PL202928B1 - New peptides and a pharmaceutical compound as well as their therapeutical applications - Google Patents

Description

Description of the invention

The invention relates to new peptides and their medical applications. The described peptides have an affinity for opioid and tachykinin receptors found in the cell membrane of nerve cells, which play an important role in regulating the level of pain signals reaching the brain. Such peptides have a strong analgesic effect by acting on opioid receptors and, at the same time, modulating the mechanisms of creating tolerance and dependence on opioid drugs by acting on tachykinin receptors. Compounds of this type have been described by Stacy E. Foran, Daniel B. Carr, Andrzej W. Lipkowski, Iwona Maszczyńska, James E. Marchand, Aleksandra Misicka, Martin Beinborn, Alan S. Kopin, Richard M. Kream in the article p.t. Substance P - opioid chimeric peptide as a novel non-tolerance forming analgesic published in Proccedings of the National Academy of Sciences USA, vol. 97, pp. 7621-7626, in 2000 and Stacy E. Foran, Daniel B. Carr, Andrzej W. Lipkowski, Iwona Maszczyńska, James E. Marchand, Aleksandra Misicka, Martin Beinborn, Alan S. Kopin, Richard M. Kream , in the article entitled Inhibition of morphine tolerance development by a substance P-opioid peptide chimera, published in the Journal of Pharmacology and Experimental Therapy vol. 295, pp. 1142-1148, in 2000. However, the proposed structures showed a low affinity for opioid receptors, and as a result cannot be components of effective pharmaceutical preparations.

The aim of the invention is to provide new compounds that would show high affinity for opioid receptors and complementary to tachykinin receptors. Such compounds could be used for the preparation of new drugs, especially analgesic preparations, especially for the treatment of chronic pain. It would be particularly advantageous to obtain substances that would also exhibit other therapeutic properties, e.g. could be used in the therapy and prevention of opioid dependence and / or tolerance.

Surprisingly, the objective thus defined has been realized in the present invention.

The invention relates to novel peptide amides containing the following amino acid sequence in the N- to C-terminus direction:

^A 1 ^A 2 ^A 3 ^A 4 ^A 5 ^A 6 ^A 7 ^A 8 ^A 9 ^A 10 ^A 11 ^A 12 ^A 13 ^-NH 2 where:

A1 is Tire

A2 is D-Ala, D-Leu-, D-Met, D-Arg, D-Lys, D-Ile, D-Val, D-Ser, D-Cys, D-Asn, or D-Gln,

A3 is Phe, Trp,

A4 is Gly, Lys, His, Phe, Pro, Ala, Cys, Thr, Ser, Trp, Met, Asp, Asn, Glu, Gln, Tyr

A5 is Gly, Lys, His, Phe, Pro, Leu, Ile, Ala, Cys, Thr, Ser, Trp, Met, Asp, Asn, Glu, Gln, Tyr or is eliminated,

A6 is Gly, Lys, His, Phe, Pro, Leu, Ile, Ala, Cys, Thr, Ser, Trp, Met, Asp, Asn, Glu, Gln, Tyr or is eliminated,

A7 is Gly, Lys, His, Phe, Pro, Ala, Cys, Thr, Ser, Trp, Met, Asp, Asn, Glu, Gln, Tyr or is eliminated,

A8 is Gly, Lys, His, Phe, Pro, Ala, Cys, Thr, Ser, Trp, Met, Asp, Asn, Glu, Gln, D-Tyr, D-Lys, D-His, D-Phe, D- Ala, D-Cys, D-Thr, D-Ser, D-Trp, D-Met, D-Asp, D-Asn, D-Glu, D-Gln, D-Tyr or is eliminated,

A9 and A10 is the same or different amino acid selected from Phe, D-Phe, Trp, D-Trp, Tyr, Ile, D-Ile, Leu, Val, Thr, Met,

A11, there is Gly, Pro, D-Phe, D-Trp, D-His, D-Tyr,

A12 is Leu, Phe, Ile, Val, D-Phe, D-Trp, or is eliminated,

A13 is Met, D-Met, Leu, D-Leu, Ile, D-Ile.

The term "is eliminated" means that a given amino acid may be omitted from some peptides of the invention.

Preferably the C-terminal carboxyl group is blocked, especially by amino substitution as shown in the general sequence.

As noted, the amino acids of the peptide according to the invention can exist in various forms of chiral varieties, with L chirality naturally occurring in natural protein amino acids or also with D-chiral optical isomers thereof.

Exemplary peptide structures according to the invention are shown in Table 1.

