HRP920204A2 - Galanin antagonist - Google Patents
Galanin antagonist Download PDFInfo
- Publication number
- HRP920204A2 HRP920204A2 HRP920204A HRP920204A2 HR P920204 A2 HRP920204 A2 HR P920204A2 HR P920204 A HRP920204 A HR P920204A HR P920204 A2 HRP920204 A2 HR P920204A2
- Authority
- HR
- Croatia
- Prior art keywords
- gly
- leu
- pro
- galanin
- tyr
- Prior art date
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Description
Ovaj patent odnosi se na antagonist galanina koji je ligand galaninskog receptora. Odnosi se i na peptide Galanin (1-12)-Pro-Supstanciju P(5-11), Galanin(1-12)-Pro-Bradikinin(2-9), Galanin(1-12)-Pro-Pro-Pro-(Leu5-Enkefalin(5-1)), Galanin (1-12)-Pro-Lys-(ε-NH)-Pro-(Leu5-Enkefalin(5-1)) i na njihove funkcije derivate koji pokazuju u biti isti galaninski oprečni učinak kao navedeni peptidi. Antagonist galanina upotrebljava se u tretmanu poremećaja na sisavcu koje ovise o fiziološkoj funkciji galanina na galaninskom receptoru. This patent relates to a galanin antagonist that is a ligand for the galanin receptor. It also refers to the peptides Galanin (1-12)-Pro-Substance P(5-11), Galanin(1-12)-Pro-Bradykinin(2-9), Galanin(1-12)-Pro-Pro-Pro -(Leu5-Enkephalin(5-1)), Galanin (1-12)-Pro-Lys-(ε-NH)-Pro-(Leu5-Enkephalin(5-1)) and on their functions derivatives that show essentially the same galanin antagonistic effect as the above peptides. A galanin antagonist is used in the treatment of mammalian disorders that depend on the physiological function of galanin at the galanin receptor.
Temeljno Fundamentally
Neuro prijenosnici i hormoni mogu indicirati celularne učinke vezivanjem za i aktiviranjem receptora vezanih za opnu. Neuropeptid izoliran je 1983 iz tankog crijeva svinje. Analizom je nađeno 29 aminokiselinskih ostataka (Tatemoto, k., et al, FEBS Lett., 164 (1983) 124-128). Opisane sekvence galanina su iz druga dva sisavca, štakora i krave (Vrontakis M. E. et al, J. Biol. Chem. 262 (1987) 16755-16758; Kaplan L. M. et al, Proc. Natl. Acad). Neurotransmitters and hormones can signal cellular effects by binding to and activating membrane-bound receptors. Neuropeptide was isolated in 1983 from the small intestine of a pig. The analysis found 29 amino acid residues (Tatemoto, k., et al, FEBS Lett., 164 (1983) 124-128). Described galanin sequences are from two other mammals, rat and cow (Vrontakis M. E. et al, J. Biol. Chem. 262 (1987) 16755-16758; Kaplan L. M. et al, Proc. Natl. Acad).
Sci. U.S.A. 85(1988) 1065-1069 I Rkeaus A. and Carlquist M. FEBS Lett. 234 (1988) 400-406). Uspoređivanje peptidne sekvence galanina iz štakora, svinje i vola otkriva kako su N- terminalne 1-15 aminokiseline identične. Stoga je najvjerojatnije kako će se spomenuti očuvani segment naći u galaninu iz drugih sisavaca, uključujući i čovjeka. Sci. USA 85(1988) 1065-1069 and Rkeaus A. and Carlquist M. FEBS Lett. 234 (1988) 400-406). Comparison of the peptide sequence of rat, pig and ox galanin reveals that the N-terminal 1-15 amino acids are identical. Therefore, it is most likely that the mentioned conserved segment will be found in galanin from other mammals, including humans.
Galanin ima široku shemu raspodjele, koja često korelira sa višestrukim efektima neuro-formule ispoljenim od različitih sustava. Još nisu objavljeni galaninski antagonisti koji su ligandi galaninskih receptora. Galaninski antagonisti bit će korisni pri određivanju fiziološkog značenja galanina i za razvoj fiziološke funkcije galanina na galaninskom receptoru. Galanin has a broad distribution pattern, which often correlates with multiple neuro-formula effects exerted by different systems. Galanin antagonists that are ligands of galanin receptors have not yet been published. Galanin antagonists will be useful in determining the physiological significance of galanin and for developing the physiological function of galanin at the galanin receptor.
