A LACTOFERRIN CONTAINING THERAPEUTIC AGENT FOR RHEUMATISM AND DERMATOLOGICAL AND COSMETIC COMPOSITIONS CONTAINING SUCH AGENT

Abstract: The present invention concerns a therapeutic agent for rheumatism which contains lactoferrin as an active ingredient. Such a therapeutic agent shows excellent inhibitory effect against collagenase activity and is useful for treatment for rheumatism.

Description: A LACTOFERRIN CONTAINING THERAPEUTIC AGENT FOR RHEUMATISM AND DERMATOLOGICAL AND COSMETIC COMPOSITIONS CONTAINING SUCH AGENT This invention relates to a therapeutic agent for rheumatism, which contains lactoferrin as an active ingredient. Lactoferrin, a protein existing in milk or tears of human being, bovine, etc., is known to have pharmacological effects such as antibacterial effect and proliferating effect of lymphocytes (Japanese ϋnexamined Patent Publication 48534/1990 etc.). However, it is desired to study potential effects of lactoferrin, a natural product, and expand the applications thereof. Thereupon, we studied to find new pharmacological effects of lactoferrin and found that lactoferrin was useful for treatment of rheumatism. This invention relates to a therapeutic agent for rheumatism, which contains lactoferrin as an active ingredient. It is expected that lactoferrin, a natural protein, has a possibility to be applied for treatment of various diseases. However, as the pharmacological effects of lactoferrin, only antibacterial effect and proliferating effect of lymphocytes etc. have been reported. Therefore, we studied to find new pharmacological effects of lactoferrin and found that lactoferrin was useful for treatment of rheumatism. An inhibitory effect of a medical substance against collagenase activity can be used as an index to examine the utility thereof for antirheumatic agent. The relationship of inhibitory effect against collagenase activity and rheumatism has been reported (W. H. Johnson et. al., J. Enzyme Inhibition, 2., 1 (1987) ; Woolley D. E. et. al., Arthritis Rheum., 2J), 1231 (1977)). Therefore, we examined the inhibitory effect of lactoferrin against collagenase activity. As the result, we found that lactoferrin significantly inhibited the degradation of collagen caused by collagenase and showed excellent inhibitory effect against collagenase activity. The detailed experimental data are shown in the article of pharmacological test. Lactoferrin is usually administered directly to knee joint etc. by injection, but it may be administered orally. The dosage is adjusted depending on symptom, dosage form etc. In case of injection, the usual concentration of lactoferrin is 0.1% - 10%, preferably 0.5% - 5%, and suitable volume of the solution is injected to a rheumatoid joint. In case of oral form, the usual daily dosage is 1 to 5000 mg, preferably 50 - 1000 mg in one or a few divided doses. The preparations of lactoferrin can be prepared by usual methods. Injection can be prepared by dissolving lactoferrin in distilled water for injection and, if necessary, usual excipients such as isotonic agent, pH adjusting agent and viscosity agent can be added. Tablet can be prepared by using usual excipients such as binding agent and lubricant. Examples of formulations are shown below. Examples of Formulations 1 ) Injection formulation 1 lactoferrin 1g sodium chloride 0.9g distilled water for injection q.s. total 100ml formulation 2 lactoferrin 2g sodium chloride 0.9g distilled water for injection q.s. total 100ml formulation 3 lactoferrin 5g sodium chloride 0.7g distilled water for injection q.s. total 100ml formulation 4 lactoferrin 0.5g sodium chloride 0.9g distilled water for injection q.s. total 100ml formulation 5 lactoferrin 10g sodium chloride 0.5g distilled water for injection q.s. total 100ml formulation 6 lactoferrin 0.1g sodium chloride 0.9g methyl cellulose 0.5g distilled water for injection q.s. total 100ml Tablet formulation 7 lactoferrin 100mg lactose 50mg crystalline cellulose 30mg hydroxypropylcellulose 5mg magnesium stearate 5mg total 190mg PHARMACOLOGICAL TEST We examined the inhibitory effect of lactoferrin against collagenase activity according to the method of Nagai et. al. (Japanese Journal of Inflammation, §_, 123 (1984)). Inhibitory effect against collagenase derived from microorgan (Experimental Method) Collagen labeled with