WO2005007682A2

WO2005007682A2 - A peptide and a pharmaceutical composition, and their medical applications

Info

Publication number: WO2005007682A2
Application number: PCT/PL2004/000057
Authority: WO
Inventors: Andrzej W. Lipkowski; Daniel Carr
Original assignee: Lipkowski Andrzej W; Daniel Carr
Priority date: 2003-07-20
Filing date: 2004-07-20
Publication date: 2005-01-27
Also published as: PL361364A1; WO2005007682A3; PL202927B1

Abstract

New peptides and pharmaceutical compositions are presented as are their medical application, in particular in the prevention and treatment of diseases invoked by lentiviral infections.

Description

A peptide and a pharmaceutical composition, and their medical applications

The invention involves new peptides and their medical application. The described peptides are similar in structure to those in the proteins constituting the capsid of lentiviruses, in particular of the human immune deficiency virus (HIV), and may find application in the production of new pharmaceuticals.

The goal of the invention is to provide new compounds which show would affinity to opioid receptors. Such compounds may be used to obtain new drugs, in particular analgesic medications. Particularly advantageous would be to obtain substances which would also present other therapeutic properties, for example in the prevention and therapy of lentiviral infection, in particular HIV infection. It is also advisable to become better acquainted with the course of infection by viruses of this group, which would suggest new therapeutic methods and goals.

The goal thus defined was unexpectedly realized in this invention.

The subject of the invention are new peptides containing the following sequence of amino acids, from the N- to the C-terminal: Al A2A3A4A5A6A7A8A9, where:

Al is eliminated or is an amino acid selected from among Glu, Gin, Asp, and Asn,

A2 is eliminated or is an amino acid selected from among He, Leu, Thr, and Val,

A3 is an amino acid selected from among Tyr and nitrophenylalanine,

A4 and A5 are the same or different amino acids selected from among Lys, ornithine, and Arg,

A6 is an amino acid selected from among Tip, Phe, 1 -naphtylalanine, and 2-naphtylalanine.

A7, A8, and A9 are the same or different amino acids selected from among He, Leu, Thr, and

Val.

The term "is eliminated" means that the given amino acid may be omitted in some of the peptides in accordance with the invention.

Advantageous is that the N-terminal amino group Al be blocked, in particular by substitution with an acetyl (Ac) or dansyl (DNS) group. Also advantageous is that the C-terminal carboxyl group be blocked, in particular by substitution with an amino group.

The amino acids which constitute the peptide according to the invention may appear in different chiral variants, in particular the use of isomers of R- or S-amino acids on α-carbon is considered advantageous.

Sample structures of the peptides according to the invention are presented in Fig. 1.

Advantageous is that the peptide according to the invention is a peptide selected from among: Ac-Glu-Ile-Tyr-Lys-Arg-Trp-Ile-Ile-Leu-NH2 Ac-Gln-Ile-Tyr-Arg-Arg-T -Ile-Ile-Gln-NH2 Ac-Asρ-Ile-Tyr-Lys-Arg-Trp-Ile-Ile-Leu-NH2 DNS-Asn-Ile-Tyr-Arg-Arg-Phe-Ile-Ile-Leu-NH2 Ac-Thr-Ile-Tyr-Lys-Lys-Phe-Ile-Ile-Leu-NH2 Ac-Gln-Ile-Tyr-Orn-Orn-Npa-Leu-NH2 DNS-Leu-Tyr-Arg-Arg-Phe-Ile-NH2 Ac-Val-Tyr-Lys-Arg-Phe-Ile-Leu-NH2 DNS-Tyr-Lys-Lys-Phe-Leu-NH2

The synthesis of the peptides according to the invention should not present any problems for the specialist with knowledge of the available technical methods of the synthesis of peptides. The subject of the invention is also the application of the peptides according to the invention, as defined above, to develop an analgesic medication. It was unexpectedly discovered that the peptides according to the invention show high affinity to opioid receptors on the surface of nerve cells.

The subject of the invention is also the application of a compound showing affinity to opioid receptors, of advantage the μ-receptor, for the treatment or prevention of lentiviral infection, in particular HIV.

