WO2011045081A1

WO2011045081A1 - Use of lactoferrin and lactoferrin-containing compositions

Info

Publication number: WO2011045081A1
Application number: PCT/EP2010/006335
Authority: WO
Inventors: Kurt S. ZÄNKER
Original assignee: Vczs Venture Capital Zentralschweiz Ag
Priority date: 2009-10-18
Filing date: 2010-10-18
Publication date: 2011-04-21
Also published as: DE102009049702A1

Abstract

The present invention relates to novel uses of lactoferrin and lactoferrin-containing compositions.

Description

description

Use of lactoferrin and lactoferrin containing

compositions

The present invention relates generally to the use of lactoferrin and lactoferrin-containing compositions. More particularly, the invention relates to the use of recombinant human lactoferrin and human lactoferrin and compositions containing the same.

Lactoferrin, more precisely Lactotrans ferrin, is a protein that is found in the secretions of all mammals, especially in milk, and on the one hand acts as a protease, on the other hand as an iron-binding protein. It is also localized in white blood cells.

As a protease lactoferrin is capable of destroying several colonization proteins of the pathogen Haemophilus influenzae. In particular, the concentration of lactoferrin occurring in human breast milk can prevent respiratory tract infections that often occur in infants with this germ. It also affects the type III secretion system in Shigella and pathogenic Escherichia coli. Liberated by leukocytes, it is also part of the immune system. As shown in a recent study by the Journal of the American Medical Association, the use of lactoferrin on underweight infants (<1500 g birth weight) may significantly and clinically reduce the incidence of sepsis and sepsis-associated mortality (Manzoni P, Rinaldi M, Cattani S, et al.: Bovine lactoferrin supplementation for prevention of late-onset sepsis in very low birth-weight neonates: a randomized trial. In: JAMA. 302, No. 13, October 2009, p. 1421-1428).

A recombinant human lactoferrin 1-1 1 was tested in a clinical study in healthy volunteers to develop a safety profile; This is the intention to use the lactoferrin in patients with hematopoietic stem cell transplantation as infection protection. An i.v. Administration with a dose range of 0.005, 0.05, 0.5 and 5 mg was found to be relatively safe with the exception that possibly transaminases can be increased by the administration of lactoferrin (WJ, van Iersel TM, Blijlevens NM, Donnelly JP, (2009 BMC Med. 7, 44). Recombinant human lactoferrin is structurally identical to native human lactoferrin for all relevant traits. Lactoferrin has a role as an antibacterial drug in dentistry, in which lactoferrin suppresses the initial adherence of S. gordonii and S. gordonii co-aggregates by its secretion of iron; no pathological biofilm can form in the oral cavity (Arsian SY, Leung KP, Wu CD (2009) Oral Microbiol Immunol 24, 41 1-416).

Bovine lactoferrin as a cationic antimicrobial peptide also has a cytotoxic potential against cancer cells; a truncated lactoferrincinB in a carrier platform of fusogenic liposomes has cytotoxic activity against T-leukemia as well as against breast cancer cells (Richardson A, de Antueno R, Duncan R, Hoskin DW (2009) Biochem. Biophys. Res. Commun. 388, 736-741 ).

Lactoferrin is also produced in mucosal epithelial cells and in granulocytes. It is involved in the regulation of the immune response, it has an antioxidant potential, it has anti-carcinogenic and anti-inflammatory properties and remains a protective molecule against various microbial infections. In particular, it seems to represent a molecular protective mechanism against lipopolysaccharide (LPS) -induced inflammatory processes (SIRS) (Kruzel ML, Actor JK, Radak Z, et al. (2009), Innate Imun., Sept. 1 (Epub ahead of print). Lactoferrin is also involved in modulation in the adjuvant setting in the generation of antigen-specific T cells by stimulation of dendritic cells (Hwang SA, Actor JK (2009) Int Immunol 21, 185-175-1197).

Interestingly, bovine lactoferrin is growth-stimulant for various bifidobacterium strains, the most abundant microorganism in the human gut (Rahman MM, Kim WS, Ito T, et al. (2009) Anaerobic 15, 133-137).

More and more work is being done on truncated lactoferrin molecules for the large 80kDa lactoferrin molecule to demonstrate and clinically exploit their antiviral, antifungal and antitumoral properties in lactoferrin-derived molecules (Lizzi AR, Carnicelli V, Clarkson MM et al. (2009) Mini Rev Med Chem, 9, 687-695).