Opis patenta Description of the patent
Jedan dio patenta usmjeren je na galaninski antagonist koji je ligand galaninskog receptora. Tako se antagonistički učinak antagonista galanina glede izuma očituje na galaninskim receptorima. U realizaciji ovog dijela patenta antagonist se bira iz grupe koja sadrži peptide: One part of the patent focuses on a galanin antagonist that is a ligand for the galanin receptor. Thus, the antagonistic effect of the galanin antagonist according to the invention is manifested on galanin receptors. In the implementation of this part of the patent, the antagonist is selected from the group containing peptides:
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-X, H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-X,
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-X, H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-X,
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Pro-Leu-Phe-Gly-Gly-Tyr-X, H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Pro-Leu-Phe-Gly-Gly-Tyr-X,
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly- H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-
Pro-Lys-X, Pro-Lys-X,
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Pro-Leu-Phe-Gly-Gly-Tyr-H Pro-Leu-Phe-Gly-Gly-Tyr-H
Gdje je X -NH2 ili –OH (amid ili slobodna kiselina), i njihovi funkcijski derivati i analozi. Where X is -NH2 or –OH (amide or free acid), and their functional derivatives and analogs.
Prijavom i zahtjevima željelo se ‘funkcionalnim analozima’ peptida iz patenta, između ostalog, obuhvatiti peptide kraćeg ili dužeg lanca, sa ili bez zamjene jednog ili nekoliko aminokiselinskih ostataka sa drugim aminokiselinskim ostacima, sve dok takovi analozi temeljno čine isti farmakološki učinak kao i peptidi iz patenta, tj. to su galaninski antagonisti na receptoru galanina. Zatim, namjera je da ‘funkcijski derivati’ peptida glede patenta obuhvate bilo koji spoj koji temeljno pokazuje istu farmakološku funkciju kao peptidi iz patenta, tj., to je antagonist galanina na galaninskom receptoru. With the application and claims, it was intended that the 'functional analogues' of the peptides from the patent, among other things, include peptides of shorter or longer chains, with or without replacement of one or several amino acid residues with other amino acid residues, as long as such analogues fundamentally produce the same pharmacological effect as the peptides from of the patent, i.e. they are galanin antagonists on the galanin receptor. Then, the patent's 'functional derivatives' of the peptides are intended to include any compound that fundamentally exhibits the same pharmacological function as the peptides of the patent, i.e., it is a galanin antagonist at the galanin receptor.
Osebujno, kao spoj može biti onaj koji je izveden iz jednog peptida iz patenta, ali u kojem je nekoliko aminokiselinskih ostataka zamjenjeno sa drugim kemijskim grupama, tj., organskim ili anorganskim molekulama ili elementi koji nastaju u nekom peptidomimetiku. In particular, the compound can be one that is derived from one peptide from the patent, but in which several amino acid residues are replaced with other chemical groups, i.e., organic or inorganic molecules or elements produced in some peptidomimetic.
Vjeruje se kako je baš strukturna komformacija na galaninskom receptoru peptida iz patenta temeljna za njegovu farmakološku ulogu galinskog antagoniste i za njegov afinitet prema galaninskom receptoru (koji je na taj način ligand galaninskog receptora). It is believed that the structural conformation on the galanin receptor of the peptide from the patent is fundamental for its pharmacological role as a galin antagonist and for its affinity to the galanin receptor (which is thus a ligand for the galanin receptor).
Vjeruje se kako funkcijski derivati i funkcijski analozi koji su obuhvaćeni patentom imaju sličnu strukturnu konformaciju na galaninskom receptoru kao peptidi iz patenta. Okružujući galaninski receptor može biti mimikriran, npr. krvlju ili fiziološkom otopinom soli. The functional derivatives and functional analogs covered by the patent are believed to have a similar structural conformation at the galanin receptor as the patent peptides. The surrounding galanin receptor can be mimicked, eg with blood or saline.