It was unexpectedly discovered that the peptides according to the invention show structural similarity to fragments of the proteins which constitute the protein capsid of lentiviruses, in particular to the p24 protein isolated from sera of HIV-infected persons. p24 protein is an element of the capsid surrounding the RNA molecule of HIV. Therefore, it is advantageous to use a peptide according to the invention, defined above, as a compound showing affinity to the opioid receptor.

The subject of the invention is also a means of treatment or prevention of lentiviral infection characterized in that patients requiring such treatment shall be administered a drug containing a compound showing affinity to an opioid receptor, advantageously a μ-receptor. Of advantage is that the lentivirus be HIV. In the particular realization of this aspect of the invention, a peptide according to the invention, as defined above, is used as the compound showing affinity to an opioid receptor. The subject of the invention is also a pharmaceutical composition containing an active ingredient and a possible pharmaceutically acceptable carrier characterized in that the active ingredient contains a peptide according to the invention, defined above.

"Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or inummoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; saltforming counterions such as sodium; and/or nonionic surfactants such as TWEEN, polyethylene glycol (PEG), and PLURONICS. To better illustrate the essence of the invention and the possibility of its realization, this description is supplemented by figures. Figure 1 presents example peptides according to the invention.

Figure 2 presents the life cycle of HIV- 1: A: The generally known infection route of the mature virus, including its replication in the infected cell, B: the alternative development of infection according to the invention, in which the immature virus, having the form of a mature capsid, is recognized by the surface receptor of the infected cell, allowing its intemalization and replication.

Figure 3 presents the results of experiments aimed at investigating the activity of peptides derived from p24 protein towards the opioid receptors on the nerve cell membranes of rats. Details are provided in Example 2. Example 1 : investigation of the affinity to opioid receptors

Investigation of the affinity to opioid receptors was conducted by the method of displacing selected marked ligands, as described by, for example, Aleksandra Misicka, Andrzej W. Lipkowski, Lei Fang, Richard J. Knapp, Peg Davis, Thomas Kramer, Thomas F. Burks, Henry I. Yamamura, Daniel Carr and Victor J. Hruby in the article entitled "Topographical requirements for delta opioid ligands: Presence of a carboxyl group in position 4 is not critical for deltorphin high delta receptor affinity and analgesic activity" published in the journal Biochemical and Biophysical Research Communication, vol. 180, pp. 1290-1297, in 1991:

Mature Wistar rats weighing 250 - 350 g were sacrificed and their brains immediately removed and chilled on ice. All the brains were homogenized. The hemogenates were kept for 30 min at 25°C, then centrifuged twice for 15 min at 48,000 x g and fixed before examination. The basic investigation was conducted using a fast filtration method and [3H]deltorphine radioligands in the δ-receptor affinity test, and [3H]naltrexon[*naloxone?] in the μ-receptor affinity test. For the study, 10 μl of homogenate was taken and incubated with 0.75 nmol of the respective radioligand and 1 ml TRIS buffer, pH7.4, containing a 5 mmol MgCL2 solution, 1 mg/ml bovine serum albumin, and 100 μl phenylmethanesulfonyl fluoride. The appropriate quantity of the compound under investigation was added to the sample and incubated for three hours at 25°C. Then the cell membranes were filtered and washed in saline solution. The filter papers were placed in a scintillation solution and their radioactivity measured. A specific bond was recognized as the change in total radioactivity of the cell membranes with only the radioligand and that which remained after applying 10 μmol of the hydrochloride of naltrexon as displacing compound. With the data on the radioactivities of the individual samples and their concentrations, the IC50 value was determined, which is the concentration of the compounds under investigation able to displace 50% of the radioligand from its binding site on the receptors. The results, comparable with the known values for opioid compounds, are presented in Table 1.

Npa: L-2-naphtylalanine group Example 2

It is generally recognized [1] that HIN penetrates human cells (e.g. T lymphocytes) through interaction with the CD4 receptor and an additional cell-surface co-receptor (Fig. 2 A). This mechanism, though, does not explain the infection of cells not possessing such receptors (e.g. neurons). It was determined unexpectedly that the peptides according to this invention appear on the capsids of many lentiviruses. Based on studies which compared known protein sequences, a structural motif of the general pattern -AA-Tyr-AA2-Arg/Lys-Trp/Phe- was identified which appears in the peptides according to the invention and also on the capsids of lentiviruses (Table 2).

