WO1996018652A1

WO1996018652A1 - Hexapeptides possessing antitumor activity

Info

Publication number: WO1996018652A1
Application number: PCT/IB1994/000430
Authority: WO
Inventors: Augusta A. Mikhailova; Rem V. Petrov; Larissa A. Fonina; Leonid A. Strelkov; Sergei A. Guriyanov; Galina K. Gerassimova; Elena M. Treshchalina
Original assignee: Individual
Current assignee: Individual
Priority date: 1994-12-16
Filing date: 1994-12-16
Publication date: 1996-06-20
Anticipated expiration: 1997-06-16
Also published as: AU1117595A

Abstract

The present invention provides hexapeptides of the formula (1): X?1-Y1-Y2-Y3¿-Tyr-Pro-Trp-X2 in which X1 is H, Form, Ac or any amino acid, or an acid addition salt or complex thereof; X2 is H, any salt, amide, substituted amide or any amino acid; Y1 is Leu, Ile, Val, Nle, Nva, Ala, Gly; Y2 is Leu, Ile, Val, Nva, Ala; and Y3 is Leu, Ile, Val, Nva, Ala. The hexapeptides possess antitumor activity.

Description

Hexapeptides possessing anti umor activity.

Background of the Invention

Tumor cells of various origin produce substances inhibiting or disrupting the normal functions of the immune system (Miescer et al.. Functional properties of tumor-infiltrating and blood lymphocytes in patients with solid tumors, J. Im¬ munol. 136, 1899-1907 (1986)). In particular the dysfunc¬ tion of T-lymphocytes characteristic of acute myeloid leuke¬ mia is connected with the suppressive effect of leukemic cell products (Chiao et al.. Suppression of lymphocyte acti¬ vation and functions by a leukemia cell-derived inhibitor, Proc. Natl. Acad. Sci. USA 83, 3432-3436 (1986)). Administration of cytokines such as IL-2, CSF(s), IFNs, re¬ sults in many cases in antitumor effects. Cytokines are polypeptides with a molecular weight of 15 - 70 kDa. Admi¬ nistration of such comparatively large polypeptide substan¬ ces at effective doses to the patients induces as a rule harmful side effects. It would be more preferable to use clinically low molecular weight peptide compounds, particu¬ larly ones of endogenic origin (Fridman & Michon, Pathophy- siology of cytokines, Leukemia Res. 14, 675-677 (1990)).

Furthermore, the antitumor action of most of the cytokines is based on an enhancement of their reduced activity in the body of the tumor carrier. However, positive effects of cytokines are short-termed and require supportive injections. It is therefore necessary to administer these substances many times and at rather high doses.

Bone marrow cells produce a group of bioregulatory molecules - myelopeptides (MPs) . MPs display various biological acti¬ vities: immunoregulatory, cell differentiating, neurotropic activity etc. (Petrov et al., Myelopeptides-bone marrow me¬ diators with immunostimulating and endorphin-like activity, Scand. J. Immunol. 24, 237-243 (1986)). A mixture of un¬ identified MPs isolated from the supernatant of porcine bone marrow cell culture has been used in Russia in both human and veterinary applications for a variety of effects. In particular, this mixture inhibited the suppressive effect of leukemic cells HL-60 on T-lymphocyte functional activity (Strelkov & Mikhailova, Myelopeptides abolish T-lymphocyte suppression induced by human leukemia cells, Immunologia 6 (in Russian), 32-35 (1990)).

The hexapeptide Leu-Val-Val-Tyr-Pro-Trp together with the hexapeptide Phe-Leu-Gly-Phe-Pro-Thr were isolated from the supernatant of porcine bone marrow cell culture as substance modulating pain sensitivity. It induced hypoalgesia in high pain sensitivity threshold and hyperalgesia in low pain sen¬ sitivity threshold (Fonina et al.. Structures of two myelo¬ peptides affecting pain sensitivity, Doklady Academy Nauk 319 (in Russian), 755-757 (1991)). No further biological activities of these peptides are known.

It is the object of the present invention to provide sub¬ stances which possess antitumor activity but exhibit no or only low side-effects.

Summary of the Invention

The present invention provides new hexapeptides possessing antitumor activity as well as the use of known hexapeptides for the preparation of pharmaceutical compositions having antitumor activity.