Drugi aspekt izuma usmjeren je na peptide: Another aspect of the invention is directed to peptides:
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-X, H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-X,
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-X, H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-X,
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Pro-Leu-Phe-Gly-Gly-Tyr-X, H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Pro-Leu-Phe-Gly-Gly-Tyr-X,
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly- H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-
Pro-Lys-X, Pro-Lys-X,
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Pro-Leu-Phe-Gly-Gly-Tyr-H Pro-Leu-Phe-Gly-Gly-Tyr-H
gdje X predstavlja –NH2 ili –OH (amid ili slobodna kiselina), i njihove funkcijske derivate i analoge, koji u biti ispoljavaju isti galaninski efekt kao navedeni peptidi. where X represents –NH2 or –OH (amide or free acid), and their functional derivatives and analogues, which essentially exhibit the same galanin effect as the mentioned peptides.
Kao što je gore spomenuto peptidi se također mogu imenovati kao Galanin(1-12)-Pro-Supstanca P(5-11), Galanin(1-12)-Pro-Bradikanin(2-9), Galanin(1-12)-Pro-Pro-Pro-(Leu5-Enkefalin(5-1) i Galanin(1-12)-Pro-Lys(ε-NH)-Pro-(Leu-Enkefalin-(5-1). As mentioned above the peptides can also be named as Galanin(1-12)-Pro-Substance P(5-11), Galanin(1-12)-Pro-Bradykanin(2-9), Galanin(1-12) -Pro-Pro-Pro-(Leu5-Enkephalin(5-1) and Galanin(1-12)-Pro-Lys(ε-NH)-Pro-(Leu-Enkephalin-(5-1).
U pokusnom dijelu ove prijave gore navedeni peptidi u amidnom obliku (tj. X= NH2) imenovani su M15, M35, M36,A, odnosno M34,A. In the experimental part of this application, the above-mentioned peptides in the amide form (i.e. X=NH2) are named M15, M35, M36,A, and M34,A, respectively.
Funkcionalni analozi mogu biti i peptidi kao što su: Functional analogs can also be peptides such as:
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-(D-Arg)-Pro-Lys-Pro-Gln-Gln-(D-Trp)-Phe-(D-Trp)-Leu-Leu-X H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-(D-Arg)-Pro-Lys-Pro-Gln-Gln-(D-Trp)- Phe-(D-Trp)-Leu-Leu-X
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Pro-Gln-Phe-Phe-Gly-Leu-Met-X H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Pro-Gln-Phe-Phe-Gly-Leu-Met-X
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Leu-Ala-Ala-Leu-Ala-Leu-Ala-X, H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Leu-Ala-Ala-Leu-Ala-Leu-Ala-X,
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Pro-Ala-Leu-Ala-Leu-Ala-X H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Pro-Ala-Leu-Ala-Leu-Ala-X
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Gly-Phe-Phe-Ser-Pro-Phe-Arg-X H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Gly-Phe-Phe-Ser-Pro-Phe-Arg-X
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-X H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg- Tyr-X
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Ala-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-X H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Ala-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg- Tyr-X
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Pro-Tyr-Gly-Gly-Gly-Pge-Leu-X H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Pro-Tyr-Gly-Gly-Gly-Pge-Leu-X
H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Pro-Leu-Phe-Gly-Gly-Gly-Tyr-X, H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-Pro-Pro-Leu-Phe-Gly-Gly-Gly-Tyr-X,
gdje X predstavlja –NH2 ili –OH (amid ili slobodna kiselina). where X represents –NH2 or –OH (amide or free acid).
Daljnji dio patenta usmjeren je na uporabu galaninskog antagonista koji je ligand galaninskog receptora za izradu farmaceutskog preparata. Kako su gore navedeni peptidi topivi u vodi, to olakšava proizvodnju farmaceutskih preparata za injekcije. The further part of the patent focuses on the use of a galanin antagonist, which is a ligand of the galanin receptor, for the production of a pharmaceutical preparation. As the above peptides are water soluble, it facilitates the production of pharmaceutical preparations for injection.
Novi daljnji dio pronalaska upravljen je na galaninski antagonist koji je ligand galaninskog primača za uporabu u farmaceutskim preparatima. A new further part of the invention is directed to a galanin antagonist which is a galanin receptor ligand for use in pharmaceutical preparations.