Table 2. Opioid motifs identified in the p24 protein of the viruses HIV-1, HIV-2 and SIV

(located in the Gag protein, at approx. the 255 amino-acid position)

Peptide Viral strain structural motif

GEIYKRWIJLG GAG ΪN1A2; HV1BI; HV1B5; HV1BR; HV1C4; HV1H2; HV1J3; HV1JR; HV1LW; HV1Ν5; HV1ND; HV1OY; HV1PV; HV1RH; HV1W2; HV1Y2 GEIYKRWIIVG GAG_HV1EL GDIYKRWIILG GAGJrlNlMA GDIYRRWIILG GAG_HV1U4 GΝIYRRWIQIG GAG TV2BE; HV2CA; HV2D1; HV2G1; HV2KR; HV2SB; HV2RO; HV2ΝZ GNIYRRWIQLG GAG_HV2D2; SINM1 ; SIVMS; SINS4; SINSP GSIYRRWIQIG GAG_HV2ST; GAIYRRWIILG GAG_SIVA1 ; S1NAG; S1YAT GDVYRRWVILG GAG_SIVCZ GΝIYRRWIQLR GAG_SIVMK GTIYKSWIILG GAG_SINGB

For the experimental verification that the structural motifs appearing in the capsids of lentiviruses may also interact with an opioid receptor, the peptides presented in Table 2 were synthesized using standard techniques. Gly(l) and Gly(l 1) were replaced by an acetyl and amide group, respectively, to simulate the intra-protein locations of these peptides. The affinities of the synthetic peptides to opioid receptors in the rat brain homogenate were determined (see Example 1 for methods). It was ascertained that all the synthetic peptides showed high affinity to the μ - receptor (on a level of 0.5 - 1.5 μmol), while not showing high affinity to δ-receptors (over 50 μmol).

The functional effectiveness of the interaction of the peptides according to this invention with opioid receptors was also investigated by measuring the stimulation to [35S] binding. Experiments were carried out on two selected peptides: Ac-AIYRRWIILK-NH2 (I) and Ac- SYRRWIIL-NH2 (II). Both showed activity confirming their ability to appear as agonists (Figure 3). Both peptide I and peptide II were administered to rats intrathecally in quantities corresponding to 5 mg of protein and showed significant anti-nociceptive activity. The compounds were given in the form of complexes with cyclodextrane due to their low solubility in aqueous solutions. Surprisingly, their stimulatory activity was not blocked by administration of naloxone (Fig. 3B), which may suggest that another, non-opioid, receptor also interacts with the peptides.

To recapitulate, one can see that the peptides according to the invention may find application not only in analgesic medications, but also in drugs for the treatment of lentivirus infections, in particular HIV. Current technical data do not offer an explanation for the H1N infection observed in cells which have no CD4 and/or co-receptor (e.g. nerve cells). The data presented in this invention are the first to allow for the existence of an alternative infectious route of HIV. It was unexpectedly determined that some protein motifs appearing in p24 protein may effectively interact with opioid and other nerve cell surface receptors. p24 protein has been identified in the sera of HIV-infected patients. The level of this protein often correlates better with disease course [5] than does HIN particle level (evaluated from the protein level of the lipid envelope). Earlier studies [6] showed that p24 protein alone can form RΝA-containing capsids. The discovery of the interaction between p24 protein and an opioid receptor and a possible other, yet unidentified, surface receptor offers an alternative route of penetration of HIV RΝA into infected cells (Fig. 2B) and explains the above-described phenomena accompanying the development of infection. The presented results indicate that the p24 capsid, without a surrounding lipid-protein envelope, is also a virulent factor, especially in the body of an infected host (the lipid envelope of the mature virus increases its stability in the environment). Because p24 presents structural elements on its capsid surface different from those on the surface of a mature virus, one must assume that this allows it to interact with other surface receptors (e.g. an opioid receptor), thus representing a new route of infection. As the p24 capsid is much smaller than a mature virus, the simple mechanism of pinocytosis of the surface receptor and capsid complex may be used to penetrate the infected cell.

The proposed new mechanism of the development of lentiviral infection provides new possibilities for the development of treatments for infections by such viruses. This may be AIDS therapy, but also the treatment of other diseases accompanying lentiviral infections, such as cancer. All the elements participating in the proposed mechanism are potential therapeutic targets which may be exploited in designing new drugs.