Preferably, the peptide of the invention is a peptide selected from:

PL 202 928 B1

T a b e l a 1

No The rest of the amino acid in the peptide sequence A1 A2 A3 A4 a ₅ A6 A7 As A9 A10 A11 A12 A13 1 Tyr D-Ala Phe Gly Tyr Pro Arg D-Ala Phe Phe Gly Leu Underworld NH2 2 Tyr D-Ala Phe Gly Tyr Pro Cheese D-Cys Phe Phe Gly Leu Underworld NH2 3 Tyr D-Ala Phe Gly Tyr Pro Cheese D-Ser Phe Phe Gly Leu Underworld NH2 4 Tyr D-Ala Phe Gly Tyr Pro Cheese D-Ala Phe Phe Gly Leu Underworld NH2 5 Tyr D-Arg Phe Lys - - Main Main Phe Phe Gly Leu Underworld NH2 6 Tyr D-Ala Phe Gly Tyr Pro Cheese - Phe Phe Gly Leu Underworld NH2 7 Tyr D-Arg Phe Lys - - - - Phe Phe Gly Leu Underworld NH2 8 Tyr D-Arg Phe Lys - - Main Main Phe Phe Gly Leu Underworld NH2 9 Tyr D-Arg Phe Lys - - - - D-Phe Phe D-Phe Leu Underworld NH2 10 Tyr D-Arg Phe Lys Cheese Cheese - - D-Phe Phe D-Phe - Underworld NH2 11 Tyr D-Ala Phe Lys Pro - Main Main D-Phe Phe D-Phe Leu Leu NH2 12 Tyr D-Ala Phe His Leu Leu Asp - D-Phe Phe D-Phe Leu Leu NH2 13 Tyr D-Met Phe Glu Leu Leu Main Main D-Phe Phe D-Phe Leu Leu NH2 14 Tyr D-Ala Trp His How much How much Glu D-Ser D-Trp Phe D-Trp D-Trp Leu NH2

The syntheses of the peptides of the invention can be carried out by the solution method or on a solid support, and should not present any problem to those skilled in the art of peptide synthesis available in the art.

The invention also relates to the use of the peptides of the invention as defined above for the preparation of an analgesic and / or medicaments for the treatment of mental and physical dependence on opioid compounds. It has surprisingly been found that the peptides according to the invention have a high affinity for opioid receptors found on the surface of nerve cells and show agonistic or antagonistic activity in tests determining the activity of substance P.

It has surprisingly been found that the peptides of the invention possess opioid and tachykinin pharmacophores in a structural relationship that allows the compounds to have independent agonistic interaction with opioid receptors and agonistic or antagonistic interaction with tachykinin receptors.

In a particular embodiment of this aspect of the invention, a peptide according to the invention as defined above is used as the compound showing affinity for the opioid receptor. The invention also relates to a pharmaceutical composition containing the active ingredient and, optionally, a pharmaceutically acceptable carrier, characterized in that the active ingredient is a peptide according to the invention as defined above. Both the selection of an appropriate carrier and the route of administration of the pharmaceutical composition of the invention are within the skill of the art. The particularly advantageous routes of administration should be mentioned both to the sites of expected action (subarachnoid, and directly to the postoperative wound or accident wound) as well as systemically and systemically (e.g. intravenously). When administered by inhalation (e.g. by inhalation), a suitable pharmaceutical composition may be in the form of an aerosol formed from a suitable droplet size of a solution containing the peptide compounds of the invention as well as a gas suspension containing dry (e.g. obtained by lyophilization) particles of appropriate size containing the peptide compounds according to the invention.

PL 202 928 B1

To better illustrate the essence of the invention and enable its implementation, the description has been supplemented with tables.

Table 1 shows exemplary peptides according to the invention.

P r z k ł a d 1.

The affinity study for opioid receptors was carried out by the method of displacing labeled selective ligands, which was described, inter alia, by 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 an 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 Biochemical and Biophysical Research Communication, vol. 180, pp. 1290-1297, in 1991, while the methodology of affinity testing for tachykinin receptors was described, inter alia, by Iwona Maszczyńska Bonney, Stacy E. Foran, James E. Marchand, Andrzej W. Lipkowski, Daniel B. Carr, in the article p.t. Spinal antinociceptive effects of AA501, a novel chimeric peptide with opioid receptor agonist and tachykinin receptor antagonist moieties, published in the European Journal of Pharmacollogy vol. 488, pp. 91-99, in 2004.

