NEW OLIGOPEPTIDES WITH AFFINITY TO OPIOID RECEPTORS

Abstract: Disclosed are novel oligopeptides with opioid receptor affinity which are straight or cyclic pentapeptides with the primary sequence backbone Tyr-X-Phe-Leu-Z, where X and Z denote amino acids or amino acid derivatives and/or analogues and where X and Z can be covalently coupled to provide a heterocyclic structure wherein Z is chosen among Cys, Glu, Gln or amino derivatives of Glu and Gln; X will be chosen among the amino acids or amino acid analogues Ser, Gly, Pro, AMCA, D-Ala, D- or L-ornithine, D- or L-2,4-diaminobutyric acid, D- or L-Lys and D- or L-Cys. Also disclosed are pharmaceutical preparations comprising said oligopeptides, which are potentially useful as analgesics for alleviating pain, for the treatment of depressions and for increasing the comfort of individuals suffering from extraordinary stress or shock.

Description: New oligopeptides with affinity to opioid receptors Field of invention The invention is related to new oligopeptides with opioid receptor affinity, which can be straight or cyclic pentapeptides with the primary sequence backbone Tyr-X-Phe-Leu-Z, where X and Z denotes amino acids or amino acid derivatives and/or analogues and where X and Z can be covalently coupled to provide a heterocyclic structure wherein Z is chosen among Cys, Glu, Gin or derivatives of Glu and Gin; X will be chosen among the amino acids or amino acid analogues such as Ser, Pro, Gly, D-Ala, D- or L-2,4-diaminobutyric acid, AMCA, Cys or derivatives thereof. The invention is also directed to pharmaceutical preparations containing the new oligopeptides, which are potentially useful as analgesics for alleviating pain and for increasing the comfort for individuals suffering from extraordinary stress or shock as well as for reducing depressions and for the possible treatment of individuals addicted to opiates. Background of the invention The physicians treating growth hormone deficient (GHD) patients with human growth hormone have frequently reported that patients experience an increased quality of life in comparison with a control group not subjected to the treatment. Statistically significant differences were found between the groups regarding social isolation, physical mobility, sleep and emotional status (see Acta Paed Scand (Suppl), v356, p55-59, 1989, S Bjork et al. and Acta Paed Scand (Suppl), v356, p70-72, 1989, GA McCauley) In clinical treatment with human growth hormone some secondary effects on the central nervous system (CNS) have been observed. It has been suggested that opioid active peptide fragments released from growth hormone may reach the CNS if the human plasma contains proteolytic activity for releasing them. If such fragments can pass the blood-brain barrier, they may affect the CNS. These previous studies indicate that human growth hormone affect the CNS and suggests that enzymatically released fragments may interfere with opioid receptors. A number of studies have shown that enzymatically treated preparations of proteins can include peptides with opioid activity such as β-casomorphin, cytochrophins and hemomorphins. β-casomorphin originates from degraded beta-casein peptone and is previously disclosed in Physiol Chem, v360, p 1211- 16, 1979, N Brantl et al.; Pharmacol Sci, v4, pl93, 1979, V Brantl et al.; Eur J Pharmacol, vl06, p213-21 , 1984, V Brantl et al. and J Clin Endocrinol Metab, v68, p283-9, 1989, F Νyberg et al. It is also shown that enzymatically derived fragments of mitochondrial cytochrome b contain cytochrophins, another opioid peptide (see Eur J Pharmacol, vlll, p293, 1985, V Brantl et al). For a reference of hemomorphins see Eur J Pharmacol, 125, p 309-10, 1986, V Brantl et al. The opioid activity of these peptides were confirmed by testing their inhibition of the electrically induced contractions of the guinea-pig ileum myentric plexus longitudinal muscle preparation (GPI-assay) as well as by receptor assay. Certain cyclic oligopeptides with opioid receptor activity