Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

• the present invention concerns dermorphin analogs with analgesic activity. More specifically, the invention concerns some new opioid heptapep- tides having by a potent analgesic action that takes place substantially at the level of the receptors for peripheral opioid pain stimuli (nociceptors) without involving the more serious central side effects typical of opiates.
• opiate alkaloids - of which morphine is the main ex- ponent - are substances of plant origin that have powerful narcotic and analgesic properties long used in pain therapy, but subject to considerable limitations due to their many critical side effects. Morphine and its synthetic analogs may be administered as analgesics only for limited time periods due to the onset of gastrointestinal problems (constipation), or a growing tolerance to the drug (requiring ever greater doses in order to obtain the desired analgesic effect), or even other adverse reactions such as respiratory depression and catalepsy. Opiate alkaloids are also notorious for their addictive property.
• endogenic peptides found in various regions of the central nervous system, show similar or correlated ac- tivities to those of opiates - bonding to or influencing in some other way the opiate receptors and causing both agonistic and antagonistic effects to those exerted by opiates.
• endogenic peptides are the enkephalins, two natural pentapeptides found in the brain, spinal chord and gastroenteric tract, that have opioid-like effects.
• Other examples are the dynorphins, opioid peptides present both in the central and peripheral nervous system, some of which are involved in the regulation of pain, others in the hypothalamic regulation of hunger and thirst stimuli.
• opioids Similar molecules of a peptidic type, both endogenous and derived from endogenous ones through synthetic modifications, that have activities similar to those of opiates, are generically referred to as opioids.
• ⁇ receptors show a selective affinity for enkephalins
• ⁇ receptors show greater selectivity for morphine and for other poly-cyclic alkaloids
• K receptors show a comparable affinity for both the aforesaid groups of ligands and a preferential affinity for dynorphins.
• ⁇ receptors are more often involved with analgesic effects, even if these effects may at times also be mediated by the ⁇ or K receptors.
• opioid peptides offer a model of drugs that are agonistic of the opioid receptors and unable to pass through the blood-brain barrier (hemato-encephalic barrier). This inability has been related to the poor capacity of the peptidic molecule to pass through the lipid membrane of cell walls.
• hemato-encephalic barrier blood-brain barrier
• Some peptides pass through the hemato-encephalic barrier and can thus enter the central nervous system after peripheral administration because they are recognised by specific transportation systems located on blood vessel endothelia (Banks, WA, Kastin, A.J., Am. J. Physiol. 259, E1-E10, 1990); others, glycosilated, use the sugar tranfer systems (Negri, L. et al., Br. J. Pharmacol. 124, 1516-1522, 1998); others still, containing basic aminoac- ids, can pass through the barrier via the endothelial pinocytosis mechanism. As is known, the latter represents the mechanism through which cells engulf external fluid and its contents by forming invaginations in the cell membrane, which later close and open so as to form cytoplasmic vacuoles full of liquid.
• Dermorphin is the first example, in an animal organism, of a D- aminoacid, essential for biological activity, inserted in a peptide molecule. It has been hypothesised that the conversion of L-alanine into D-alanine is post- transchptional.
• dermorphin represented the most potent natural peptide described until then, acting selectively on the ⁇ receptors. Either intracerebroventricularly or intratecally, its analgesic and catatonizing power was 2000-4000 times greater than that of morphine, 40 times greater than that of ⁇ -endorphine and 10,000 times greater than that of the enkephalins. However, also dermorphin entails a rapid development of addiction and the inevitable withdrawal symptoms when a chronic administration of the substance is interrupted.
• the analgesic activity of two of them is presented as assessed in rats according to the "tail pinch" pharmacological test (Bianchi, C. & Franceschini, J., Br. J. Pharmacol., 9, 280, 1954), after subcutaneous administration, with no comparison with morphine as a rererence drug and with no data on the duration of the analgesic action found.
• the in- vitro degradation of dermorphin firstly produces hydrolysis in the Gly-Tyr bond of the peptide of formula (A), dermorphin(1-4) being the main metabolyte.