The present invention is directed to a hexapeptide of the formula (1) X¹-Y¹-Y²-Y³-Tyr-Pro-Trp-X² in which

X¹ is H, Form, Ac or any amino acid, or an acid addition salt or complex thereof,

X² is H, any salt, amide, substituted amide or any amino acid,

Y¹ is Leu, lie, Val, Nle, Nva, Ala, Gly,

Y² is Leu, lie, Val, Nva, Ala,

Y³ is Leu, He, Val, Nva, Ala, except the compound Leu-Val-Val-Tyr-Pro-Trp.

Particularly preferred is the new compound OHC-Leu-Val-Val- Tyr-Pro-Trp which is named Bivalfor. In comparison with IL- 2 it has a similar effect (Chiao et al., supra). Bivalfor has a low molecular weight (927 Da) . It is able to stimu¬ late IL-2 production in T-lymphocytes. The presence of the N-terminal formyl group of the peptide molecule increases its stability to aminopeptidases and provides a prolongation of its action in the body. These properties allow the admi¬ nistration of this peptide at lower doses than the IL-2 doses and avoid toxic effects.

The present invention is further directed to pharmaceutical compositions comprising the hexapeptides and to a method of reducing tumor growth in a mammal and to inhibit the sup¬ pressive effect of tumor cells on T-lymphocyte functional activity.

In a preferred embodiment, the present invention is directed to the use of the hexapeptide Leu-Val-Val-Tyr-Pro-Trp for the preparation of a pharmaceutical composition for reducing tumor growth in a mammal and to inhibit the suppressive ef¬ fect of tumor cells on T-lymphocyte functional activity. Preferably, the hexapeptide comprises at least 10%, 25%, 50% or 90% of the pharmaceutical composition of the invention.

The peptides of the invention are synthesized according to usual methods, e.g. the solid phase method (see, e.g. "Aminosauren, Peptide, Proteine", Eds. H.D. Jakubke and H. Jeschkeit, Akade y Verlag, Berlin, 1982) .

Detailed description of the Invention.

Example 1. Synthesis of Leu-Val-Val-Tyr-Pro-Trp and Bivalfor

OHC-Leu-Val-Val-Tyr-Pro-Trp.

The peptide was synthesized by the solid phase method on Biosearch 9600 (USA) . The hexapeptide was prepared by car- bodiimide method using poly(4-hydroxymethyl) phenylacet- amidomethyl resin.

During the synthesis the N-terminal amino groups were blocked by Fmoc groups, the OH group of Tyr was protected as O-tert.-butyl ether. The Fmoc groups were removed by treat¬ ment with piperidine-toluene-dimethylformamid (30:35:35). The final peptide was separated from the resin by treatment with trifluoroacetic acid containing 2.5% ethane diol and 2.5% water (0 °C, 1.5 hours).

The free peptide was purified by HPLC using Diasorb-130 C-16 T column, 10 μ (250x26 mm) with an acetonitrile gradient (10 - 100%) in 0.05 M phosphate buffer, pH 3.0. Its homogeneity was checked by HPLC on an Ultrasphere ODS-3 column (500x4.6 mm) with an acetonitrile gradient (20 - 80%) in 0.05 M phos¬ phate buffer, pH 3.0. Amino acid analysis: Leu 0.95 (1), Val 1.87 (2), Tyr 1.03 (1), Pro 0.96 (l). 1 mg of this pep¬ tide was dissolved in 0.5 ml 98% HCOOH and treated with 0.25 ml (CH₃CO)2θ. After stirring for 30 in at 18°C the solvent was evaporated, Bivalfor was isolated by HPLC on Ultrasphere C-18 ODS (4.6x250 mm) with an acetonitrile gradient (5-60%) in 0.1% trifluoroacetic acid. The yield of Bivalfor was 47%.

Biological activity of the hexapeptides of the invention

Example 2. Ability of Bivalfor to restore T-lymphocyte acti¬ vity inhibited by tumor cells

The human leukemic HL-60 cell line originally derived from bone marrow cells of patients with acute myeloid leukemia produces protein(s) inhibiting T-lymphocyte functions. Such inhibition results in the decrease of T-lymphocyte prolife- rative response to mitogen- phytohe agglutinin (PHA) (Chiao et al., supra). Bivalfor is able to abolish this inhibitory effect of leukemic cells on T-lymphocyte mitogenic response.

T-lymphocytes from peripheral blood of healthy donors were cultured at lxlO⁶ cells per ml and stimulated with PHA (3 μg/ml) during 3 days. PHA-stimulated proliferation was de¬ termined by the ³H-thymidine incorporation procedure (Chiao et al., supra). HL-60 cells conditioned media (DM) with in¬ hibitory activity collected from cells during logarithmic growth phase (on the 3rd - 4th day of growth) and various concentrations of Bivalfor were added to the cultures at the start of cultivation. ^JH-thymιdιne pulsing was for 4 hours using 2 μCi/ml on the 3rd day of PHA stimulation. The cells were harvested and their radioactivity was measured in a li¬ quid scintillation counter. The typical results of one of four experiments are presented in Table 1. Table 1.