have previously been disclosed by J De Maio et al. in Proc. Νatl. Acad. Sci., Vol. 77(12), 1980, p 7162-6, in which article a number of cyclic enkephalin analogues are discussed and prepared. Cyclic oligopeptides with opioid receptor affinity are also disclosed in J Med Chem, Vol. 35, 1992, p 3956-3961, P Schiller et al. The U.S. patent 4254024 (J Stewart et al.) discloses a class of tetrapeptides demonstrated to have opiate activity with a guinea pig ileum strip test having the general formula H-Tyr-X-Y-Z. Another reference which cites opioid receptor binding straight tetrapeptides is the European patent application EP 350 221. In a previous study two different peptidases were investigated for their ability to release fragments of human growth hormone, which may interfere with opioid receptors. The enzymes were a commercially available trypsin and an endopeptidase partially purified from human plasma. The fragments were separated on reversed phase HPLC and subsequently analyzed by an opioid receptor assay performed with synaptic plasma membranes from rat brains (without cerebellum). The results indicated that receptor active fragments were released both by trypsin and the endopeptidase. However, in a guinea-pig ileum myenteric plexus/longitudinal muscle preparation (GPI) assay, these fragments were found to be less potent than beta-casomorphins (fragments of casein peptone). These findings were published at the IV Meeting of the European Νeuroendoαine Association (Santiago de Compostela 28-30 June 1989). Description of the invention When the sequences of the peptides obtained from tryptic digest of human growth hormone were examined, it was noticed that some subsequences in certain aspeds were related to those of beta-casomorphin. These new oligopeptides with a proposed opioid activity were terra- or pentapeptides with the sequences Tyr-Gly-Leu-Leu and Tyr-Ser-Phe-Leu-Gln. The oligopeptides were synthesized and somewhat modified in that Gin is substituted for Glu in the pentapeptide. The peptides were subsequently tested for their opioid receptor affinity in comparison with β-casomorphin (see Table 1 below). The tests showed surprisingly, that the new oligopeptide Tyr-Ser- Phe-Leu-Glu have an opioid receptor affinity of about the same magnitude as β-casomorphin. This result has consequently lead us to a wider scope of new oligopeptides which are straight or cyclic analogues of Tyr-Ser-Phe-Leu-Glu. These new peptides are surprisingly shown to have high affinity to opioid receptors when compared to structurally related tetrapeptides peptides disdosed by the above-mentioned U.S. patent 4254024 (see also Table 2 below). The present invention is direded to new oligopeptides with opioid receptor affinity which are derived from sequences naturally occurring in human growth hormone. The new oligopeptides are described by the general formula Tyr-X-Phe-Leu-Z, wherein X and Z denotes amino adds, derivatives or analogues thereof, wherein X and Z can be covalently linked to form the cydic compounds : Tyr - X - Phe I I Z - Leu If the oligopeptide is straight, X is Ser, Gly, Pro, AMCA (trans-(4- aminomethyl)-cydohexan carboxylic add) or D-Ala and Z is Glu or Gin or their amino derivatives. Some preferred straight oligopeptide according to the invention are Tyr- Ser-Phe-Leu-Glu, Tyr-Ser-Phe-Leu-Glu-NH 2 , Tyr-D-Ala-Phe-Leu-Glu, Tyr-D-Ser-Phe-Leu-Glu, Tyr-Pro-Phe-Leu-Glu, Tyr-Ser-Phe-Leu-Gln and Tyr- AMCA-Phe-Leu-Glu. The straight oligopeptides are readily prepared according to the examples below. The cydic oligopeptides of the present invention will be cyclizised either by a side chain of an amino add or by oxidation of two cystein groups. For obtaining analogues with cydic structures to Tyr-Ser-Phe-Leu-Glu amino add number two, serine, will be substituted by D- or L-2,4-diaminobutyric add (D- or L-Dab), D- or L-ornithine, D- or L-lysine or D- or L-Cysteine. Amino add number five can be substituted by glutamine, glutamic add or their amino derivatives or cysteine. In the general