of LF to shift the immune response toward a Th1 type may explain its anti-asthmatic effect. Immunomodulatory Functions There is strong evidence that LF affects cell-mediated host responses. LF has been shown to enhance the delayed type hypersensitivity (DTH) response in mice mediated by macrophages and T cells, up-regulating IL-15, MIP-1alpha, and MIP-2.[36] LF has a high affinity for surface receptors found on activated, but not resting, lymphocytes in vitro.[37] LF also accelerates T-cell maturation by inducing the expression of the CD4 surface marker in vitro.[38] LF has been shown to activate natural killer (NK) cells, induce colony-stimulating activity, activate polymorphonuclear (PMN) cells, regulate granulopoeisis, enhance antibody-dependent cell cytotoxicity, stimulate lymphokine-activated killer cell (LAK) activity, and potentiate macrophage toxicity.[39–41] There is a strong possibility that some of the immunomodulatory properties attributed to LF follow stimulation of IL-18, a key Th1 cytokine and a strong inducer of interferon-gamma and GM-CSF release (Figure 2).[42–44] Innate and cell-mediated immunity are both known to be critical for protection against viruses and some diseases, such as cancer. Indeed, LF can exert an anticancer effect. LF has a significant effect on the cytotoxicity of NK cells against hematopoietic and breast epithelial cell lines.[45] Kuhara, et al.,[46] discovered the link between the oral administration of LF, induction of IL-18, immunomodulation, and protection against experimental cancer metastasis (lung colonization by colon 26 carcinoma). LF increased CD4+, CD8+, and NK cells in peripheral blood of tumor-bearing mice (pDisclosure. The editors of WOUNDS wish to disclose that Aristidis Veves, MD, DSC, Section Editor of Biology and Treatment of Diabetic Foot Ulcers for WOUNDS, has received a research grant from Agennix, Inc.