• the present invention lies within the aforesaid research field, with a project (No. 10966 of 20 July 2000) funded by the Italian Ministry of Universities and of Scientific and Technical Research (MURST), dealing with the "pharmacological design, synthesis and evaluation of peptides resistant to proteolytic degradation and having a high penetration through the hemato- encephalic barrier".
• MURST Italian Ministry of Universities and of Scientific and Technical Research
• Within said resarch project a total of 68 different peptides were studied. These were obtained via various modifications starting from already known peptide molecules. These modifications were aimed at improving the activity of the known peptide agents in order to obtain the pharmacological effects of the reference analgesic product - morphine - but at the same time avoiding the already mentioned side effects.
• the present invention thus aims to provide new pharmacologically active opioid peptides having a good penetration capacity through the hemato-liquoral barrier, in order to enter the spinal chord fluid, but - above all - being devoid of the known effects on the central nerv- ous system of the opiate drugs currently in use.
• the synthesis of opioid peptides resistant to enzymatic degradation and having a sufficiently long half-life provides, according to the present invention, new analgesic drugs having peripheral activity that are devoid of the side effects on the central nervous system that are typical of the opiate drugs hitherto in use (constipation, respiratory depression, addiction, tolerance, catalepsy). Therefore, the present invention specifically provides a peptide analog of dermorphin having the following general formula:
• B 2 represents a D- ⁇ -aminoacid
• a 3 , A 4 , A 5 and A 6 each represent an L- ⁇ -aminoacid, optionally substituted in the side chains with the substituent groups commonly found in natural amino- acids, such as linear or branched alkyl groups, or phenyl or benzyl groups, in turn possibly substituted either with t-butoxycarbonyl (Boc) protective groups or fluorenyl-methyl-oxycarbonyl (Fmoc) groups, or a pharmaceutically acceptable salt thereof.
• Boc t-butoxycarbonyl
• Fmoc fluorenyl-methyl-oxycarbonyl
• B 2 is selected from among D-AIa, D-Arg, D-VaI, D-IIe, D-Leu, D-Pro, D-Ser, D-Thr, D-Met, D-Lys or D-Om, and more preferably it represents D-alanine.
• a 3 preferably represents phenylalanine
• a 4 preferably represents glycine
• a 5 preferably represents tyrosine
• a 6 preferably represents proline.
• the most deeply studied version of the peptide according to the present invention corresponds to Arg 7 -dermorphin and has the following gen- eral formula:
• arginorphin This product has been called arginorphin.
• the present invention obviously also extends to the pharmaceutically acceptable salts of arginorphin.
• all the peptides according to the present invention are characterised by the fact that in position 1 they contain a link element made up of tyrosine, in position 2 an aminoacid in destrorotatory configuration, resistant to enzymatic degradation, and in position 7 the aminoacid presenting the maximum basicity, i.e. arginine.
• the present invention also concerns the use of a peptide analog of dermorphin as defined above, or one of its pharmaceutically acceptable salts, for the preparation of an opioid drug for pain therapy and/or for the treatment of gastrointestinal disorders, and/or having antipsychotic or anxiolytic activity.
• the proposed drug has a peripheral analgesic activity.
• the present invention further specifically provides, according to a preferred embodiment thereof, the use of Arg 7 - dermorphin (arginorphin), having the following general formula:
• the present invention also concerns a pharmaceutical composition for pain therapy and/or for the treatment of gastrointestinal disorders, and/or having antipsychotic or anxiolytic activity, containing a peptide analog of dermorphin, as defined above, or one of its pharmaceutically acceptable salts, along with any phar- maceutically acceptable carrier or excipient.
• the present invention specifically concerns a drug composition with peripheral analgesic activity and containing as active ingredient Arg 7 -dermorphin or a pharmaceutically acceptable salt thereof.