cydic formula Tyr -X -Phe the following I I Z - Leu amino adds will be used according to the present invention: X = D- or L-2,4-diaminobutyric add(D or L-Dab),D-Orn, D- or L-Lys or D- or L- Cys and Z = Glu, Gin or thdr amino derivatives or Cys with the proviso that if X = D- or L-Cys then Z = Cys. Preferred cyclic oligopeptides according to the present invention are: Tyr-cydo-(-Lys-Phe-Leu-Glu-) with a peptide bond between alpha carboxy Tyr-cydo(-D-Lys-Phe-Leu-Glu-), Tyr-cydo(-D-Orn-Phe-Leu-Glu-), Tyr-cydo-(D-Dab-Phe-Leu-Glu-), Tyr-cydo-(D-Dab-Phe-Leu-Gln-) and the cydic peptides S S S S Tyr --CCvyss -- PPhhee -- LLeeuu -- CCyyss aanndd TTvyrr -- DD--CCvyss -- PPhhee -- LLeeuu -- CCvys which are cydizised by oxidation of the two cysteine groups. The cyclic oligopeptides have heterocydic structure related to enkephalins and those cyclic peptides described by J De Maio et. al., in the above mentioned artide. In the tests as disdosed in Example 3 and Tables 1 and 2, below it is dearly demonstrated that the oligopeptides according the present invention have a high affinity to opioid receptors and espedally to the μ- and δ-receptors. It is also demonstrated, in Table 2, that the cydic oligopeptides are functional agonists and have a high biological activity as opioids when measured on stimulated guinea pig ileum strips in relation to normorphine. The straight peptides show, in comparison to the cydic peptides, a considerably lower biological activity which may give them properties to ad as potential partial opioid receptor agonists or under certain drcumstances as antagonists. It is notable that the biological activity of the straight peptides is considerably reduced even for those with high binding affinity profiles to the μ-receptor. The suggested partial agonists would inhibit the agonistic responses of normorphine and the said cyclic peptides in equivalence with fundional antagonists. The straight and cydic oligopeptides according to the present invention represent a new possibility for preparing drugs for alleviating pain as an analgesic, for increasing comfort for patients suffering from shock or stress or for treating depressions, for administration to individuals suffering from disorders in the levels of endogenous morphine and opiate analogues or potentially for the treatment individuals addided to opiates. The person skilled in the art will readily find additional pharmacological uses of seleded oligopeptides according to the present invention which are linked to their properties as agonists, partial agonists or antagonists to the opioid receptors. Several administration forms of the new oligopeptides are conceivable. Both oral, transdermal and/or parenteral administration forms with appropriate carriers and/or diluents and conventionally used stabilizers and enhancers can be prepared. The new oligopeptides will readily be formulated and modulated to suitable administration forms such as oral, nasal, parenteral, enteral or buccal preparations. Various modifications of the invention and equivalents, such as salts and derivatives of the inventive oligopeptides will be apparent for anyone skilled in the art and shall be regarded as covered by the appended daims. It is also to be understood that the present invention is not intended to be limited to the specific examples and embodiments herein. Figure 1 shows a typical HPLC-diagram of a tryptic digest of human growth hormone (Crescormon®). The distribution of opioid receptor activity is represented by dark bars. Figure 2 shows the HPLC-pattern of receptor activity when Genotropin® was used as a substrate for tryptic digest. The distribution of opioid receptor activity is represented by dark bars. Figures 3 and 4 show binding studies performed with the invented oligopeptides. The figures show displacement curves for the binding of Tyr-Ser- Phe-Leu-Glu and Tyr-GIy-Leu-Leu to rat membranes with (3H)-DAGO (Figure 3) and (~Η)-OAOL (Figure 4) as labelled ligands. Table 1 presents initial binding data for an oligopeptide