Peptide-immunoregulator (options), medicinal products, including peptide-immunoregulator

 

(57) Abstract:

The invention relates to peptides-immunoregulators and their therapeutic use. New peptides are a type of peptides, including 4-8 amino acids. The peptides of the present invention can be manufactured in the form of pharmaceutical compositions of any suitable type using carriers or diluents. Medicinal products containing new peptides, intended for the treatment or prophylaxis of viral or bacterial infection or disease in need of immunostimulation. New drug extends the Arsenal of tools for a similar purpose. 7 S. p. f-crystals.

The technical field to which the invention relates

The invention relates to mielopeptides and their therapeutic use.

Background of the invention

According to Mikhailova et al., Immun. Lett. 47:199-203 (1995), and in WO-A-9618652, the bone marrow cells of different animals and humans produce a group of bioregulatory peptides called mielopeptide (MPs). MPs have a broad spectrum of functional activities: immunoregulatory, differentiating and opiate-like. They call 2-3-fold stimulation of the production of antibodies is etoc bone marrow and peripheral blood obtained from healthy donors and donors, patients with leukemia. They induce terminal differentiation in leukemic cells IL-60 person and affect pain sensitivity.

Specifically, Mikhailova et al. reported two specific Hexapeptide, i.e., FLGFPT (Mr-1) and LVVYPW (Mr-2), with immunoregulatory properties. WO-A describes Hexapeptide with antitumor activity, of the formula Y1-Y2-Y3-Tyr-Pro-Trp. An example is the Mr-2.

Summary of invention

Using solid phase extraction and HPLC of the supernatant obtained from the culture of bone marrow cells pigs were allocated additional peptides. These new peptides are a type of peptides, including 4-10 amino acids, including the EMG-X and/or X-Pro, where X is a hydrophilic amino acid, and which, as a rule, otherwise include hydrophobic amino acids.

These new peptides have therapeutic applicability. For example, they can be used in cases when it is necessary to immunostimulation or antiviral activity. In particular, they can induce the production of interferon(s), to inhibit the replication of viruses, including HIV, confectii.

The description of the present invention

As stated above, from natural sources have obtained new peptides using traditional techniques. These or other peptides of the present invention can also be obtained using the methods of synthesis that are known to those of ordinary skill in this field, for example using the well known solid phase method.

Some preferred characteristics of these new peptides are defined in the claims. They may be Tetra-, Penta-, hexa-, hepta-, OCTA-, Nona - or Decapeptide. Specific examples are LVCYPQ (here Mr-3), FRPRIMTP (MP-4), VVYPD (Mr-5) and VDPP (Mr-6), and data for these peptides are shown below. As follows from the results, these peptides do not possess the same characteristics. However, it is also clear that it is possible to carry out appropriate tests to determine their greatest suitability for any specific application.

The peptides of the present invention can be manufactured in the form of pharmaceutical compositions of any suitable type using carriers or diluents, for example in the form of solutions or dispersions for injection with or without adjuvant. The amount of peptide can be the Qualified doctors are able to choose the appropriate quantity, for example, on the basis of the effective dose given in the following results.

Mr-3 stimulates the phagocytosis of macrophages

Phagocytosis opsonizing sheep erythrocytes (SRBC) murine peritoneal macrophages was measured in NBT-test (recovery narasinga of tetrazole superoxide anions released during the oxidative burst). Peritoneal cells obtained from mice (CBAxC57BI)F1, was placed in a well of 96-well flat-bottomed tablet (1106cells/well) in medium 199. After 2 h incubation at 37oIn an atmosphere of air with 5% CO2unattached cells were removed by intensive washing with a mild balanced Hanks solution (BSS). To each well was brought to 100 μl of NBT solution (1 mg/ml), 50 μl of 1% suspension opsonizing SRBC and MP-3, MP-1 or MP-2 at a concentration of from 10-6up to 10-18g/ml of the Control wells did not contain the MPs. After 1 h incubation at 37oWith cells were washed with BSS and fixed with 10% formalin solution. After 10 min the cells were washed with distilled water and dried. Insoluble blue formazan was solubilizers adding first 60 μl/well of 2M KOH, and then 70 μl/well of dimethyl sulfoxide (DMSO). The contents of these holes were then mixed to p who was cityval on the spectrophotometer ELISA Multiskan MCC/340. The level of phagocytosis in each well is treated with MPs, compared with the level of phagocytosis of the control wells (100%).