• HBTU/HOBt/ DIEA O-benzotriazolyl-N,N,N',N'-tetramethyluronio hexafluoro- phosphate/1 -hydroxybenzotriazole/N-ethyl-diisopropylamine
• NMP N- methylpyrrolidone
• the final peptide-resin was unblocked in amine nitrogen by treatment with 20% piperidine in NMP, carefully washed with NMP and dried.
• the detachment of the resin and the simultaneous removal of the protective groups in the side chain was carried out by treatment with a mixture of trifluo- roacetic acid (TFA), water and triethyl silane (95/2,5/ 2,5 in vol.) (4 ml/100 mg of peptide-resin, 2 hours at room temperature).
• the acidic solution was concentrated under vacuum and the peptide precipitated with an excess of tert- buthyl-methyl ether, collected by centrifugation, dissolved in a small volume of trifluoroacetic acid and reprecipitated twice with terf-buthyl-methyl ether, dried and further purified by semipreparatory HPLC (high performance liquid chromatography) and finally characterised by analytical HPLC, composition determination and molecular weight determination. The final yield was in the region of 65-70%.
• the milliQ water was obtained by purifying deionised water with the Reagent Grade Water System of Millipore.
• the fractions containing the product were collected and dried to a constant weight.
• the purity of the single fractions was determined by analyti- cal HPLC on a Vydac C18 column (4,6x250 mm, 10 ⁇ ), using a Perkin Elmer series 410 chromatograph complete with solvent pressuhsation box, mod.
• the exact aminoacid composition of the peptides was assessed by analysing the aminoacids after hydrolysis with 6N HCI (with 0.5% phenyl act- ing as a scavenger for the tyroxine), under vacuum, at 110 0 C for 24 hours.
• the instrument used was the Carlo Erba automatic analyser mod. 3A30.
• the tryptofan was de- graded during acid hydrolysis.
• the mass spectra were obtained with the Perspective Biosystem Mariner 5220 instrument manufactured by Applied Biosystem, with the ionisa- tion technique ESI-MS (electronspray ionisation mass spectrometry), and a TOF detector.
• ESI-MS electrospray ionisation mass spectrometry
• a 1 % TFA solution in diclo- romethane (DCM) was used; the resin underwent repeated short-term treatments (5-10 min) with the unblocking mixture and monitoring was carried out via TLC (thin-layer chromatography) of the filtrate composition.
• the reaction progress was assessed by TLC; the reaction was completed in 3 hours and the solvent was then concentrated in small volume and the residue was taken again with AcOEt. Washes were carried out on the organic phase, first with H 2 O and later with a saturated solution of NaCI, and the solvent was then concentrated in order to obtain a solid residue.
• the product was purified through preparatory HPLC; the fractions containing the required compound were collected together and dried obtaining 18.8 mg of clean product.
• the analgesic activity of synthesised peptides has been studied in rats by using the tail flick test with a thermal stimulus (D'Amour F. E & Smith D. L., J. Pharmacol. Exp. Ther. 72, 74, 1941).
• a ray of light is concentrated on the animal's tail and an electronic device calculates the tail flick time to the nearest one-tenth of a second.
• the chosen test is universally accepted as the ideal analgesic test for drugs of analgesic activity at the central, spinal chord or supra-spinal chord level, typical of opioid drugs.
• the intravenous administration of the peptides in the study confirmed their analgesic properties.
• the analgesic potency of the studied peptides, relative to morphine in rats, turned out to be the following:
• the duration of the analgesic effect varies depending on the derivative: for an E 50 dose, the mean durations relative to morphine turn out to be the following.
• the most potentl analgesic peptide turns out to be the agonist of the ⁇ opioid receptors according to the present invention: Arg 7 -dermorphin, with an action duration that is approximately 50% higher than the one of morphine.
• the glycosilate derivates of endomorphins have very weak analgesic properties, never exceeding 1% of morphine activity.
• the action duration of H-Tyr-Pro-Trp-Phe-Glc is about 30% higher than the one of morphine.
• Subcutaneous administration of the peptides under study provided power and analgesic duration data very similar to the ones obtained by intravenous administration.