according to the invention in comparison with β-casomorphin. Table 2 presents opioid receptor affinity and activity assays for oligopeptides according to the invention in comparison to prior art tetrapeptides. Example 1 Enzymatic degradation of growth hormone Human growth hormone preparations (Crescormon® and Genotropin®, m22000 u) were supplied by Kabi Pharmada AB (Stockholm, Sweden). Crescormon® was purified from fresh frozen human pituitaries whereas Genotropin® was prepared by gene technology. TPCK-treated trypsin from bovine pancreas was obtained from Sigma (St. Louis, MO, U.S.A.). The chroma tographic materials Sephadex G-100 and Sephadex G-25 (PD-10) were from Pharmada (Uppsala, Sweden). All other chemicals and solvents were of analytical grade from commerdal sources. Preparation of plasma endopeptidase A 20 ml plasma sample colleded from a non-pregnant, non-puerperal woman of fertile age, was fractionated on a Sephadex G-100 column (5 cm x 90 cm). The column was eluted with 20 mM Tris-HCl, pH 7.4, and fractions of 20 ml were colleded maintaining a flow rate of 80 ml/h. Aliquots (1 ml) of the fractions were desalted on Sephadex G-25 (PD-10 columns) and lyophilized prior to enzyme assay. The active fractions were collected, pooled and kept frozen before further studies. Further purification was obtained by chromatography on DEAE- Sepharose CL-6B. The column (2 cm x 12 cm) was equilibrated with 20 mM Tris- HCl, pH 7.4, and following sample application it was eluted with a linear gradient of NaCl (0-0.5M) containing the same Tris-HCl buffer. Fractions of 10 ml were colleded at a flow rate of 100 ml/h and treated as above before enzyme assay. High performance liquid chromatography Reversed phase HPLC was performed using a Pharmada/LKB instrument (see F Nyberg et al J. Chromatogr. v359, 1986, p 541-551) equipped with a Spherisorb TSK-ODS-120 DT column (4.6 mm x 250 mm, particle size 5 μ). The column was developed with a linear gradient of acetonitrile (15-60 %) containing 0.04 % trifluoroacetic add (TFA). The sample was dissolved in 200 μl of starting buffer. Fractions of 0.5 ml were colleded at a flow rate of 0.5 ml/min and evaporated before receptor assay. Enzymatic digestion Lyophilized trypsin or plasma enzyme fractions were dissolved in 100-200 μl of 0.4 M ammonium bicarbonate (pH 7.8) and incubated with 0.2-1.5 mg of human growth hormone at 37 C for 5-8 h in a final volume of 250 μl. The reaction was terminated by the addition of 1 ml ice-cold methanol. The sample was evaporated in a Savant Vac concentrator (Hicksville, N.Y., NY; U.S.A.) before further analysis by reversed phase HPLC. Results Figure 1 shows a typical HPLC chromatogram of a tryptic digest of human growth hormone (Crescormon®). As can be seen, at least two receptor active peaks were recorded. When Genotropin® was used as substrate the HPLC pattern of receptor activity showed some differences (Figure 2). The large peak eluting in fraction 31 was still dominating. However, the peak observed in fraction 18 in the digest of the Crescormon® preparation (Figure 1) was almost depleted as shown in Figure 2. One explanation to this may be the fad that the Crescormon® preparation contain higher degree of deamidated forms of the hormone and such forms may be more susceptible towards enzymatic degradation as discussed elsewhere (Biochem. Biophys. Acta, v625, 1980, p255- 260, F Nyberg et al). The plasma enzyme gave rise to several receptor-active fragments recorded by the HPLC-radioreceptor assay procedure. Following separation on Sephadex G-100 this enzyme was found to elute in a position corresponding to protein with a molecular mass of 100-110 ku. Further purification of the plasma enzyme was achieved by ion exchange chromatography on DEAE-Sepharose, where the enzyme eluted at a NaCl concentration of around 0.1 M. The receptor-active fragments released by the plasma enzyme differed from the active tryptic fragments with regard to their behaviour in HPLC chromatography. It was noted that some