ITMI981189A1 - PEPTIDES WITH CHEMOTACTIC AND ANTI-HIV ACTIVITIES - Google Patents

Description

The present invention relates to peptides derived from the MIP-Ια chemokine sequence capable of interacting with chemokine receptors.

BACKGROUND OF

"Macrophage inflammatory protein-1 alpha" (MIP-Ια), was discovered as an inhibitor of hematopoietic stem cell growth (Graham, G.J., et al., Identification and characterization of an inhibitor of haemopoietic stem celi proliferation. Nature. 344 (6265 : 442-4, 1990) and later also revealed myeloprotective properties (Dunlop, D.J., et al., Demonstration of stem celi inhibition and myeloprotective effects of SCI / rhMIPl alpha in vivo. Blood. 79 (9): 2221-5, 1992).

MIP-Ια also possesses proinflammatory activity by acting as a chemoattractant for monocytes and neutrophil granulocytes (Cook, D.N., The role of MIP-Ια in inflammation and hematopoiesis. J. Leuckoc Biol.

59 (1): 61-66, 1996), its main function appears to be that of mediator of the inflammatory response during viral infections (Cook, D.N., et al., Requirement of MIP-Ια for an inflammatory response to virai infection . Science. 269 (5230): 1583-1585, 1995). Recently, this chemokine has also been proposed as an effective candidate for cancer gene therapy, having been shown to reduce tumorigenicity and induce immune protection in a mouse model (Nakashima, E., et al., A candidate for cancer gene therapy: MIP- Ια gene transfer to an adenocarcinoma celi line reduced tumorigenicity and induced protective immunity in immunocompetent mice. Pharm.Ses.13 (12): 1896-1901, 1996).

The family of MIPs (and mainly MlP-la, MIP-Ιβ and RANTES) is also able to inhibit HIV infection, in fact some chemokine receptors (mostly CCR5, CXCR4 and CCR3) are used by the virus as favoring coreceptors fusion and therefore infection (Cocchi, F., et al., Identification of RANTES, MIP-Ια, and MIP-Ιβ as the major HIV-suppressive factors produced by CD8 + T cells. Science 270 (5243): 1811- 5, 1995). MIP-Ια is the natural ligand of CCR1, CCR4 and CCR5 receptors, and is therefore particularly active on HIV strains with macrophage tropism that typically use CCR5 (Alkhatib, G., et al., CC CKR5: a RANTES, ΜΙΡ- Ια, MIP-Ιβ receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272 (5270): 1955-1958, 1996).

SUMMARY OF

It has now been found that peptides with sequence corresponding or homologous to the region comprising the second and third cysteine of the MIP-Ια sequence, are able to interact with the CCR receptors of chemokin.

The invention also relates to pharmaceutical compositions containing such polypeptides as well as the use of the peptides for the preparation of anti-retro-viral medicaments, in particular anti-HIV.

The invention, in a further aspect thereof, also relates to DNA or RNA sequences which code for the polypeptides of the invention, expression vectors which contain such DNA or RNA sequences, and cells transformed with such expression vectors.

DETAILED DESCRIPTION OF THE

The peptides of the invention comprise from 10 to 40 amino acids, preferably from 15 to 30 amino acids.

By homologous sequences it is meant amino acid sequences which have at least 60%, preferably at least 80% and even more preferably at least 90% of homology with the region comprising the second and third cysteine of MlP-1a.

Particularly preferred peptides of the invention have the following sequence:

where X1-X15 represent the amino acid residues corresponding to the positions of the MIP-Ια sequences or conservative substitutions of the same amino acids.

By conservative substitution, we mean the substitution of an acidic, neutral or basic amino acid with another amino acid belonging to the same category.

A particularly preferred peptide has the PTACCFSYTSRQIPQNFIADYFETSS sequence. Equally preferred are peptides which differ from the latter by substitution, deletion or addition mutations of 1-5 amino acids, preferably 1-3 amino acids.

The polypeptides of the invention can be prepared with conventional methods of peptide synthesis, both in the solid phase and in the homogeneous phase. Equally conventional are the stabilization methods with which the polypeptides of the invention can be made stable to metabolic degradation, for example by work of the protected.