Jedan aspekt pronalaska upravljen je na farmaceutski preparat koji obuhvaća galaninski antagonist koji je ligand galaninskog receptora kao aktivne supstance, zajedno sa farmaceutski prihvatljivim aditivom(ima). Prema posebnostima tipa farmaceutskog preparata kojeg treba proizvesti biraju se takovi aditivi za formiranje željenog preparata. Adekvatan(i) aditiv(i) za tip preparata koji se želi proizvesti, npr. otopine za injekcije, tablete ili flasteri, nalaze se u U. S. Farmakopeji. One aspect of the invention is directed to a pharmaceutical preparation comprising a galanin antagonist that is a galanin receptor ligand as an active substance, together with a pharmaceutically acceptable additive(s). According to the peculiarities of the type of pharmaceutical preparation to be produced, such additives are chosen to form the desired preparation. Adequate additive(s) for the type of preparation to be produced, eg, injectable solutions, tablets, or patches, are found in the U.S. Pharmacopoeia.
Sadašnji preparat osigurava i preparate koji sadrže efikasnu količinu spoja iz sadašnjeg patenta, uključujući njihove netoksične adicidne soli amide i estere, koji mogu samostalno služiti za osiguravanje gore navedenih terapeutskih prednosti. Takovi preparati mogu se osigurati skupa sa nekom fiziološki podnošljivom tekućinom sa geleom ili sa čvrstim razblaživačima, dodacima i sastojcima. The present preparation also provides preparations containing an effective amount of the compounds of the present patent, including their non-toxic acid amide salts and esters, which can independently serve to provide the above-mentioned therapeutic benefits. Such preparations can be provided together with some physiologically tolerable liquid with gel or with solid diluents, additives and ingredients.
Ovi spojevi i preparati mogu se davati sisavcima za veterinarsku uporabu, domaćim životinjama, kao i ljudima u kliničkoj uporabi slično kao druga terapeutska sredstva. Efikasna terapeutska doza varira od oko 0.01 do 1000 mcg/kg, češće 0.1 do 1000 mck/kg tjelesne težine pacijenta. Alternativno se doze unutar navedenih granica mogu davati kontinuirano infuzijom sve do dosegnuća željenog terapeutskog efekta. These compounds and preparations can be administered to mammals for veterinary use, domestic animals, as well as humans in clinical use similar to other therapeutic agents. The effective therapeutic dose varies from about 0.01 to 1000 mcg/kg, more often 0.1 to 1000 mcg/kg of the patient's body weight. Alternatively, doses within the specified limits can be administered continuously by infusion until the desired therapeutic effect is achieved.
Tipično se pripremaju preparati kao što su sastojci za injekcije, ili kao čvrsta otapala ili kao suspenzije. Također se mogu pripraviti čvrsti oblici podesni za otapanje ili pripremu suspenzije. Preparat se također može emulgirati ili inkorporirati u sustav za dovođenje lijeka kao što su lipozomi ili mikrosfere. Preparations such as injectable ingredients are typically prepared either as solid solvents or as suspensions. Solid forms suitable for dissolution or suspension preparation can also be prepared. The preparation can also be emulsified or incorporated into a drug delivery system such as liposomes or microspheres.
Aktivni sastojak se često miješa sa otapalima ili sastojcima koji su konpatibilni i fiziološki sinenergični. Podesna otapala i činitelji su, na primjer, voda, otopine soli, dakstroza, glicerol i kombinacije istih. Ako se želi, preparati mogu sadržavati minorne količine sredstava za vlaženje ili emulziranje sredstava za stabiliziranje pH i slično. The active ingredient is often mixed with solvents or ingredients that are compatible and physiologically synergistic. Suitable solvents and agents are, for example, water, saline solutions, dextrose, glycerol and combinations thereof. If desired, the preparations may contain minor amounts of wetting agents or emulsifying agents for stabilizing pH and the like.