All reviews on lactoferrin, which focus on bovine lactoferrin, now view lactoferrin as a multifunctional protein of particular importance. Its properties are emphasized in the immune defense (viruses, bacteria, fungi), its anti-inflammatory and anti-tumoral properties (Pierce A, Legrtand D, Mazurier J (2009) Med Sei (Paris) 25, 361-369; Rodrigues L, Teixeira J, Schmitt F et al. (2009) Crit Rev Fodd Sein Nutr. 49, 203-217) and bovine lactoferrin therefore play a special role in nutrition (prevention) (Lönnerdal B (2009) Curr Opin Clin Nutr Metab Care 12, 293-297).

The review article by Tomita M, Wakabayashi H, Shin K, Yamauchi K, Yaeshey T, Iwatsuki K (2009) summarizes the results of 25 years of research on Lac- Together, toferrin (Twenty-five years of research on bovine lactoferrin applications, Biochimie 91, 52-57) and Baker EN, Baker HM (2009), provides a structural framework for understanding the multifunctional nature of lactoferrin (A structural framework to understanding the multifunctional character of lactoferrin, Biochimie, 91, 3-10).

Starting from the above-described prior art, it is an object of the present invention to provide further uses of lactoferrin, as well as compositions containing same for such uses.

In this context it should be mentioned that in the context of the present invention a reference to lactoferrin always means a reference to recombinant human lactoferrin and / or to human lactoferrin and also includes equivalent derivatives thereof.

In the investigations carried out, it has now surprisingly been found that lactoferrin influences the level of adiponectin in the blood. The adiponectin level in the blood can be regulated, in particular increased, as a function of the dosage of the lactoferrin.

While the use of human and recombinant human lactoferrin to treat diabetes, typically diabetes type II, obesity, and to treat arteriosclerosis has already been discussed in the literature, it is now also possible by administering the adiponectin level by administration human or recombinant human lactoferrin to treat other diseases and processes.

In particular, these indications range from inflammatory processes, to supporting cardiac function and the protection of the heart muscle or heart in general, hypertension, ischemia, ischemic heart disease up to Cancer therapy. Lactoferrin finds use in both the direct and in the supporting therapy in combination with one or more further active ingredients. Adiponectin is an adipocytokine first described in 1995/1996 (other names are GBP-28, apMl, AdiopQ, Acrp30). It is expressed in fat cells. Adiponectin stimulates fatty acid oxidation by stimulating AMP kinase activity, reducing free fatty acids in the blood, and improving insulin sensitivity in liver and skeletal muscle fat cells. Furthermore, vasoprotective and anti-inflammatory effects of adiponectin were described.

Noteworthy for a hormone is the relatively high plasma level of 5 - 30 g / ml (0.01% of the total plasma protein, for comparison: FT3 to 4.2 ng / ml, testosterone to 1, 5 ng / ml). Adiponectin shows no significant circadian rhythm and can therefore be determined independently of the time of day. The plasma levels of women are higher than in men. Low levels are found in rare lipoatrophy and then reflect the absence of fat cells. Elevated plasma levels are found in chronic renal insufficiency, diabetes mellitus type 1 and anorexia nervosa. Adiponectin correlates inversely with the degree of obesity, especially visceral obesity, hyperinsulinemia, insulin resistance and CHD risk.

Adiponectin is also thought to be a "biomarker of insulin resistance." It has low levels in obese patients and type 2 diabetics or patients with metabolic syndrome. After weight loss or initiating therapy with glitazone ("insulin sensitiser"), plasma levels rise. A risk assessment for insulin resistance and arteriosclerosis is given: Adiponectin serum levels: <7 μg / \ high to very high risk, 7-10 μg / l normal risk and> 10 low risk. Since the adiponectin level in the blood can be influenced by the ingestion of lactoferrin, new approaches for the use of lactoferrin in the context of the present invention arise. The invention therefore particularly relates to the use of recombinant human lactoferrin or of human lactoferrin for the manufacture of a medicament or a dietary supplement for regulating adiponectin levels in the blood or in a supplemented balanced diet. An advantage of this use is that lactoferrin is a native human protein that is readily available and generally acceptable to the human organism. Lactoferrin is found in its highest concentration in milk and colostrum, while recombinant human lactoferrin is available via organisms such as Aspergillus niger or transgenic animals such as transgenic cows.

In a particular and preferred embodiment, the present invention relates to the use of recombinant human or human lactoferrin for the prevention or treatment of obesity, diabetes, for the treatment of metabolic syndrome, arteriosclerosis or for weight loss by influencing adiponectin levels in the blood. Furthermore, the present invention relates to the use of recombinant human or human lactoferrin for the treatment of inflammatory processes, for the promotion of cardiac function and the protection of heart muscle or heart in general, for the treatment of hypertension, ischemia, ischemic heart disease and in cancer therapy, both in the direct, as well as in the supporting therapy in combination with one or more further active ingredients.