Such methods may be based, for example, on the use of non-natural amino acids of the D series, retro-inverted bonds or functionalizations of the terminal NH2 or COOH bonds or of other functional bonds of the amino acids of the sequence.

The polypeptides of the invention can optionally be inserted within the sequence of known proteins, for example albumin, so as to provide chimeric proteins which can offer desired characteristics of transport, stability and pharmacokinetics.

The amino acid sequences can be altered by resorting to conservative amino acid substitutions, as described for example in H. Neurath and R.L. Hill "The proteins", Academic Press, N.Y. (1979).

Alternatively, the peptides of the invention can also be obtained with recombinant DNA methods. For this purpose, suitable DNA sequences, obtained synthetically or using POR with suitable primers on the ΜΙΡ-Ια gene sequences, are inserted into expression vectors for prokaryotic or eukaryotic cells, according to known methods.

The peptides of the invention have proved to be able to stimulate the chemotaxis of monocytes, to activate the chemokine receptors and to induce angiogenesis.

The same peptides have also been shown to inhibit HIV infection.

The peptides of the invention or their derivatives, or chimeric proteins containing them, can be used in the therapy or prophylaxis of AIDS.

For this purpose, they will be administered appropriately formulated in pharmaceutical compositions as described for example in "Remington's Pharmaceutical Sciences Handbook", Mack Publishing Company, New York, U.S.A.

The compositions according to the invention will contain an effective amount of the peptides (or their derivatives and chimeric proteins), for example from 0.1 to 100 mg and will preferably be administered parenterally, in particular by the intraperitoneal, subcutaneous or intramuscular route. The daily dosage will obviously depend on several factors, such as severity of the disease, weight, sex and age of the patient and will be chosen from time to time on the basis of the toxicological, pharmacokinetic and pharmacodynamic studies of each individual peptide or derivative. In principle, the daily dosage of peptide can in any case be between 10 and 1500 pmol / kg of body weight and the treatment can be continued even for long periods. Other routes of administration are also possible, for example topical, transdermal, intranasal or inhalation or oral, by resorting to the formulation of peptides in liposomes or other known techniques for the administration of peptides or proteins via the gastrointestinal tract, such as those described in W093 / 25583 .

The following Example illustrates the invention in greater detail.

EXAMPLE

The peptide derived from ΜΙΡ-Ια, defined pM (PTACCFSYTSRQIPQNFIADYFETSS) was synthesized while keeping the cysteine residues protected to avoid the random formation of intra or inter-catenary disulfide bridges, the purification took place by HPLC. The purity of the compound was verified by mass spectrometry. The lyophilized peptide was stored at -20 ° C; for the experiments, the peptide was dissolved in PBS 0.1% BSA (containing 10 mM of DTT, used as a reducing agent) at the final concentration of 250 μΜ and aliquoted in microvials in volumes of 10 μΐ. For infectivity tests, pM was dissolved in the same buffer, but devoid of DTT. These diluted stocks were stored at -80'C.

Total PBMCs were prepared from buffy coats by Ficoll gradient centrifugation. Monocytes are prepared using two successive centrifugations on Ficoll and Percoli (bringing the latter to a density of 1.062). Dendritic cells were obtained by differentiating primary monocytes by culture in RPMI 10% FCS in the presence of GM-CSF (50 ng / ml) and IL-4 (1000 U / ml) for one week. Total polymorphonuclear granulocytes (FMNs) were prepared by eliminating PBMCs by centrifugation on Ficoll followed by lysis of the erythrocytes contained in the pellet by means of an ammonium chloride solution.

For chemotaxis experiments, purified cells are immediately washed and resuspended in serum-free RPMI containing 0.1% BSA and used as recently published (Benelli, R., et al., Manocyte-derived dendritic cells and monocytes migrate to HIV-Tat RGD and basic peptides. AIDS. 12: 261-268, 1998). To test the ability to activate calcium channels we applied the method of (Grynkiewicz, G., et al., A new generation of Ca <++> indicators with greatly improved fluorescence properties. J. Bioi.Chem.