Preparati se konvencionalno daju parenteralno, injekcijom, na primjer, ili potkožno, intramuskularno, intraperitonealno ili intravenozno. Dopunske varijacije podesne za druge načine davanja uključuju supozitorije, vaginalete, intranazalne aerosole, bukalne forme kao što su adhezivne tablete, gele ili kreme i u nekim slučajevima oralne forme. Supozitorije i vaginalete mogu imati tradicionalna veziva i sastojke, na primjer, polialkilenglikole ili trigliceride; takovi supozitoriji mogu se formirati iz smjese koja ima 0.5 do 10%, bolje 1 do 2%, aktivne supstance. Oralne forme mogu imati uobičajeno rabljene sastojke i to: laktoze, manitol, škrob, magnezijum-stearat, natrijum-saharin, celuloze, magnezijum karbonat i ostali, sličnih farmaceutskih odlika. Ovi preparati su u obliku otopina, suspenzija, tableta, pilula, kapsula, formi sa usporenim aktiviranjem ili praha i sadrže 10%-95% aktivne supstance; poželjno je 25%-70%. The preparations are conventionally administered parenterally, by injection, for example, or subcutaneously, intramuscularly, intraperitoneally or intravenously. Additional variations suitable for other routes of administration include suppositories, vaginalettes, intranasal aerosols, buccal forms such as adhesive tablets, gels or creams, and in some cases oral forms. Suppositories and vaginalettes may have traditional binders and ingredients, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from a mixture that has 0.5 to 10%, preferably 1 to 2%, of active substance. Oral forms can have commonly used ingredients, namely: lactose, mannitol, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and others, of similar pharmaceutical properties. These preparations are in the form of solutions, suspensions, tablets, pills, capsules, forms with delayed activation or powder and contain 10%-95% of active substance; preferably 25%-70%.
Peptidne spojeve moguće je formirati u neutralne ili preparate u obliku soli. Farmaceutski prihvatljive netoksične soli uključuje adicidne soli sa kiselinama (formirane sa slobodnim amino grupama) i koje se formiraju sa takovim anorganskim kiselinama kao što su, na primjer, klorovodična ili fosforna kiselina, ili sa organskim kiselinama kao što su vinska, octena, oksalna, bademova i slične. Soli formirane sa slobodnim karboksilnim grupama mogu se izvesti iz anorganskih baza kao što su, na primjer, natrij-, kalij-, amonijak-, kalcij- ili fero-hidroksidi i takovih organskih baza kao što su izopropilamin, trimetilamin, 2-etilaminoetanol, histidin, prokain i slične. Peptide compounds can be formed into neutral or salt preparations. Pharmaceutically acceptable non-toxic salts include acid acid salts (formed with free amino groups) and formed with such inorganic acids as, for example, hydrochloric or phosphoric acid, or with organic acids such as tartaric, acetic, oxalic, mandelic and the like. Salts formed with free carboxyl groups can be derived from inorganic bases such as, for example, sodium, potassium, ammonia, calcium or ferrous hydroxides and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine , procaine and the like.
Daljnji dio pronalaska upućen je na postupak liječenja poremećaja kod sisavaca koji zavisi od fiziološke funkcije galanina na galaninskom receptoru, koji obuhvaća davanje sisavcu učinkovite količine galaninskog antagoniste koji je ligand galaninskog receptora. Budući da je fiziološka funkcija galanina na galaninskom receptoru osebujna za svakog pojedinca, farmakološki učinkovita količina galaninskog antagoniste potrebna za liječenje poremećaja na sisavcu bit će određena od strane liječnika ili veterinara za svaki pojedini slučaj. A further part of the invention is directed to a method of treating a disorder in mammals that depends on the physiological function of galanin on the galanin receptor, which includes administering to the mammal an effective amount of a galanin antagonist that is a ligand of the galanin receptor. Since the physiological function of galanin at the galanin receptor is unique to each individual, the pharmacologically effective amount of galanin antagonist required to treat the disorder in a mammal will be determined by the physician or veterinarian on a case-by-case basis.
Minimalno galaninski antagonist učinkovit je u reguliranju slijedećih poremećaja: Minimal galanin antagonist is effective in regulating the following disorders:
oslobađanju inzulina, insulin release,
oslobađanje hormona rasta, release of growth hormone,
oslobađanju acetiholina, release of acetylcholine,
oslobađanju dopamina, release of dopamine,
oslobađanju tvari P, the release of substance P,
oslobađanu somatostatina, released by somatostatin,
oslobađanju noradrenalina. release of noradrenaline.