Adult Wistar rats weighing in the range 250-350 g were sacrificed and their brains were immediately removed and chilled on ice. Whole brains were homogenized. The homogenates were kept for 30 min at 25 ° C and then centrifuged twice for 15 min at 48,000 x g before the actual binding study. The main study was performed using the rapid filtering method and [3H] deltorphine radioligands in the delta affinity study, [3H] naltrexone in the mu receptor affinity study, and [ ¹²⁵ I] substance P in the tachykinin affinity study. For the study, 10 microliters of homogenate were collected, incubated with 0.75 nanomoles of the appropriate radioligand, and the sample volume was supplemented to 1 ml with TRIS buffer at pH 7.4 containing 5 mmolar solution of MgCl2, bovine serum albumin (1 mg / mL) and phenylmethanesulfonium fluoride (100 microL). An appropriate amount of the test compound was added to the sample and incubated for three hours at 25 ° C. The cell membranes were then filtered and washed with saline. The filters were transferred to a scintillation solution and the radioactivity was measured. The specific binding was the change in the total radioactivity of the cell membranes with only the radioligand and that which remained after the use of 10 micromoles of naltrexone hydrochloride as a displacement compound for mu and delta opioid receptors, and 10 micromoles of substance P acetate for tachykinin receptors. From the obtained radioactivity data of individual samples and the concentration of the tested samples, the IC50 values were determined, i.e. the concentrations of the tested compounds, which are capable of displacing 50% of the radioligand from the binding sites in the receptors. The affinity level for individual receptors is presented in Table 2.

T a b e l a 2.

Relationship number in table 1 Receptor affinity, +++ means an IC50 <30 nM; ++ means 30 nM <IC50 <300 nM; + is 300 nM < IC50 < 3000 nM; on. means 3000 nM < IC50 Mu receptors Delta receptors Tachykinin receptors 1 2 3 4 1 +++ + ++ 2 +++ + ++ 3 +++ + ++ 4 +++ + ++ 5 ++ ++ +++ 6 +++ + + 7 ++ ++ ++ 8 ++ ++ +++

PL 202 928 B1 cont. table 2

1 2 3 4 9 ++ + +++ 10 +++ + + 11 ++ + ++ 12 + +++ ++ 13 + +++ ++ 14 + +++ ++

P r z k ł a d 2

In order to experimentally verify the hypothesis that the compounds according to the invention may have analgesic effects, tests were carried out on the analgesic effect after subarachnoid administration to the experimental Wistar rats. For this purpose, a cannula was inserted into the rats' core under anesthesia. The methodology used is described in detail by Stacy E. Foran, Daniel B. Carr, Andrzej W. Lipkowski, Iwona Maszczyńska, James E. Marchand, Aleksandra Misicka, Martin Beinborn, Alan S. Kopin, Richard M. Kream in the article entitled Substance P - opioid chimeric peptide as a novel non-tolerance forming analgesic published in Proccedings of the National Academy of Sciiences USA, vol. 97, pp. 7621-7626, in 2000. The day after the operation, the tail-flick test was investigated and then the same effect after administration of 2 nanomoles of the test compounds. The mean tail draw time without compound administration was 2.7 seconds. All compounds in Table 1 at 15 minutes after dosing extended the tail draw time to the maximum test time, ie 7 seconds.

P r z k ł a d 3.

To demonstrate the possibility of using peptides as analgesics in long-term administration, a test was carried out in which 5 micromoles of a peptide with the sequence Tyr-D-Ala-Phe-Gly-TyrPro-Ser-D-Ser-Phe-Phe-Gly-Leu-Met-NH2 ( compound number 3 in table 1) was administered intravenously to Wistar rats. At 15 minutes after dosing, rats were completely unresponsive to the thermal stimulus on the tail as described in Example 2. Subsequently, 5 micromoles of peptide were intravenously administered to the rats for five consecutive days. After one day of interruption, 5 micromoles of peptide was re-administered and a pain test was performed. At 15 minutes after peptide administration, complete analgesia was obtained. Tests carried out according to the identical pattern of morphine led to the induction of tolerance manifested by a decrease in the degree of analgesia. After chronic administration of morphine, the reduction in tail withdrawal time after administration of 5 micromoles of morphine was achieved, to an average of 3.5 seconds.

P r z k ł a d 4

Wistar rats administered morphine as described in Example 3 developed tolerance to morphine with an average response of 3.5 seconds in the tail flick test. After three days without morphine, the response to this analgesic remained unchanged. In a parallel experiment, rats who developed tolerance to morphine were administered 5 micromoles of the peptide indicated by number 3 in Table 1 for two days. Then, for one day, the animals were not treated with any drug, and the next day with 5 micromoles of morphine. It was surprisingly found that the animals fully regained sensitivity to the administered morphine as evidenced by an extended tail flick time of up to 7 seconds over a period of 15 minutes after morphine administration.