• Intrathecal administration yielded the following results.
• the tail flick test in rats is sensitive to the effects of powerful analgesics acting at the spinal chord level, but it does not measure the analgesic effects at a cerebral level or at the level of the peripheral nociceptive nerve endings.
• the test is carried out after peripheral administration (intra- venous or subcutaneous) and spinal administration (intrathecal) of the drug.
• the ratio between intratecal ED 50 and peripheral ED 5O is a penetration index of the drug in the spinal chord through the spinal hemato-liquoral barrier.
• the opioid drugs capable of activating the receptors of the central nervous system produce analgesia, catalepsy, respiratory depression, constipation, tolerance and addiction in rodents (rats, mice and guinea pigs). These effects are all considered an expression of the activation of the central opioid receptors, except for the constipation effect, which is partly due to action on the opioid receptors of the intestine.
• Catalepsy was studied by using the bar test, by measuring the time of maintaining an unusual posture obtained by placing both front legs of a rat on a bar situated 10 cm above the plane of the rat's hind legs. The rats which maintained this posture for over a minute were considered to be cataleptic.
• a dose CD50 of Arg 7 -dermorphin was defined as the dose which determined catalepsy in 50% of the animals concerned.
• the respiratory depression produced by the drug was assessed by measuring respiratory volume and frequency with the differential pletismo- graphic method in rats, which were free to move about in the measurement chambers, before and after respiratory stimulation with a concentration of 5% of CO 2 in the air they breathed.
• a dose RD 50 of Arg 7 -dermorphin was defined as the dose enabling a 50% reduction of the increase of respiratory volume produced by inhaling CO 2 in 50% of the animals concerned.
• Constipation was studied by measuring the transit time inside the intestines of coal particles ingested by the rats before and after Arg 7 - dermorphin administration.
• a dose SD 5O of Arg 7 -dermorphin is the one producing double the ideal transit time of the ingested coal in 50% of the animals concerned.
• Tolerance of the analgesic effect of Arg 7 -dermorphin was studied by injecting the respective AD 50 doses via the two administration routes twice a day for 30 days, at the same time measuring the time course of the analgesic reaction.
• the tolerance level is expressed as the number of administration days necessary for extinguishing the analgesic response.
• the addiction induced by the drug was measured with the place preference test by assessing the degree of preference of a rat for the cage compartment in which the animal had been injected with Arg 7 -dermorphin the day before, compared to the other compartment in which it was injected with a physiological solution.
• the preference is expressed as a percentage of visits made by the rats to the drug compartment out of the total number of visits to the two compartments within an observation time of one hour.
• Peripherally administered Arg 7 -dermorphin shows a constipation effect at a dose 80 times higher than the analgesic dosage.
• morphine has a constipating effect at the usual analgesic dosages. This confirms what has also been observed with other opioid peptides: the opioid re- ceptors of the gastrointestinal tract are difficult to reach for opioids of a pep- tidic structure.
• the analgesimetric pressure test on the rat's paw is sensitive to peripheral analgesic action, which is performed by pure analgesic drugs (some opioids) and anti-inflammatory analgesic drugs (e.g., COX inhibitors) at the level of the peripheral nociceptor nerve endings, after local or systemic administration (oral, subcutaneous or intravenous).
• pure analgesic drugs e.g., me opioids
• anti-inflammatory analgesic drugs e.g., COX inhibitors
• arginorphin has a powerful peripheral analgesic activity on skin peripheral nociceptors, like the one performed by COX inhibitor drugs, but exerted on the peripheral opioid receptors of the nociceptive nerve endings, as shown by the results obtained with the aforesaid pharmacological test.
• Arg 7 -dermorphin does not have any cerebral side effects after subcutaneous administration, in this way demonstrating that it entails little or no penetration through the cerebral hemato- liquoral barrier. Therefore, the proposed peptide represents a new opioid drug of peripheral activity, devoid of the more serious side effects of opiates, and is not classifiable among the drugs subjected to legal restrictions on the use of stupefacient substances.

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