In general, the daily dose of lactoferrin to be administered according to the invention is from 25 mg to 1000 mg, preferably from 100 mg to 800 mg, more preferably from 200 mg to 500 mg. The daily dose to be administered is basically in addition to the body weight of the person to be treated and the intended use. It is usually assumed that, when used as a dietary supplement, the lactoferrin is administered approximately in an amount per day which corresponds to up to three times the maximum daily intake of a baby during lactation, preferably in an amount which corresponds to the mean daily intake of lactoferrin in the first week of lactation or up to twice that amount.

In a further embodiment of the present invention, the daily dose is generally administered as a single dose divided into up to five single doses or, preferably, divided into two to three single doses. From the point of view of patient acceptance, a single dose is preferred, especially when the lactoferrin is administered in the form of tablets, capsules, dragees or the like. Depending on the age of the person taking the lactoferrin, it may also be preferable to divide the daily dose into two to three single doses. This usually ensures a sufficiently constant saturation.

Regardless of the number of individual doses to be administered, there is an equitable distribution of the daily dose to be ingested, especially when the lactoferrin is given to infants during lactation as an adjunct to breastmilk or as an adjunct to regular bottle feeding instead of breastfeeding , For this purpose, the lactoferrin is advantageously added in the appropriate amount of the bottle food or the milk powder. The amount thereof is e.g. 1 10 mg, 220 mg or 330 mg lactoferrin per 250 grams of milk powder or bottle concentrate, with about one liter of bottle food requiring about 180 g of concentrate or milk powder.

Preferably, the administration of lactoferrin over a continuous period of one to nine months, preferably 2 to 8 months, more preferential over a period of 3 to 6 months. This is especially true when administering the lactoferrin to infants, as these periods correspond approximately to the usual breastfeeding periods. However, in adults it is quite possible to extend the intake period and in particular to measure the administration of lactoferrin in both the dose and the duration of the treatment success, ie, for example, the achieved weight reduction or improvement of the overall condition of the patient or other relevant parameters known to those skilled in the art , The present invention further relates to a pharmaceutical composition or a dietary supplement containing recombinant human or human lactoferrin, wherein the pharmaceutical composition or dietary supplement contains 25 mg to 1000 mg of recombinant human lactoferrin or human lactoferrin in a single dose, preferably 50 mg to 400 mg, more preferably 100 mg to 250 mg.

The pharmaceutical composition or the dietary supplement according to the invention is preferably in the form of a tablet, a hard or soft capsule, a dragee of a powder or a solution for oral, parenteral or topical administration.

It is also preferred if the pharmaceutical composition contains, in addition to the recombinant human lactoferrin or human lactoferrin, one or more excipients or additives selected from the group consisting of fillers, binders, coating agents, lubricants and disintegrants. The concrete formulation is fundamentally not critical and the person skilled in the art will select the appropriate auxiliaries depending on the purpose from the generally customary auxiliaries. The above-mentioned findings will be explained in an exemplary manner with the following experiments. Human visceral preadipocytes and adipocytes (Lonza AG, Cologne) were used for this purpose, and the preadipocytes were supplied with preadocyte growth medium (PromoCell, Heidelberg) at 37 ° C. + 5% CO 2.

Real Time RT PCR:

The isolation of the RNA from adipocytes was carried out with the RNeasy Micro Kit (Qiagen, Hilden). For the reverse transcription the First Strand cDNA Synthesis Kit (Fermentas GmbH, St. Leon-Rot) was used.

The following primers (Invitrogen GmbH, Karlsruhe) were used for Real Time PCR:

EBPa: SEQ. ID.

number 1

Forward (5 '-3'): TGG ACA AGA ACA GCA ACG AG

Reverse (5'-3 '): TTG TCA CTG GTC AGC TCC AG SEQ. ID.

No. 2

In each case 300 ng were used.

PPARy 2: SEQ. ID.

No. 3

Forward (5 '-3'): GAT ACA CTG TCT GCA AAC ATA TCA CAA

Reverse (5'-3 '): CCA CGG AGC TGA TCC CAA SEQ. ID.

No. 4

In each case 300 ng were used.

Ubiquitin: SEQ. ID. No. 5 Forward (5'-3 '): ATT TGG GTC GCG GTT CTT G

Reverse (5'-3 '): TGC CTT GAC ATT CTC GAT GGT SEQ. ID.

No. 6

Were used in each case 100 ng. β-glucuronidase: SEQ. ID.

No. 7

Forward (5 '-3'): CTC ATT TGG AAT TTT GCC GAT T

Reverse (5'-3 '): CCG AGT GAA GAT CCC CTT TTT A SEQ. ID. No. 8

In each case 300 ng were used.

The following TaqMANTM probes (Eurogentec GmbH, Cologne) were used for Real Time PCR:

ΕΒΡα (5'-3 '): CAC CTT CTG CTG CGT CTC CAC CTT SEQ. ID.