First and foremost, though, the invention presented indicates new applications of opioid-like ligands in the prevention and treatment of diseases invoked by lentiviruses, such as AIDS. The publications below represent a supplement of this description.

1. Kilby J.M., Eron, J.J., N. Engl. J. Med. 348, 2228-2238 (2003).

2. Tang, J.L., Lipkowski, A.W., Specter, S., Int. J. Immunopharmacol. 20, 457-466 (1998).

3. Peterson, P.K., Gekker, G., Lokensgoral, J.R., Bidlack, J.M., Chang, A.C., Fang, X., Portoghese, P.S., Biochem Pharmacol. 61, 1145-1151 (2001).

4. Misicka, A., Lachwa, M., Carr, D.B., Hruby, V.J., Yoshikawa, M., Lipkowski, A.W., Proc. "Opioid Mimetic Analgesics 2002" (Y. Okada, L.H. Lazarus, eds.), Kobe 2002, pp. 53-54.

5. Ofori, H., Prokop, J., Jagodzinski, P.P., Biomed. Pharmacother. 57, 15-19 (2003).

6. Gross, I., Hohenberger, H., Wilk, T., Wiegers, K., Grattinger, M., Muller, B., Fuller, S., Krausslich, H.G., EMBO J. 19, 103-1 13 (2000).

Claims

1. Peptides containing the sequence: A1A2A3A4A5A6A7A8A9, where:

A2 is eliminated or is an amino acid selected from among He, Leu, Thr, and Val,

A3 is an amino acid selected from among Tyr and nitrophenylalanine,

A6 is an amino acid selected from among Trp, Phe, 1-naphtylalanine, and 2-naphtylalanine.

Val.

2. A peptide according to claim 1, whereby the N-terminal amino group Al is blocked, in particular by substitution with an acetyl (Ac) or dansyl (DNS) group.

3. A peptide according to claim 1, whereby the C-terminal carboxyl group is blocked, in particular by substitution with an amide group.

4. A peptide according to claim 1, whereby it possesses the structure presented in Figure 1.

5. A peptide according to claim 1, whereby it is a peptide selected from among: Ac-Glu-Ile-Tyr-Lys-Arg-Trp-Ile-Ile-Leu-NH2 Ac-Gln-Ile-Tyr-Arg-Arg-Trp-Ile-Ile-Gln-NH2 Ac-Asp-Ile-Tyr-Lys-Arg-Trp-Ile-Ile-Leu-NH2 DNS-Asn-Ile-Tyr-Arg-Arg-Phe-Ile-Ile-Leu-NH2 Ac-Thr-Ile-Tyr-Lys-Lys-Phe-Ile-Ile-Leu-NH2 Ac-Gln-Ile-Tyr-Orn-Orn-Npa-Leu-NH2 DNS-Leu-Tyr-Arg-Arg-Phe-Ile-NH2 Ac-Val-Tyr-Lys-Arg-Phe-Ile-Leu-NH2 DNS-Tyr-Lys-Lys-Phe-Leu-NH2

6. Use of a peptide according to one of the claims 1 to 5 to produce an analgesic medication.

7. Use of a compound showing affinity to an opioid receptor to produce medications for the treatment or prevention of lentiviral infection.

8. The use according to claim 7, whereby the opioid receptor is a μ-receptor.

9. The use according to claim 7, whereby the lentivirus is HIV.

10. The use according to claim 7, whereby a peptide according to one of the claims 1 to 5 is used as the compound showing affinity to an opioid receptor.

11. A method of treatment or prevention of lentiviral infection, whereby the patient requiring such therapy is administered a medication containing a compound showing affinity to an opioid receptor.

12. The method according to claim 11, whereby the lentivirus is HIV.

13. The method according to claim 11, whereby a peptide according to one of the claims 1 to 5 is used as the compound showing affinity to an opioid receptor.

14. The method according to claim 11, whereby the opioid receptor is a μ-receptor.

15. A pharmaceutical composition containing an active agent and a possibly pharmaceutically acceptable carrier, whereby the active agent contains a peptide according to one of the claims 1 to 5.

16. A pharmaceutical composition for the treatment or prevention of lentiviral infection, or of diseases connected with it, containing an active agent and possibly pharmaceutically approved vehicle, whereby the active agent contains a compound showing affinity to an opioid receptor, advantageously a peptide according to one of the claims 1 to 5.