Pretpostavljena su trenutno područja medicinske indikacije endokrinologija, neurologija i psihijatrija: senilna demencija Alzheimer typa, shizofrenija, analgezija, bolesti crijeva. Currently, the fields of medical indication are assumed to be endocrinology, neurology and psychiatry: senile dementia of the Alzheimer type, schizophrenia, analgesia, intestinal diseases.
Dobivanje galaninskog antagoniste prema patentu Obtaining a galanin antagonist according to the patent
Za proizvodnju galaninskih antagonista iz patenta koriste se konvencionalne metode za proizvodnju peptida, modificirani na odgovarajući način ako je antagonist peptidomimetik. Pored sinteze faze liganda, konvencionalne metode sinteze novih spojeva iz ovog patenta uključuju postupak sinteze peptida u čvrstoj fazi. U takovom postupku sinteza novih spojeva izvodi se sekvencionalno unkorporiranje željenih aminokiselinskih ostataka, jedan po jedan, u rastućem peptidnom nizu prema općim načelima postupka u čvrstoj fazi. Conventional peptide production methods are used to produce the galanin antagonists of the patent, modified accordingly if the antagonist is a peptidomimetic. In addition to ligand phase synthesis, conventional methods of synthesizing the novel compounds of this patent include solid phase peptide synthesis. In such a procedure for the synthesis of new compounds, the sequential unincorporation of the desired amino acid residues, one by one, in a growing peptide sequence is performed according to the general principles of the procedure in the solid phase.
Rabljene su sljedeće kraćenice: The following abbreviations were used:
NMP N-Metilpirolidon NMP N-Methylpyrrolidone
HOBt N-hidroksibenzotriazol HOBt N-Hydroxybenzotriazole
MBHA P-Metilbenzhidrilamin MBHA P-Methylbenzhydrylamine
PAM p-Acetoksimetil PAM p-Acetoxymethyl
Boc terc-Butiloksikarbonil Boc tert-Butyloxycarbonyl
DMSO Dimetilsulfoksid DMSO Dimethylsulfoxide
DIEA diizopropiletilamin DIEA diisopropylethylamine
Tos tozil That tozil
OcHex cikloheksil estar OcHex cyclohexyl ester
4-MeBzl 4-metilbenzil 4-MeBzl 4-methylbenzyl
OBzl benzil estar OBzl benzyl ester
Bom Benziloksilmetil Bom Benzyloxylmethyl
Clz 2-Klorobenziloksikarbonil Clz 2-Chlorobenzyloxycarbonyl
Bzl O-benzil Bzl O-benzyl
For formil For formula
Brz O-benzil Fast O-benzyl
TFA trifluoroctena kiselina TFA trifluoroacetic acid
DMS dimetilsulfid DMS dimethylsulfide
DCM diklormetan DCM dichloromethane
DMF N,N-dimetilformamid DMF N,N-dimethylformamide
Peptidi “M15”, “M35” i “M36,A” iz ovog patenta sintetizirani su postupno na čvrstom nosaču uporabom peptidnog sintetizatora Applied Biosystem Model 341A uz standardnu HMP/HOBt. Otopinsko-aktivirajuća strategija u omjeru 0.1 mmola (mala sinteza). Peptides “M15”, “M35” and “M36,A” of this patent were synthesized stepwise on a solid support using an Applied Biosystem Model 341A peptide synthesizer with standard HMP/HOBt. Solvent-activating strategy in the ratio of 0.1 mmol (small synthesis).
Peptid “M34,A” istovjetno je sintetiziran, osim što je zaštitna grupa bila ε-Fmoc i Boc za Lys. Prema tome jedan dio peptida sintetiziran je uporabom Boc-kemije, a drugi dio je sintetiziran uporabom Fmoc-kemije. Peptide “M34,A” was synthesized identically, except that the protecting group was ε-Fmoc and Boc for Lys. Accordingly, one part of the peptide was synthesized using Boc-chemistry, and the other part was synthesized using Fmoc-chemistry.