260: 3440-3450, 1985) by labeling cells with Fura2-AM. For the in vivo angiogenesis experiments we used the mouse model with matrigel sponges as previously described in (Albini, A., et al., Angiogenic potential in vivo by Kaposi sarcoma cell-free supernatants and, HIVl-tat product: inhibition of KS-like lesions by TIMP-2. AIDS. 8: 1237-1244, 1994). For HIV infection tests, we cultured PBMCs for five days in the presence of 0.5 pg / ml of PHA and 20 U / ml of IL-2; on the sixth day the cells were washed and resuspended in culture medium containing 5 or 1.25 µM of µM; after 30 minutes we added the virus: HIV-1 strain 2028 which uses the coreceptors CCR5, CCR3 and CXCR4; SF162 using only CCR5; 2005 using only CXCR4 or HIV-2 strain ROD / B using coreceptors CXCR4, CCR3, BOB, BONZO, CCR2b and V28. After three hours the cells were washed four times and resuspended in the presence of pM and IL-2. Infection tests were also performed on U87 CD4 + cells transfected with the individual CCRs to verify the specific inhibitory potential of the peptide on each chemokine receptor.

RESULTS

Description of the Figures:

Figure 1: chemotactic response of monocytes to pM, the migration index was obtained by direct densitometric reading of the chemotaxis filters; the values found are expressed on a relative scale.

Figure 2: induction of calcium fluxes in monocytes treated with pM or MIP-Ια; the dosages are reported in the abscissa, the entity of the calcium fluxes (nM) in the ordinate. The Ca <++> fluxes proved to be rapid and transient.

Figure 3: Studies of cross inhibition of calcium fluxes. The pM was always used at the 5 μΜ dose, preceded by increasing doses of chemokines (A = MIP1-α; B = MCP-1; C = MCP-3; D = RANTES). MCP-3 appears to be the most effective inhibitor of the monocyte response to pM.

Figure 4: the pM peptide is also active in the induction of calcium fluxes in neutrophil granulocytes; the use of the controls consisting of MCP-3 and Eotaxin excludes that it is an eosinophil response.

Chemotaxis: pM has been shown to induce chemotaxis of monocytes when used at a high concentration (1-5 μΜ). The response is dose-dependent (Figure 1).

Activation of calcium channels mediated by chemokine receptors: pM is able to induce a rapid and transient calcium flux in monocytes, using it at the same effective doses in chemotaxis tests (Figure 2). The cross-inhibition studies have shown that pM has a broad spectrum interaction capacity with chemokine receptors (Figure 3), in fact the pretreatment with MCP-1, MlP-la, MCP-3 or RANTES (covering the main CCR receptors -1, 2, 3, 4 and 5) is able to partially inhibit the calcium flux mediated by pM, MCP-3 exerts the most marked inhibitory activity. pM has also been shown to activate calcium fluxes in granulocytes (Figure 4).

HIV infectivity: PBMC supernatants, collected five days after infection, demonstrate the inhibitory activity exerted by pM; the data are reported in the following Table containing the percentage of inhibition compared to untreated controls:

Table

PM peptide

The data indicate that pM is an active inhibitor of numerous HIV strains using different coreceptors: CXCR4 or fusin (HIV-1, 2005); CCR5 (HIV-1, SF162), CCR3 (HIV-1, 2028) and other receptors of minor relevance in HIV infection: CCR-2b, BOB, BONZO and V28 (HIV-2, ROD / B).

Claims (10)

1. Peptides with sequence corresponding or homologous to the region comprising the second and third cysteine of the ΜΙΡ-Ια sequence, capable of interacting with the CCR receptors of chemokines. 2. Peptides according to claim 1 comprising from 10 to 40 amino acids. 3. Peptides according to claim 2 comprising from 15 to 30 amino acids. 4. Peptides according to claim 1 having the sequence: where X1-X15 represent the amino acid residues corresponding to the positions of the MIP-Ια sequences or conservative substitutions of the same amino acids. 5. Peptide according to claim 4 having the sequence 6. Pharmaceutical compositions containing as an active principle a peptide of claims 1-5, in admixture with an acceptable vehicle. 7. Use of the peptides of claims 1-5 for the preparation of a medicament with anti-HIV activity. A nucleotide sequence encoding a polypeptide of claims 1-5. 9. The expression vector comprising the sequence of claim 8. 10. A cell transformed with the vector of the claim