fluorescein isothiocyanate and collagenase derived from microorgan (Clostridium histolyticum) were dissolved in Tris—HC1 buffer (0.05M, pH7.5), which contains sodium chloride (0.2M), calcium chloride (0.005M) and sodium azide (0.02%), in a concentration of 0.025% and 12.5 unit/ml respectively. To 0.4ml of this solution, lactoferrin was added and the mixture was incubated for 1hr at 35°C in a brown test tube. 10μl of water-ethanol solution (1:1 V/V) dissolving o-phenanthroline (80mM) was added to stop the reaction and the mixture was incubated for Ihr at 35°C. 400μl of a mixture of Tris-HCl buffer (0.05M, pH9.5), which contains sodium chloride (0.2M), and ethanol (3:7 V/v) was added and the mixture was stirred and centrifuged. Degradated collagen was assayed by measuring the fluorescence intensity of the supernatant (excitation wavelength : 495 nm, emission wavelength : 520nm) . In the control, it was treated by the same manner as the above except the addition of lactoferrin. (Result) Inhibitory percentage against the degradation of collagen by the addition of lactoferrin was shown in Table 1. Table 1 fluorescence intensity inhibition(%) Inhibitory effect against collagenase derived from .tissue (Experimental Method) The inhibitory effect against tissue collagenase, which was purified from cornea of rabbit according to the method reported by Burns et al. (Invest. Ophthalmol. Vis. Sci., .30 / 1569 (1989)), was examined by the similar method as for collagenase derived from microorgan. In this experiment, the amount of collagenase to be added was 2.5mg protein/ml. (Result) The experimental results were shown in Table 2 Table 2 fluorescence intensity inhibition(%) As shown in Tables 1 and 2, we found that lactoferrin inhibited the degradation of collagen in a dose—dependent manner and showed an inhibitory effect against collagenase activity in the both experiments using collagenase derived from microorgan or tissue. As shown in the article of pharmacological test, lactoferrin shows excellent inhibitory effect against collagenase activity and is useful for treatment for rheumatism. As it was shown that lactoferrin is able to inhibit bacterial and tissue collagenase activity in a dose dependent manner, it is evident that this activity is of interest to cosmetic application. Skin ageing is a complex process, but it is well known that enzymatic hydrolysis of tissue specific macromolecules, such as collagen and elastine, can contribute to premature scenescence. Natural, inoffensive and efficacious collagenase inhibitors are therefore of interest. Lactoferrin can therefore be used as an agent for the prevention of premature ageing due to its contribution to the preservation of the integrity of the skin. The concentrations at which lactoferrin is active against these collagenases are of reasonable range for dermatology and cosmetic applications. Cosmetic applications include oral hygiene also, where lactoferrin may be of use in the inhibition of bacterial collagenases : prevention of bacteriolitic destruction of the gums. Therefore, in dermatological and cosmetic compositions, lactoferrin is used as inhibitor of bacterial and tissues collagenases, in concentrations between 0.005 % and 5 % (preferably between 0.05 % and 0.5 %). Lactoferrin may also be used for dermatological and cosmetic compositions in the form of creams, lotions, milks, tooth paste, mouthwash rinses, gels, for application in skin care, oral hygiene and prevention of tissue degradation.

Claims: CLAIMS 1/ - A therapeutic agent for rheumatism, which contains lactoferrin as an active ingredient. 2/ - Dermatological and cosmetic compositions containing lactoferrin as inhibitor of bacterial and tissue collagenases, in concentrations between 0.005 % and 5 %, preferably between 0.05 % and 0.5 %. 3/ - Dermatological and cosmetic compositions according to claim 2, characterised in that they are : creams, lotions, milks, tooth paste, mouthwash rinses, gels, for application in skin care, oral hygiene, prevention of tissue degradation.

Inventors: DEGRE MICHEL FRANCOIS, HAYASHI MASAAKI, HIKIDA MITSUSHI, DEGRE, MICHEL, FRANCOIS, HAYASHI, MASAAKI, HIKIDA, MITSUSHI
Applicants: BIO SERAE LAB, BIO SERAE LABORATOIRES S.A.
Application year: 1993
Main classification: A61K37
IPC codes: A61K8, A61K38, A61P29, A61P43, A61Q11, A61Q19