Funkcijski derivati iz izuma načinjeni su na analogan način, terc-Boc-aminokiseline vezane su za terc-Boc-aminokiselinskuPAM (Nova Chemical Company Ltd., UK) smolu za slobodnu kiselinu (X=-OH) ili MBHA (Bachem Feinchemikalien AG. Switzerland) smolu za amid (X=NH2) kao hidroksibenzotriazol-(HOBt) estri. The functional derivatives of the invention are made in an analogous way, the tert-Boc-amino acids are attached to the tert-Boc-amino acid PAM (Nova Chemical Company Ltd., UK) resin for the free acid (X=-OH) or MBHA (Bachem Feinchemikalien AG. Switzerland ) amide resin (X=NH2) as hydroxybenzotriazole-(HOBt) esters.
Pojedini ciklus za svaku aminokiselinu uključuje odstranjivanje zaštite terc-Boc-grupe sa 50%-nom trifluoroctenom kiselinom u CH2Cl2 (DCM) tijekom 19 minuta, i aciliranje sa 10-strukim viškom (u poređenju s količinom amino grupa na smoli) zaštićenih aminokiselina u smjesi 15% DMSO u N-metilpirolidinu (NMP) tijekom 35 minuta. Između takovih zahvata učinjeno je nekoliko ekstezivnih ispiranja sa CH2Cl2, DIEA i NMP. Poslije svakog spajanja izvedeno je acetiliranje i 5% DIEA i NMP tijekom 5 minuta. A separate run for each amino acid involves deprotection of the tert-Boc group with 50% trifluoroacetic acid in CH2Cl2 (DCM) for 19 min, and acylation with a 10-fold excess (compared to the amount of amino groups on the resin) of the protected amino acids in the mixture. 15% DMSO in N-methylpyrrolidine (NMP) for 35 minutes. Between such interventions, several extensive washings with CH2Cl2, DIEA and NMP were performed. After each coupling, acetylation was performed with 5% DIEA and NMP for 5 minutes.
Sa izuzetkom Boc-Lys(Fmoc) u slučaju peptida 34mA, sve upotrijebljene aminokiseline bile su terc-Boc-zaštićene na N-terminalu (dobiven od Nova Chemical Company Ltd., UK), a njihovi bočni reaktivni lanci zaštićeni su sa Tos u Arg. OcHex u Asp, 4-MeBzl u Cys, OBzl u Glu, Bom u His, Clz u Lyz, Blz u Ser Thr, For u Trp i Brz u Tyr. With the exception of Boc-Lys(Fmoc) in the case of the 34mA peptide, all amino acids used were tert-Boc-protected at the N-terminus (obtained from Nova Chemical Company Ltd., UK) and their reactive side chains were protected with Tos in Arg . OcHex to Asp, 4-MeBzl to Cys, OBzl to Glu, Bom to His, Clz to Lyz, Blz to Ser Thr, For to Trp and Brz to Tyr.
Sva otapala i reagensi za automatsku sintezu peptida bili su od Applied Biosistems. All solvents and reagents for automated peptide synthesis were from Applied Biosystems.
Rabljeni reagensi u fazama skidanja zaštite i prekidanja su analitički zadovoljavali i upotrijebljeni su bez dodatnog pročišćavanja: The reagents used in the deprotection and termination phases were analytically satisfactory and were used without additional purification:
1,2-etandiotiol, acetanhidrid, trifluorometilsulfonska kiselina (TFSMA), dimetilsulfid (DMS) i p-krezol od firme Fluka, dietileter i acetonitril od Mercak-a i HF od AGA Gas. 1,2-ethanediothiol, acetic anhydride, trifluoromethylsulfonic acid (TFSMA), dimethylsulfide (DMS) and p-cresol from Fluka, diethyl ether and acetonitrile from Mercak and HF from AGA Gas.