No. 9

PPARy 2 (5'-3 '): AGA GAT GCC ATT CTG GCC CAC CAA CTT SEQ. ID. No. 10

β-glucuronidase (5'-3 '): TGA ACA GTC ACC GAC GAG AGT GCT GG

SEQ. ID. No. 11

Modification: 3 'TAMRA, 5' FAM

Ubiquitin (5'-3 '): TCA AGT GAC GAT CAC AGC GAT CCA CAA

SEQ. ID. No. 12

Modification: 3 'TAMRA, 5' JOE

In each case 100 nM were used.

The quantitative PCR was started with the qPCR Core Kit (Eurogentec GmbH, Cologne). Supernatant measurement of adiponectin:

The FlowCytomixTM technology (Bender Medsystems GmbH, Vienna) was used to determine the adiponectin concentration in the adipocyte supernatant. The concentration of adiponectin was determined using a flow cytometer.

Previously, 10,000 pre-adipocytes per well (96well) were used and supplied with 200 μΐ differentiation medium (Lonza AG, Cologne) and the respective concentrations of lactoferrin (human recombinant or bovine).

Every 3 days, the medium and thus the lactoferrin were changed. After 10 days, the differentiation medium was replaced by DMEM without FCS (PAN Biotech GmbH, Aidenbach). After 48 h of incubation 25 μΐ of the supernatant were removed for determining the concentration of adiponectin.

Adiponectin curve: adiponectin was measured in the culture supernatant after incubation with rh-LF and bovine LF.

Adipocytes in DMEM release a certain amount of adiponectin without a lactoferrin (control group).

Incubated adipocytes incubated with 10 and 100 ug recombinant human lactoferrin, with graphical representation of the results, in a bell curve concentration-dependent less and then in higher concentration more adiponectin.

By comparison, adipocytes incubated with 10 and 100 μg bovine lactoferrin significantly inhibit adiponectin release, demonstrating that bovine lactoferrin does not give the results of the invention. Further confirmation of the above-mentioned facts is provided by Real Time PCR for assaying PPAR-γ and EBPa in adipocytes.

PPAR-γ and EBPa are the major transcription factors in lipid metabolism. Both are elevated in mature adipocytes after incubation with recombinant human lactoferrin. PPAR-γ at 10 μg lactoferrin by a factor of 4.7, at 100 μg by a factor of 14.7, demonstrating a corresponding dose-dependency. There is a 20-fold increase in EBPa, with no dose-dependency in this area.

Claims

claims

1. The use of recombinant human lactoferrin or of human lactoferrin for the manufacture of a medicament, a dietary supplement or in a supplemented balanced diet for the regulation of adonectin levels in the blood.

2. The use of recombinant human or human lactoferrin according to claim 1, wherein regulating the adiponectin level in the blood consists in increasing it.

3. Use of recombinant human or human lactoferrin according to claim 1 or 2, for the prevention or treatment of obesity, diabetes, for the treatment of metabolic syndrome, arteriosclerosis or for weight loss.

Use of recombinant human or human lactoferrin according to claim 1, for the treatment of inflammatory processes, for supporting cardiac function, for the protection of heart muscle or heart in general, for the treatment of hypertension, ischemia, ischemic heart diseases and in cancer therapy

5. Use according to one of claims 1 to 4, characterized in that the treatment is carried out in combination with one or more further active ingredient or active ingredients.

6. Use according to any one of claims 1 to 5, characterized in that the daily dose is from 25 mg to 1000 mg, preferably 100 mg to 800 mg, more preferably 200 mg to 500 mg.

7. Use according to one of the preceding claims, characterized in that the daily dose is administered as a single dose or distributed in up to five individual doses, or, preferably, distributed in two to three individual doses.

8. Use according to one of the preceding claims, characterized in that the administration takes place over a continuous period of one to nine months, preferably 2 to 8 months, more preferably over a period of 3 to 6 months.

Pharmaceutical composition or dietary supplement containing recombinant human or human lactoferrin for use according to one of claims 1 to 5, characterized in that the pharmaceutical composition or dietary supplement contains 25 mg to 1000 mg of recombinant human lactoferrin or human lactoferrin in a single dose 50 mg to 400 mg, more preferably 100 mg to 250 mg.

10. A pharmaceutical composition or dietary supplement according to claim 9, characterized in that the pharmaceutical composition or dietary supplement is in the form of a tablet, a hard or soft capsule, a dragee, a powder or a solution for oral, parenteral or topical administration.

11. Pharmaceutical composition according to claim 9 or 10, characterized in that it contains, in addition to the recombinant human lactoferrin or human lactoferrin, one or more excipients or additives selected from the group consisting of fillers, binders, coating agents, lubricants and disintegrants.