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Updated 16 January 2003;15 February 2000
Lactoferrin for Developing Novel Therapeutics Against Primary Infection and Inflammatory Diseases Lactoferrin (LF) is an anti-infective protein that has been associated with host defense against primary infection at mucosal surfaces through its antibacterial properties. It is also an immunoregulatory protein associated with inflammation and immunomodulation processes, including maturation of immature lymphocytes. LF was isolated from cow milk in 1939 as a red protein. It is an iron-containing glycoprotein present in most mammalian milk, and in a wide range of body fluids. In humans, LF (691 amino acid residues) is present in colostrum at 5-7 mg/ml, and at 2-3 mg/ml in the normal mother milk. The protein is also a major component of neutrophils and secreted into blood upon bacterial infection and cytokine stimulation. It has been repeatedly shown that breast-fed infants have fewer and less severe infections and atopic eczema than formula-fed infants (Kramer MS et al, 2001: uid=11242425), and it is likely that LF is a major factor for the decreased episodes upon breastfeeding. In addition, LF is under investigation for the treatment of inflammatory bowel diseases (Crohn's disease and ulcerative colitis) because it down-regulates tumor necrosis facter-alpha (TNFalpha), a pro-inflammatory cytokine important in the pathogenesis as discussed below.
Anti-microbial effects
There have been reports on the inhibitory effects, in vitro and in vivo, of LF against adenovirus, cytomegarovirus, hantavirus, hepatitis C virus (HCV), herpes simplex virus type I, human immunodeficiency virus and rhinovirus. These effects arise from the physical contact with viral particles to result in their disruption, inhibition of viral adsorption to the host cell, or inhibition of viral enzymes. The study has been sporadic. For adenovirus infection, LF (bovine, human) was able to inhibit adenovirus replication in a dose-dependent manner, and to prevent viral replication when added both before, or during the viral adsorption step, or when present during the entire replicative cycle of adenovirus (Arnold D et al, 2002: uid=11750941). LF action appears to take place on an early phase of viral replication. In the case of human cytomegarovirus infection, LF seems to inhibit virus entrance into the host cell, and the native LF was seven-fold more active in this effect than N-end cyclic lactoferricin (Andersen JH, 2001: uid=11431038). Perhaps the effect on HCV is best investigated so far. Bovine LF inhibits HCV infection when the virus is pre-incubated with LF, and the adsorption to the host cell is inhibited here (Ikeda M et al, 2000: uid=10653917). This viral interaction appears to be rather specific with LF among milk proteins. The domain of LF that interacts with HCV is not defined yet but not the one at N-terminus that is important for anti-bacterial activity (see below). In a small scale pilot study, patients with chronic hepatitis C infection received an 8-week course of bovine LF (1.8 or 3.6 g/day). At the end of LF treatment, the decrease in serum alanine transaminase and HCV RNA concentrations was apparent in 3 (75%) of 4 patients (Tanaka K et al, 1999: uid=10363572) . As good remedy for HCV infection is missing, LF may be viewed as a potential candidate to develop an anti-HCV reagent effective for the treatment of patients with chronic hepatitis.Bovine and human LF's may be digested by pepsins into small cationic domains that exhibit enhanced anti-microbial activities. These domains are in the N terminus of LF molecule, and highly effective against infections with a variety of microbes such as Staphylococcus aureus (including drug-resistant), Krebsiella pneumoniae and Candida albicans. In two strains of Candida albicans, bovine LF peptide, FKCRRWQWRM (amino acid residues 17-26), strongly suppressed cell multiplication of Candida cells, but other peptides showed only weak activities. In two strains of C. albicans, the minimum inhibitory concentration 100 of peptide 2 (MIC, ca 17 microM), and the effect was additive to that of chemical agents such as amphotericin B (Ueta E et al, 2001: uid=11298926). This peptide also prolonged the survival times of Candida-infected mice between 8 and 22 days, depending on the dose(Tanida T et al, 2001: uid=11349055).
In need of new anti-infective agents, LF cationic peptide domains and analogs have been further synthesized to fish out most effective sequence of amino acid residues. In human LF, 2 cationic domains hLF(1-11: GRRRSVQWCAV) and hLF(21-31: QWQRNMRRVRG) are important, and the former is more effective. The first 2 arginine residues in hLF(1-11) seem to play an essential role in this killing activity (Lupetti A et al, 2000: uid=11083624; Nibbering PH et al, 2001: uid=11179314).