parts of the tryptic digest fragments of growth hormone were related to the sequence of β-casomorphin. Partial amino add sequencing of the trypsin-released growth hormone fragments revealed that one peptide contained the Tyr-Ser-Phe-Leu-Gln sequence. Example 2 Preparation of the oligopeptides according to the invention The oligopeptides according to the invention were initially derived from the sequence Tyr-Ser-Phe-Leu-Gln as mentioned in Example 1. A number of straight and cydic pentapeptides with the general structure Tyr-X-Phe-Leu-Z were performed according to the following methods described in Examples 2.1 to 2.3. Example 2.1 Synthesis of Tyr-X-Phe-Leu-Glu (X = Ser I, D-Ala π, Pro HI) The solid phase method was used in the standard way for the synthesis of peptides, using a Beckman 990 synthesizer. Boc amino add were purchased from Bachem Inc. California. The phenolic group of tyrosine was protected with a 2,6 - dichlorobenzyl group, the hydroxyl group of serine was proteded with benzyl group and the gamma-carboxyl of glutamic add was esterified to cydohexylester. At the completion of the last cyde, the peptide was deaved from the resin and completely deprotected by treatment with anhydrous liquid HF at 0° for 60 min in the presence of anisole (30 ml HF and 3 ml anisole per gram resin). After removal of HF and thorough drying under vacuum the resin was washed with ether and extracted with acetic add (10 %) and the extrads were freeze dried. The crude peptide was purified by gel filtration on a Fradogel TSK HW-40 or PGM-2000 column with an acetic acid or trifluoroacetic add as the eluant. The final product was obtained as a lyophilisate. Homogeneity of the peptides was judged by TCL (silica gel 60F-254 Merck; I n- BuOH/AcOH/EtOAc/Aq: 1/1/1/1; II Pyridine/EtOAc/ AcOH/Aq: ' 5/5/1/3; X = Ser Rf I 0.74; Rf II 0.73) and analytical FPLC (column:pep-RPC, gradient A 0.1 % TFA/Aq, B 0.1 % TFA/acetonitril). The identity was established by amino add analysis (6 M HC1, 110°, 24 h) and mass spectrometric analysis, positive FAB, performed on an SX-102A double focusing masspectrometer (Jeol, Japan). Example 2.2 Synthesis of Tyr-Ser-Phe-Leu-Glu-NH2 This peptide was synthesized on 4-methylbenzyhydrylamine resin in exactly the same manner as described in Example 2.1. MBHA-resin (0.46 mmol/g, 1.74 g, Nova biochem Switzerland) was readed with Boc-Glu gammacydo hexylester. The crude peptide was purified by gelfiltration on a Fradogel TSK H W-40 column with 0.1 % TFA as the eluenf. TLC: Rf (I) 0.76, Rf (II) 0.79; amino add analysis: Tyr, 0.99; Ser, 0.99; Phe, 1.01; Leu, 1.00; Glu, 0.99; FAB-MS m/z 657.2 [M+HJ+. Example 2.3 Synthesis of Tyτ-cydo(-Lys-Phe-Leu-Glu-) The synthesis was performed by solid phase technique on a 4- alcoxybenzyl alcohol resin, using the standard Fmoc strategy protocol. The α- amino functions were proteded by the fluorenylmethyloxycarbonyl group, except for the α-amino function of Tyr which was proteded by the benzyl oxycarbonyl group. The γ-carboxyl function of Glu, the ε-amino function of Lys and the phenolic function of Tyr were proteded by benzyl, tert- butyloxycarbonyl and benzyl respectively. The peptide was deaved from the resin by 50 % trifluoroacetic add in dichloromethane leaving all the functional groups protected except for the ε-amino function of Lys and the α-carboxyl function of Glu. Cyclization was performed in DMF at a peptide concentration of 0.1 mM by benzotriazole tetramethyluronium hexafluorophosphate (HBTU) and N-ethyldiisopropylarnine.The cyclic peptide was finally deproteded by catalytic hydrogenation at atmospheric pressure in methanol with 10 % Pd on charcoal as a catalyst. The peptide was charaderized by amino add analysis, HPLC, and FAB Mass spectral analysis. Example 3 Assays of the new oligopeptides In a first assay the peptide Tyr-Ser-Phe-Leu-Gln, with a substitution of Gin for Glu, and a shorter fragment Tyr-GIy-Leu-Leu were synthezised with methods disdosed synthesized and used for binding studies to the μ- and δ- opioid