In contrast to the MP-1 and MP-2, MP-3 stimulates the phagocytosis of macrophages dose-dependent manner. Received dose curve is bimodal. Maximum stimulation of up to 250% at doses of 10-8- 10-7g/ml. There is another peak macrophage stimulation (at doses of 10-16- 10-17g/ml). This effect is less pronounced, but statistically significant (p <0,05).

Mr-3 improves the survival of mice infected with Salmonella typhimurium

Mr-3 was used for inoculation of mice (CBAxC57BL)F1 1/p in doses 0,510-4g/mouse and 110-6g/mouse. 24 hours later, these mice were infected with various doses of Salmonella typhirium 415 (102, 103, 104or 105bacterial cells/mouse). Mice of the control group was inoculable saline solution. Each group contained 10 mice. The life expectancy of each mouse was within 21 days.

A pronounced protective effect of MP-3 received both doses. When the level of the x Mr-3, 70 - 90%. When 50% mortality in the control (102bacterial cells/mouse) all mice treated with MP-3, remained alive. This gives grounds to believe that Mr-3 protects these animals from bacterial infection, due to their ability to stimulate macrophage phagocytosis.

MPs induces resistance to lethal bacterial infections

Acute bacterial infection was induced in laboratory mice derived from (Swahs)F1 using intraperitoneally injection 100 LD50Salmonella typhi, which actually contained 1000 microbial organisms. This infection was caused by rapidly progressive sepsis and 100% death of animals within 3 days with this control the infection.

Placenam control animals were injected with administered intraperitoneally or subcutaneously using 0.2 ml of 0.85% NaCl solution before infection. Then these animals were subjected to the same procedure infection with 100 LD50S. typhi. All control animals died within 3 days, because they were infected immediately without pre-treatment. In contrast, pre-treatment of mice intraperitoneally or subcutaneous injection MP-3, MP-4, MP-5 and MP-isimo.

MPs, using 1 to 10 ug doses in the mouse, defended 90 - 100% of the animals from subsequent control of infection with a lethal dose of Salmonella. Both ways injection, intraperitoneal and subcutaneous found full efficiency in the pre-treatment using MPs.

Mr-4 induces terminal differentiation of leukemic cells

Cell line HL-60 derived from bone marrow cells of patients with acute myeloid leukemia. These cells represent myelomonoblastic that intensively proliferate. They can differentiate on granuloma or monocitami way only in the presence of appropriate promoters.

Myeloid line HL-60 human maintained in standard medium: medium RPMI-1640 with the addition of 15% (vol./about.) inactivated by heating amniotic calf serum, 20 mm HEPES, 2 mm L-glutamine and 50 μg/ml gentamicin. The initial cell concentration was 2105cells/ml the cells were cultured at 37oC in air atmosphere with 5% CO2. Mr-4 was introduced into the culture at a concentration of from 110-21102g/ml After 3 days of cultivation, cells were washed and re-incubated in fresh culture medium. 3 days posnetki collected on the 6th day of cultivation and measured their radioactive DNA (3H) and protein (14C). In the three cultural samples of the culture were analyzed by the average number of counts per min (cpm).

It is known that the reduction of chromosomal DNA synthesis and increase in total protein synthesis without histone characteristic of the process of differentiation. By changing the ratio include3H/14Since you can see that the MP-4 induces the differentiation process of cells HL-60. Mr-4 acts on blastoidea cells HL-60 dose-dependent. The optimal dose is 0.1 to 5.0 μg/ml.

Morphological analysis of cells HL-60 treated with MP-4, confirms these results. Among blastoid cells found about 60% of the Mature forms (monocytes-macrophages). Differentiating action Mr-4 was compared with known differentiating factors - forborne myristicaceae and inducer of maturation (T-limfotsity differentiating factor). We can conclude that the MP-4 induces terminal differentiation in leukemic cells HL-60 monocitami way.

The increase of serum interferon in mice induced with MPs

Males weinbrenner white mice at the age of 1.5 - 2 months old, with body weight of 18-20 g, were injected with in the peritoneal cavity of a single dose of MP-3, MP-4, MP-5 is Evesham the same dose of the drug. After 4, 24, 48, 72 or 96 h after injection, scored 5 mice from each experimental group, their serum samples were pooled and stored frozen at -60oTo measure this combined serum activity of interferon. This last measurement was carried out to test serum antiviral activity in cell cultures in vitro, the virus-infected encephalomyocarditis (EMV).