Ability of Bivalfor to restore PHA responses of T-lympho¬ cytes reduced by HL-60 CM

Substances Doses H-thymidine incorporation added to the culture cpm % of control

- - 400 3.7

PHA (control) 3 μg/ml 10600 100.0

HL-60 CM 10 %, 4300 40,6 (v/v)

Bivalfor 100 μg/ml 9900 93,5

50 μg/ml 8400 79,3

5 μg/ml 6100 57,5

0.1 μg/ml 4800 45,3

Each figure is an average value of three parallel tests (in the range of +/- 200 cpm) .

HL-60 CM decreased ³H-thymidine incorporation to 40.6% of the control value. The addition of Bivalfor to the T-lym¬ phocyte culture increased the proliferative response in a dose dependent manner. The best restoration of T-lymphocyte proliferative response (at least 80% of control) took place at a dose of Bivalfor of 100 μg/ml. It was shown that the inhibitory effect of HL-60 CM on T-lymphocyte proliferative response is accompanied by a decrease of IL-2 production in T-lymphocytes (Chiao et al., supra) . Therefore, the influ¬ ence of Bivalfor on IL-2 production was studied using the experimental model of Concanavalin (Con A) - activated mouse spleen cells. Example 3. Activity of Bivalfor to enhance IL-2 production in Con A activated mouse spleen cells.

Spleen cells were isolated from (CBAxC57BL)Fl mice and incu¬ bated in RPMI 1640 medium supplemented with 10% fetal calf serum in 5% C0 at 37°C. The cell concentration was 5xl0⁶ cells/ml. Con A (5 μg/ml) and Bivalfor (1 μg/ml) were added at the start of cell cultivation. IL-2 production in T-lym¬ phocytes was determined by the ability of Con A-activated spleen cell supernatant to support the proliferation of the IL-2 dependent cytotoxic T-cell line CTLL-2. CTLL-2 cells were incubated in 96-well plates (lxio⁴ cells/well) and the tested supernatant was added to each well at final dilutions of 1/2, 1/4, 1/8, 1/32, 1/64 and 1/128. Supernatant of Con A-activated mouse spleen cells cultivated without Bivalfor was added at the same dilutions to the control wells. The proliferation of CTLL-2 cells was measured by rapid quantity colorimetric method based on the reduction of tetrazolium salt MTT to a blue farmason product (Mosmann, Rapid colori¬ metric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65, 55-63 (1986)) . Briefly, 20 μl MTT at a concentration of 5 mg/ml were added to each well. After 4 hour incubation at 37 'C the plates were centrifuged and the sediments were dissolved in DMSO. The optical density of the solution in each well was measured by reader "Multiscan" using a wavelength of 540 nm. The value of the supernatant dilution providing 50% proliferative response of CTLL cells in experimental and control wells was determined by probit analysis. This value correlated with the quantity of IL-2 in the tested supernatants. Table 2. Bivalfor enhances IL-2 production of Con A-activa¬ ted mouse spleen cells.

Substances added IL-2 production * to the culture

Con A (control) 1

Con A + Bivalfor 1.43

* Relative units characterizing the increase of supernatant dilution providing 50% proliferative response of CTLL-2 cells in experimental samples compared with control.

The data above show that the Bivalfor ability to abolish the inhibitory effect of leukemic cells on T-cell proliferation may be attributed to the stimulation of IL-2 production in T-lymphocytes.

Example 4. Toxicity of Bivalfor.

Bivalfor was administered to mice in therapeutic doses (0.5 - 4 mg/kg) and in 30-fold increased doses as compared to therapeutic doses (120 mg/kg) .

The peptide was administered 1, 2 or 5 times with 0, 96 and 24 hour intervals, respectively. The animal survival was registered on the 10th day after the last Bivalfor injec¬ tion. The results presented in the Table 3 show the full absence of Bivalfor toxicity. Table 3. Survival of (CBAxC57BL)Fl mice after Bivalfor in¬ jections.

Dose of Bivalfor Number of Number of Number of

(mg/kg) injections animals surviving animals

2.0 1 5 5

4.0 1 5 5

0.5 2 8 8

4.0 2 8 8

0.5 5 7 7

2.0 5 7 7

120.0 1 5 4

Example 5. Ability of the hexapeptide Leu-Val-Val-Tyr-Pro- Trp to reduce the tumor size in mice injected with lympho- leukemia P-388 cells.