receptors. The receptor assay was performed according to Life Sd, vol. 16, 1975, p 1979ff, L Terenius et al. and Brain Res. Vol. 259, 1983, p 267-274, F Nyberg et al. using synaptic rat plasma membranes from whole rat brain without cerebellum and with ^H-labelled dihydromorphine as the competing radioligand. Each run induded a calibration curve with Met-enkephalin and the binding activity of the tested fraction was expressed in Met-enkephalin equivalents. In the experiments noted in Figures 3 and 4 and in Table 1 (^HMD- Ala2,MePhe 4 ,Gly-ol5)-enkephalin (DAGO or DAMGO) and (3H)-(D-Ala2,D- Leu-5)-enkephalin (DADL) purchased from Amersham (Buckinghamshire, England) were used as radioligands in a binding assay performed as in Reg. Peptides, Vol. 34, 1991, pl69-179, E-L Glamsta et al. DAGO or DAMGO is a μ- receptor ligand whereas DADL is a typical δ-receptor agonist. The calculated inhibition constants are listed in Table 1. Data indicate that the pentapeptide fragment has higher affinity for both μ- and δ-receptors than the shorter growth hormone fragment. The potency of Tyr-Ser-Phe-Leu-Glu for the μ-receptor was in the same order of magnitude as β-casomorphin-5 (see Table 1). The inhibition constants, Ki, in Table 1 are defined as the concentration of the peptide that leads to a 50% blocking of the labelled ligand. In the publication β-Casomorphins and Related Peptides Ed. F Nyberg et al, pages 65-75, "Selective δ-antagonist peptides, analogues of α-casein exorphin, as probes for the opioid receptor" by S Loukas et al are various inhibiting peptides and opioid receptor activity disdosed. In Regulatory Peptides, vol 34, 1991, pag 169-179 ( E-L Glamsta et. al.) inhibition constants for β-casomoφhins are disclosed. It is thus indicated that trypsin treatment of human growth hormone results in the generation of fragments which interfere with opioid receptors. It is also evident that human plasma contains proteolytic activity which may release opioid active peptides from the hormone, which, if they pass the blood-brain barrier, may affect the central nervous system (CNS). In fact, in the dinical treatment with growth hormone some secondary effects on the CNS have been observed (unpublished). It is therefore tempting to suggest that opioid active peptide fragments released from growth hormone may reach the CNS and be responsible for these effects. For the tests accounted for in Table 2, a number of new pentapeptides and peptides according to the prior art were manufactured in accordance with Example 2, above. The peptides were employed for both receptor tests and bioassays on electrically stimulated guinea pig ileum strips (GPI-tests). The receptor assays were performed in the same manner as above with DAMGO as a radioligand to the μ-receptor. In the assays (3H)-DAMGO spedfic binding was displaced by the different tested substances ( 10"5 M to 10"10 M). The concentration of labelled ligand when 50 % was displaced, IC50 (inhibitory concentration), Ki and the binding capadties Bmax were calculated from a computer program (LIGAND, Biosoft, Cambridge, UK.) In the GPI-tests methods and material disdosed in Regul Pept , Vol.34, 1991, p 169-179 (E-L Glamsta et al.) and in The Hemorphins, Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 108, Acta Univers. Ups. Uppsala, 1993, E-L Glamsta, were used. The magnitude of activity in relation to a standard compound, normorphine was recorded. Table 2 shows that inventive oligopeptides have a high binding affinity to the μ-receptor. It is notable that the cyclic oligopeptides both have a high receptor affinity and a high opiate activity, suggesting a powerful agonist activity. The straight pentapeptides according to invention also have a high receptor affinity, but a reduced biological activity is measured by the GPI-test. These tests may suggest partial agonist or a functional antagonist activity. The differences in test value of the inhibitory constant Ki for the peptide Tyr-Ser-Phe-Leu-Glu in Tables 1 and 2 must be credited to different