In more detail, the cell culture fibroblastoid cell line L929 were grown in culture medium alpha-MEM with the addition of 10% FBS. Culture of 200,000 cells suspended in 0.1 ml of culture medium, were placed in wells of 96-well microplate, and incubated in an atmosphere with 5% CO2at 37oC. Cell culture L929 were infected with 100 TCIC50-dose adapted laboratory strain V. Cytopathologically violations of the cellular morphology was detected 24 h after infection.

Serial dilution factor of 2 in each serum sample was performed using culture medium alpha-MEM containing FBS. 0.1 ml of a particular dilution of serum sample was introduced into each microlance containing cell culture ensuring 50% inhibition of viral infection when monitoring violations in L929 cells, induced within 24 h of EMV. The opposite value of the highest dilution was taken as the titer of interferon in the serum, are presented in units/ml.

In the control groups of mice were used the most powerful of the known inducers of interferon. Namely, Newcastle disease virus (NDS), Radostin was used as positive control for comparison with the MPs on their increasing activity of interferon in the serum.

When a single injection of 0.1 µg Mr-4 in mouse serum was observed two phases apparent increase early (4 h) and late (48 h) of interferon, which reached levels of 40 and 80 units/ml of serum interferon.

Single injection of 0.01 ug MP-5 led to a very strong increase of late (48 h) of serum interferon. Titers of serum interferon reached values 320 units/ml, a level typical for Radostina, one of the strongest known inducers of interferon.

MP-3 and MP-6 weakly induced late interferon in mouse serum. Levels 20 and 40 units/ml was reached, respectively, at 48 h after a single injection of 1 - 10 µg MP-3 and 0.1 - 1 µg Mr-6.

Antiviral activity of MPs in vitro

Having SPO is terferon in the appropriate cell culture in vitro. If so, then you can follow the antiviral effect of these compounds on cell culture in vitro, which actively infected virus can replicate.

In vitro culture of murine L929 cells, dramatically infected with the virus encephalomyocarditis (EMV), used as a model for establishing active concentrations of MP-3, MP-4, MP-5 and MP-6, with regard to their ability to induce interferon synthesis in mammalian cells and, consequently, to block viral infection in these cells. Mouse fibroblastoid cell line L929 were maintained in vitro as described above. MP-3, MP-4, MP-5 and MP-6 was introduced in the triple culture using the following final concentrations: 500, 250, 125, 63, 31, 16, 8, 4, 2, 1, 0,5, 0/25 μg per 1 ml of culture. Radostin used as control inducer of interferon.

After 24 or 48 h after injection of the culture of L929 cells were infected with 100 TCID50EMV. Over the next 24 hours this viral infection was induced in cells L929 sharp violations. If these cultures pre-incubated in the presence of Mr-3, it did not protect the resulting cellular layers from wide violations due to subsequent viral infection using 100 TCID50E the impact of this virus on L929 cells. Likewise for the Mr-6 when added at a concentration of 4 μg/ml or more. Antiviral effect of MP-5 and MP-6 is comparable with the action of Radostina added at 0.5 μg/ml or more. Mr-4 also protects L929 cells from disturbances, induced by the virus after 48 h of incubation of these cells in the presence of 32 µg/ml (or higher concentrations) of this drug before they control the infection by the virus.

Induction of interferon in vitro human cells using MPs

Opportunities for MPs to induce interferon has been studied in cultures of cell lines L41 person and peripheral blood cells (PBC) donors. Cells L-41 were maintained in medium 199 supplemented with 10% FBS and antibiotics. 200,000 cells per 1 ml of culture medium were placed in wells of 24-hole plastic culture and tablets were grown at 37oC in an atmosphere with 5% CO2. After the formation of the cell monolayer in these cultures, preferably centered, introducible control and experimental interferon inducers. After 24 h after induction of interferon collected cultural supernatant and analyzed them on the content of interferon.

RVS healthy donors were incubated in the wells of 96-hole culturemore of the drug.

For measuring the activity of interferon in the collected supernatant preparing serial dilutions of the obtained supernatant and then brought them into fresh culture L-41, infected with 100 TCID50EMV. In these supernatant registered titer of interferon, as described above for the titration of interferon mouse serum in cultures of murine L929 cells.