Leukemic P-388 cells were inoculated in hybrid mice BDF^ subcutaneously at lxlO⁶ cells per mouse. The tested peptide was injected intraperitoneally once, twice (with a 96 hour interval) or 5 times (with 24 hour intervals) . The start of the peptide treatment was 48 hours after leukemic cell in¬ oculation.

The antitumor effect of the peptides was examined by the in¬ hibition of tumor growth (ITG) which was calculated as the difference between the average volume of tumors in experi¬ mental and control groups, expressed in percent. The con¬ trol group without peptide treatment contained 29 mice. Each experimental group contained from 7 to 10 mice. The results of one of three such experiments are presented in Table 4. We can see the suppressive effect of hexapeptide on growth of tumor. This scheme of peptide treatment had no effect on the life span of the animals (data not shown) . Table 4. Effect of Leu-Val-Val-Tyr-Pro-Trp on the tumor growth of lympholeukemia P-388 cells in mice.

Doses of Quantity ITG peptide of peptide 1st day a:fter 7th day after

(mg/kg) injections peptide peptide treatment treatment

2.0 1 63 * 53

4.0 1 37 56

0.5 2 83 * 66 *

1.0 2 80 * 46

2.0 2 85 * 63 *

0.5 5 82 * 72 *

1.0 5 55 51 *

2.0 5 70 * 53 *

* differences of control are significant p < 0.05

Example 6. Ability of the hexapeptide Lβu-Val-Val-Tyr-Pro- Trp to decrease the tumor size in mice injected with mela¬ noma B-16 solid tumor.

Melanoma B-16 was inoculated in C57BL mice subcutaneously at 50 mg tumor tissue per mouse (cell suspension in Pasher's media) .

The peptide treatment was started at 72 hours after tumor inoculation. The interval between two peptide injections was 96 hours. The results of one of two experiments are presented in Table 5. 7-8 mice were used in each group. Table 5. Influence of Leu-Val-Val-Tyr-Pro-Trp on melanoma fi¬ le spreading.

Doses of Quantity ITG peptide of peptide 1st day after 7th day after

(mg/kg) injections peptide peptide treatment treatment

1.0 1 55 * 65 * 2.0 1 55 64 * 4.0 1 40 42 1.0 2 61 57 * 2.0 2 82 * 70 * 4.0 2 42 76 *

* differences of control are significant p < 0.05

We can see a significant inhibition of tumor growth at the 7th day after the peptide treatment. The peptide had no ef¬ fect on the life span of the animals (data are not shown) .

It should be noted that the injected peptide in the doses used had no toxic effect. There were special animal groups consisting of 8 to 10 healthy mice for each peptide dose. Not one mouse died after peptide treatment.

Claims

1. A hexapeptide of the formula (1)

X¹-Y¹-Y²-Y³-Tyr-Pro-Trp-X² (1)

in which

X² is H, any salt, amide, substituted amide or any amino acid,

Y¹ is Leu, He, Val, Nle, Nva, Ala, Gly,

Y² is Leu, He, Val, Nva, Ala,

Y³ is Leu, He, Val, Nva, Ala, except the compound Leu-Val-Val-Tyr-Pro-Trp.

2. Hexapeptide according to claim 1 which is Form-Leu-Val- Val-Tyr-Pro-Trp.

3. A pharmaceutical composition which comprises as an active ingredient a hexapeptide of the formula (1) as defined in claim 1 or 2 or analogs thereof and optionally a pharmaceutically acceptable carrier.

4. A pharmaceutical composition according to claim 3 having antitumor activity.

5. A method of reducing tumor growth in a mammal comprising administering to said mammal an effective amount of a hexapeptide according to claim 1 or 2, including the pep¬ tide Leu-Val-Val-Tyr-Pro-Trp, to slow up growth of tumors in mammals and to inhibit the suppressive effect of tumor cells on T-lymphocyte functional activity.

6. Method according to claim 5 wherein said hexapeptide is Leu-Val-Val-Tyr-Pro-Trp.

7. Use of a hexapeptide according to claim 1 or 2, including the peptide Leu-Val-Val-Tyr-Pro-Trp, for the preparation of a pharmaceutical composition for reducing growth of tumors in mammals and to inhibit the suppressive effect of tumor cells on T-lymphocyte functional activity.

8. Use according to claim 7, wherein the hexapeptide is Leu- Val-Val-Tyr-Pro-Trp.