In summary, it can be considered that the peptides of the invention should find application not only as analgesics, especially for the treatment of chronic pain, but also for the prevention and treatment of opioid tolerance and opioid dependence. Prior art data did not fully predict the properties of the synthesized peptides. Only the data presented in the present invention can fully indicate the applications of new peptides for the design and production of new drugs.

Claims (6)

1. Peptide containing the sequence: A1A2A3A4A5A6A7A8A9A10A11A12A13-NH2 where: A1 is Tire A2 is D-Ala, D-Leu-, D-Met-D-Arg, D-Lys, D-Ile, D-Val, D-Ser, D-Cys, D-Asn, D-Gln, A3 is Phe, Trp, A4 is Gly, Lys, His, Phe, Pro, Leu, Ile, Ala, Cys, Thr, Ser, Trp, Met, Asp, Asn, Glu, Gln, Tyr A5 is Gly, Lys, His, Phe, Pro, Leu, Ile, Ala, Cys, Thr, Ser, Trp, Met, Asp, Asn, Glu, Gln, Tyr or is eliminated, A6 is Gly, Lys, His, Phe, Pro, Leu, Ile, Ala, Cys, Thr, Ser, Trp, Met, Asp, Asn, Glu, Gln, Tyr or is eliminated, A7 is Gly, Lys, His, Phe, Pro, Ala, Cys, Thr, Ser, Trp, Met, Asp, Asn, Glu, Gln, Tyr or is eliminated, A8 is Gly, Lys, His, Phe, Pro, Ala, Cys, Thr, Ser, Trp, Met, Asp, Asn, Glu, Gln, D-Tyr, D-Lys, D-His, D-Phe, D- Ala, D-Cys, D-Thr, D-Ser, D-Trp, D-Met, D-Asp, DAsn, D-Glu, D-Gln, D-Tyr or is eliminated, A9 and A10 is the same or different amino acid selected from Phe, D-Phe, D-Trp, Trp, Tyr, Ile, D-Ile, Leu, Val, Thr, Met, A11 is Gly, Pro, D-Phe, D-Trp, D-His, D-Tyr, A12 is Leu, Phe, D-Phe, Trp, D-Trp, Ile, Val or is eliminated, A13 is Met, D-Met, Leu, D-Leu, Ile, D-Ile. 2. The peptide according to claim 1 The process of claim 1, wherein the C-terminal carboxyl group is blocked, especially by substitution with an amide group. 3. The peptide according to claim 1 The method of claim 1, which is a peptide selected from: Tyr-D-Ala-Phe-Gly-Tyr-Pro-Arg-D-Ala-Phe-Phe-Gly-Leu-Met-NH2 Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-D-Cys-Phe-Phe-Gly-Leu-Met-NH2 Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-D-Ser-Phe-Phe-Gly-Leu-Met-NH2 Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-D-Ala-Phe-Phe-Gly-Leu-Met-NH2 Tyr-D-Arg-Phe-Lys-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-Phe-Phe-Gly-Leu-Met-NH2 Tyr-D-Arg-Phe-Lys-Phe-Phe-Gly-Leu-Met-NH2 Tyr-D-Arg-Phe-Lys-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 Tyr-D-Arg-Phe-Lys-D-Phe-Phe-D-Phe-Leu-Met-NH2 Tyr-D-Arg-Phe-Lys-Ser-Ser-D-Phe-Phe-D-Phe-Met-NH2 Tyr-D-Ala-Phe-Lys-Pro-Gln-Gln-D-Phe-Phe-D-Phe-Leu-Leu-NH2 Tyr-D-Ala-Phe-His-Leu-Leu-Asp-D-Phe-Phe-D-Phe-Leu-Leu-NH2 Tyr-D-Met-Phe-Glu-Leu-Leu-Gln-Gln-D-Phe-Phe-D-Phe-Leu-Leu-NH2 Tyr-D-Ala-Trp-His-Ile-Ile-Glu-D-Ser-D-Trp-Phe-D-Trp-D-Trp-Leu-NH2. 4. Use of a peptide according to claim 1 1 - 3 for the manufacture of a pain reliever, particularly in the treatment of chronic pain. Use of a compound having affinity for the opioid and tachykinin receptor according to claim 1 1 - 3 for the manufacture of medicaments for the treatment of tolerance developed to drugs acting on opioid receptors. 6. A pharmaceutical composition containing an active ingredient and optionally a pharmaceutically acceptable carrier, characterized in that the active ingredient is a peptide according to claim 1 or 2. 1 - 3.