1 µg/ml of MP-3 induced rapid formation of interferon in human cells, as in L41 and donor RVS. 0,01 µg/ml of MP-4 shows the induction of interferon synthesis, comparable to the effect of Mr-3 in cells L-41. MP-5 in small doses (0.001 microg/ml induces the formation of interferon in donor RVS, and in large doses (1 μg/ml) is active in cells L-41. MP-6 induces a relatively small number of interferon and L-41 and donor RVS.

In vitro inhibition of HIV through Mps

Antiviral activity of MP-3, MP-4, MP-5 and MP-6 was analyzed using in vitro cell culture MT4 lymphoblastoid T-cell line human, infected with HIV. As a source of infectious virus used laboratory virus strain HT 27 HIV. He has the ability to cause chronic HIV-infec infection and appropriate anti-HIV effect of MPs was evaluated in accordance with the following criteria:

(a) changes in the morphology of the normal MT4 cells induced by HIV (cytopathogenic effect);

(b) detection of HIV antigens in infected MT4 cells using fluorescent microscopy of these cells pre-labeled with fluorescent antibodies anti-HIV;

(c) identifying proteins in HIV HIV-infected in culture medium of MT4 cells using immunofluorescence assay.

The MT4 cells infected with HIV were incubated in culture medium RPMI-1640 with the addition of 10% FBS and 2 ml L-glutamine. The MT4 cells (0.5 million cells per 1 ml of culture medium) were placed in wells of 24-hole plastic culture tablets and kept for 7 days at 37oC in an atmosphere of from 4.5 CO2. Each of the analyzed MPs brought into the appropriate wells at a final concentration of 0,1, 0,5, 1, 5, 10, 50 and 100 μg per 1 ml of the culture to use. Azidothymidine (Sigma Chemical Co.) in the final concentration of 0.1 μg/ml was used as positive antiviral control in each experiment.

Testing standard cell viability was carried out after 24 h of cultivation with MP-3, MP-4, MP-5 and MP-6 that do not cause visible damage to the MT4 cells grown in vitro. With these ceterm the Oka concentrations of Mr-3 HIV replication was completely stopped in such cultures cytopathogenic effect or the formation of viral protein were not recorded, 50% suppression of HIV replication was observed in the presence of 10 μg/ml of MP-4. MP-5 and MP-6 had no effect on HIV replication in cell cultures in vitro. Azidothymidine (0.1 ág/ml) causing 100% inhibition of HIV replication in infected cells MT-4.

THE LIST OF SEQUENCES

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Primamedic Ltd.

(B) STREET: 66 Reagents Park Road

(C) CITY: London

(D) STATE: N/A

(E) COUNTRY: United Kingdom

(F) POSTAL CODE (ZIP): NW1 7SX

(ii) TITLE of INVENTION: MIELOPEPTIDE AND THEIR THERAPEUTIC USE

(iii) NUMBER of SEQUENCES: 6

(iv) the FORM of COMPUTER-based READING:

(A) DATA CARRIER TYPE: Soft disk

(B) COMPUTER: compatible with personal computers IBM

(C) OPERATING SYSTEM: PS-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(vi) the CURRENT DATA ON the APPLICATION:

(A) APPLICATION NUMBER: not yet known WO

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) structure of the CHAIN: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

< / BR>< amino acid

(C) structure of the CHAIN: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

< / BR>
(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) structure of the CHAIN: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

< / BR>
(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(C) structure of the CHAIN: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

< / BR>
(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) structure of the CHAIN: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

< / BR>
(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) structure of the CHAIN: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) a DESCRIPTION Polizovalsea as immunoregulator.

2. The peptide comprising LVCYPQ (SEQ ID NO:3), were isolated from bone marrow cells, which can be used as immunoregulator.

3. The peptide comprising FRPRIMTP (SEQ ID NO:4), which can be used as immunoregulator.

4. The peptide comprising FRPRIMTP (SEQ ID NO:4), were isolated from bone marrow cells, which can be used as immunoregulator.

5. The peptide comprising VDPP (SEQ ID NO:6), which can be used as immunoregulator.

6. The peptide comprising VDPP (SEQ ID NO:6), were isolated from bone marrow cells, which can be used as immunoregulator.

7. Drug, comprising the peptide according to any one of the preceding paragraphs, intended for the treatment or prophylaxis of viral or bacterial infection or disease in need of immunostimulation.

 

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