In another line of research where intact molecule was used, LF exhibited strong anti-fungal activity at 0.5-100 mg/ml against clinical isolates of Candida from severely immunosuppressed patients, particularly in combination with fluconazole and other antifungals (Kuipers ME et al, 2000: uid=10543740). Recombinant human LF was also shown to be effective against Helicobacter felis infection in the mouse stomach (Dial EJ et al, 2000: uid=11197084). In addition, bovine LF showed potencies to inhibit H. pylori infection in the mouse model (Wang X ett al, 2001: uid=11339250). It is conjectured that LF inhibits the attachment of Helicobacter to gastric mucosa that involves several specific structures. Taken together, there is now a growing clinical interest to develop LF as a candidate for non-antibiotic drug against H. pylori infection in man to prevent gastritis. In fact, a two-week course of treatment with human LF was sufficient to partially reverse both infection-induced gastritis, and the efficacy was comparable to the treatment with amoxicillin and standard triple therapy. Thus intact LF and/or peptidic derivatives may be used as an anti-microbial, alone or in combination with conventional agents in novel therapeutic regimens. In particular, these non-antibiotic agents may be developed against drug-resistant microbial infections that are emerging problems in the clinical arena.
Anti-inflammatory effects
Several anti-inflammatory functions have been ascribed to LF. A possible mechanism here is that LF down-regulates pro-inflammatory cytokines such as TNFalpha. In order to examine if IF is involved directly in the cytokine control, a model of cutaneous immune system was used in which it is known that epidermal Langerhans cell migration is dependent on TNFalpha. An allergen (oxazolone) induced migration of epidermal Langerhans cells (LC) from the skin, and their subsequent accumulation as dendritic cells (DC) in skin-draining lymph nodes. The intradermal injection of recombinant murine LF inhibited this LC migration and DC accumulation. As the inhibition was reversed by TNFalpha injection, and TNFalpha-dependent interleukin 1beta effect was impaired by LF, this anti-immune effect was thought to be direct inhibition of de novo TNFalpha synthesis by LF (Cumberbatch M et al, 2000: uid=10809955). In humans, oral administration of bovine LF resulted in a profound decrease of the spontaneous production of TNFalpha by cultures of peripheral blood cells (Zimecki M et al, 1999: uid=10202564). This decrease was significant, following the 7-day dose of LF (in a oral dose of 10 mg-containing capsule), and persisted for additional 14 days. Similarly, in collagen arthritis in DBA/1 mice and Staphylococcus aureus septic arthritis in Swiss mice, local LF injection showed 71% of corticosteroid-equivalent effect in the suppression of paw inflammation (Guillen C et al, 2000: uid=11014359).In addition to cytokine down-regulation, LF may prevent the activation of leukocytes by a different mechanism. LF binds to lipopolysaccharides (LPS), and is able to compete with the LPS-binding protein for LPS binding and therefore to prevent the transfer of LPS to the surface of monocytes. Hence the prophylactic effect of LF against septicemia in vivo. LF is also a major component of neutrophils and may block LPS binding to LPS receptor L-selectin on neutrophils. In fact, human LF was shown to inhibit the binding of LPS to L-selectin in a concentration-dependent manner (Baveye S et al, 2000: uid=10708745). The inhibition was maximum (87.7%) at a concentration of 50 mg/ml. This inhibition of LPS binding likely leads to the reduction (up to 55.4%) of the intracellular hydrogen peroxide production, resulting in the block of oxidative burst that would contribute to pathogenesis such as septic shock.
Inflammatory bowel diseases (Crohn's disease and ulcerative colitis) primarily affect the intestines, resulting in pain, severe diarrhea, intestinal bleeding, weight loss and fever. In ulcerative colitis, the inner lining of the colon is inflamed. Patients with Crohn's disease have similar inflammation, but it extends deeper into the intestinal wall and can also involve the small and large intestines. Because of anti-inflammatory effect probably due to TNF down-regulation, LF is now under intensive clinical studies for potential treatment of inflammatory bowel diseases and other inflammatory diseases such as topical dermatitis and asthma. LF has systemic effect, is orally administered, and most of all expected to be totally non-toxic from its origin in the mother milk and body fluids. The chances are that LF can be developed as a safe and novel therapeutic for these difficult diseases.