analytical material and procedures at the respective condition. It is, however, to be considered that the relative magnitude of activity of different compounds in each test run is the most interesting and condusive. The tests reveal the generation of new oligopeptides initially derived from tryptic fragments of human growth hormone which have an opioid receptor affinity and an opioid activity of surprisingly high magnitude. The new compounds, consequently, show a high pharmaceutical potential and a possibility to create new tissue selective opiate receptor active pharmaceuticals with potentially reduced adverse effeds. Table 1 Inhibition constants of hGH fragments for (3H )-DAGO and (3H)-DADL binding sites in rat brain homogenates Peptide Ki-values (μ M) 3H )- DAGO ( 3 H )- DADL binding site binding site Tyr-GIy-Leu-Leu 32 24 Tyr-Ser-Phe-Leu-Glu 7.4 5.8 Tyr-Pro-Phe-Val-Glu 8.9 Binding data were obtained from inhibition experiments (duplicate determination) for determination of binding parameters by the computer program EBDA/LIGAND. A one site model was chosen (Tyr-Pro-Phe-Val-Glu = human β-casomorphine-5). Table 2 Compound IC50 3 HDAMGO Ki Bmax GPI-test (nM) (M)xlO-lO (nM) (pmol) relative activity normorphine=l Tyr-Ser-Phe-Leu-Glu 7.3 24.09 2.3 17.7 <0.01 Tyr-Ser-Phe-Leu-Glu-NH2 2.6 24.09 0.82 72.5 Tyr-D-Ala-Ph -Leu-Glu T τ-Pro-Phe-Leu-Glu 0.01 Tyr-D-Ala-Phe-Leu-NH2* 0.3 Tyr-D-Ala-Phe-Met-NH2* 0.5 Tyr- D-Ala-Phe-Met* Tyr r--CCvyss--]Phe-Leu-Cys 0.8 S ~ Tyr-D- ys-Phe-Leu-Cys 11.9 23.99 3.7 11.8 Tyr-Dab-Phe-Leu-Glu 6.19 23.99 2.0 18.6 1 Tyr-L rys-Phe-Leu-Glu 0.055 24.17 0.016 1 ' These peptides were disdosed in US patent 4254 024

Claims: Qaims 1. Straight or cydic oligopeptides with affinity to opioid receptors having the primary sequence backbone Tyr-X-Phe-Leu-Z, where X and Z denotes amino adds or derivatives or analogues thereof, where X and Z can be covalently coupled to provide a heterocyclic structure according to the following conditions: i) when the oligopeptide is straight X is chosen among the amino adds Ser, Gly, Pro, AMCA and D-Ala and Z is chosen among Glu, Gin or their derivatives; ii) when the oligopeptide is cydic X is chosen among D- or L-2,4- diaminobutyric add, D- or L-lysine, D- or L-ornithine and D or L-cysteine and Z is Glu, Gin or their amino derivatives with the proviso that if X is D-or L-Cys, then Z is Cys. 2. Oligopeptides selected from daim 1 with affinity to μ- and δ-opioid receptors. 3. Oligopeptides according to daim 1 having the sequences Tyr-Ser-Phe-Leu- Glu, Tyr-Ser-Phe-Leu-Glu-NH2, Tyr-D-Ala-Phe-Leu-Glu, Tyr-Ser-Phe-Leu-Gln, Tyr-D-Ser-Phe-Leu-Glu, Tyr-Pro-Phe-Leu-Glu or Tyr-AMCA-Phe-Leu-Glu. Tyr-cydo(-D-Lys-Phe-Leu-Glu-), Tyr-cydo(-D-Orn-Phe-Leu-Glu-), 5. A pharmaceutical composition comprising at least one of the oligopeptides according to daims 1 to 4 and suitable carriers and /or diluents for adapting it to an appropriate administration route. 6. Use of an oligopeptide selected from daim 1 in the manufadure of a pharmaceutical preparation for alleviating pain, treatment of depressions, treatment of opioid addiction and /or increasing the comfort for individuals suffering from extraordinary stress or shock. 7. Method for the treatment of depressions, stress or shock, opioid addiction or for alleviating pain by administering an oligopeptide selected from daim 1.

Inventors: FHOELENHAG KARIN INGEBORG, FRYKLUND LINDA, LARSSON BO CHRISTER, LUNDIN RONNY, NYBERG FRED JARL, WESTIN-SJOEDAHL GERTRUD ELISAB, FHOELENHAG, KARIN, INGEBORG, FRYKLUND, LINDA, LARSSON, BO, CHRISTER, LUNDIN, RONNY, NYBERG, FRED, JARL, WESTIN-SJOEDAHL, GERTRUD, ELISABETH
Applicants: FHOELENHAG KARIN INGEBORG, FRYKLUND LINDA, KABI PHARMACIA AB, LARSSON BO CHRISTER, LUNDIN RONNY, NYBERG FRED JARL, WESTIN SJOEDAHL GERTRUD ELISAB, FHOELENHAG, KARIN, INGEBORG, FRYKLUND, LINDA, LARSSON, BO, CHRISTER, LUNDIN, RONNY, NYBERG, FRED, JARL, WESTIN-SJOEDAHL, GERTRUD, ELISABETH
Application year: 1993
Main classification: C07K7
IPC codes: A61K38, A61P25, C07K5, C07K7, C07K14