Anti-cancer effects
In the cancer area, there have been an increasing number of reports on LF anti-cancer effects. LF expression may be suppressed upon carcinogenesis, as Penco S et al (1999: uid=10738912) reported 31/78 of sporadic breast cancer were negative for LF mRNA, and other preliminary findings also indicate no LF expression in some other tumors. In the cell cycle of breast cancer cells MDA-MB-231, human LF inhibited the growth at the G1 to S transition (Damiens E et al, 1999: uid=10412049). This G1 arrest was associated with a dramatic decrease in the protein levels of Cdk2 and cyclin E, while Cdk4 activity was also decreased in the treated cells, both being accompanied by an increased expression of the Cdk inhibitor p21(CIP1). In addition, retinoblastoma protein pRb was kept in a hypophosphorylated form. Thus LF appears to regulate key steps in the cell cycle, and it is entirely possible that LF by itself is a tumor suppressor.Another mechanism of LF anti-cancer effect may be due to a modulation of NK cell cytotoxicity and of target cell sensitivity to lysis. LF binds to 91% of the naturally heterogeneous NK cell population, and increases the NK cell cytotoxic activity at low concentrations (10 ug/ml; Damiens E et al, 1998: uid=9606986). LF also exerts varying effect on target cells depending on the cell phenotype, particularly of the breast and colon epithelial cells. In general so far, LF anti-cancer effect seems to be pronounced on esopahrgus, lung, breast and colon. More recently, an additional mechanism of LF effect was indicated from angiogenesis study (Norrby K et al, 2001: uid=11146451). The tumor growth is angiogenesis-dependent, and in most tumors and other angiogenesis diseases, VEGF(165) plays a major role as the angiogenic factor. When treated with bovine LF, VEGF(165)-mediated responses in terms of microvessel spatial extension, overall vascularity and incidence of crossover were significantly inhibited. In vitro, LF also exerted an anti-proliferative effect on endothelial cells. How LF suppresses tumors systemically is still a matter of conjecture, but from the above findings the anti-angiogenic activity following oral LF administration might be a key mechanism, and warrants further study.
As discussed above, LF has a range of effects, namely anti-microbial, anti-inflammatory, and anti-carcinogenic. More specifically, LF may be targeted at C-type hepatitis, atopic dermatitis, H. pylori gastritis, inflammatory bowel diseases (colitis, Croh's), bacterial drug-resistance, enteric infections, and squamous cell carcinomas. The future study should be carefully focused but it can again be emphasized that LF is almost guaranteed to be non-toxic without any expected side effects, and can be orally administered repeatedly. LF, particularly recombinant human LF that everybody was once fed with, may now be produced in a large quantity in the milk of transgenic cows (Van Berkel PH et al, 2002: uid=11981562) and in Aspergillus awamori fermentation system (Agennix Inc Japanese patent nos: 2701976 and 2824332; US patent nos: 5,766,939 and 5,849,881). Natural human LF from human milk and recombinant human LF had identical iron-binding and iron-release properties, and although natural and recombinant LFs underwent differential N-linked glycosylation, they were equally effective in three different in vivo infection models employing immunocompetent and leukocytopenic mice, and showed similar localization at sites of infection.
Most LF studies have been carried out with bovine LF, simply because human LF was not available in a large quantity. Bovine LF is usually 92-95% pure, and produced under non-GMP guidelines. As long as therapeutic development is aimed at that requires GMP guidelines, bovine LF may not be the choice. However, in some cases such as oral administration, GMP guidelines may be exempted in the drug development and bovine LF may still be used. Such exemption is now considered in Japan. As a large quantity of human LF is available now that is produced under GMP guidelines, the future study of LF for therapeutic targets should use human LF.
Bovine and human LFs have exhibited similar effects in most targets so far studied in humans. Perhaps the only exception is that only human LF has anti-inflammatory effects in humans. LF study is still in its infancy, and there should be additional differential effects between bovine and human LFs in humans as the study progresses in the future. It is quite important to point out here that human LF receptor has been cloned from infant small intestinal cells, by PCR based on amino acid sequences of the purified native human LF receptor ( Suzuki YA et al, 2001: uid=11747454). Biochemical properties of the cloned receptor were similar to those of the native, and the receptor gene was expressed at high levels in fetal small intestine and in adult heart. The receptor would now make more specific and mechanistic study possible, and there would be structure-based drug design to find small molecular agonists and antagonists of LF that accelerate the study of mode of action.
Oral was the only way of administration for LF due to the lack of enough human LF. Now that human LF is available in a large quantity, i.v. and other means of administration are possible without possible risk of immunity. In fact, human LF may be injected up to 60 mg/kg in humans (XTL Biopharmaceuticals), and make it possible to study its efficacy in viral infection such as HCV, as it was shown that LF efficacy depends on the concentration in the blood.
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