Potpuno složene peptid-smole suše se u visokom vakuumu. Skidanje zaštite od formil grupe na Trp i benzil-grupi provedeno je uz uporabu niskog TFMSA postupka. Za ovo, 100 mg peptid smole tretira se sa 2 ml smjese TFMSA(10%), TFK(%50%), DMS(30%),p-krezola(8%) i demekaptoetana(2%) tijekom 2 sata na 0°C uz mućkanje i ispiranje sa EtOH, DCM, DMF, DIEA/DCM, DMF i DCM. Fully assembled peptide-resins are dried in a high vacuum. Deprotection of the formyl group on Trp and the benzyl group was performed using the low TFMSA procedure. For this, 100 mg of peptide resin is treated with 2 ml of a mixture of TFMSA(10%), TFK(%50%), DMS(30%), p-cresol(8%) and demecaptoethane(2%) for 2 hours at 0 °C with shaking and washing with EtOH, DCM, DMF, DIEA/DCM, DMF and DCM.
Poslije sušenja u vakuumu peptid se skine sa smole i oslobodi od zaštite miješanjem 10 ml tekućine HF (koji 20% 1:1 smjese p-krezola i p-tiokrezola) sa 1g peptid-smole na 0°C tijekom 60 minuta. Smola se dva puta ispire sa 10 ml Et2O, peptid se ekstrahira sa smjesom 20% acetonitril/voda i filtrira. Liofilizacija vodenog filtrata daje sirovi peptid. After drying in a vacuum, the peptide is removed from the resin and released from protection by mixing 10 ml of liquid HF (which is a 20% 1:1 mixture of p-cresol and p-thiocresol) with 1 g of peptide-resin at 0°C for 60 minutes. The resin is washed twice with 10 ml of Et2O, the peptide is extracted with a mixture of 20% acetonitrile/water and filtered. Lyophilization of the aqueous filtrate gives the crude peptide.
Pročišćavanje sirovog produkta izvodi se HPLC-metodom, gradijentnom evolucijom (45 min) na koloni C18 sa reverznom fazom, tako da se 10 mg sirovog peptida otopi u 1 ml smjese 20-60% smjese 0.1% TFA/acetonitril u 0.1% TFA/voda uz protok 2.0 ml/min. Frakcije se odvajaju na valnoj dužini 328nm. Purification of the crude product is performed by HPLC-method, gradient evolution (45 min) on a C18 column with reverse phase, so that 10 mg of the crude peptide is dissolved in 1 ml of a mixture of 20-60% mixture of 0.1% TFA/acetonitrile in 0.1% TFA/water. with a flow rate of 2.0 ml/min. The fractions are separated at a wavelength of 328 nm.
Tablica Table
Čistoća pojedinačnih peptida provjerena je analitičkom HPLC-metodom i iznosi 99%. Molekularna masa peptida određena je uporabom Plasma Desorption Mass Spectrometar (PDMS). Model Bioion 20, Appleid Biosystems, te polučeni očekivani rezultati. The purity of individual peptides was checked by analytical HPLC-method and is 99%. The molecular mass of the peptide was determined using a Plasma Desorption Mass Spectrometer (PDMS). Model Bioion 20, Appleid Biosystems, and obtained expected results.
Galanin(1-12)-Pro-SP(5-11) amid(M15): (M.masa=2199) IC50 = 0.1 nM Galanin(1-12)-Pro-SP(5-11) amide (M15): (M.mass=2199) IC50 = 0.1 nM
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IC50 je opitno određen potiskivanjem u hipotalamus štakora. The IC50 was experimentally determined by injection into the hypothalamus of rats.
Farmakološki opiti Pharmacological tests
1) Galanin inhibira glukozom inducirano inhibiranje inzulina, mjereno prema Ahren-u. B., et al (Biochemic al and Biophysikal Resarch Comunications, V ol. 140. No. 3. 1986. 1059-1063). Uporabom istog postupka kao što je citirano Ahren B., et al. dokazano je kako peptid prema patentu M15 blokira galaninom-inhibirano oslobađanje insulina u ekvimolarnoj koncentraciji. 1) Galanin inhibits glucose-induced inhibition of insulin, measured according to Ahren. B., et al (Biochemical and Biophysical Research Communications, Vol. 140. No. 3. 1986. 1059-1063). Using the same procedure as cited in Ahren B., et al. the peptide according to the M15 patent has been shown to block galanin-inhibited insulin release at an equimolar concentration.
Životinje i izgradnja stanica Animals and cell construction
Odrasli debeli hiperglikemični miševi (ob/ob) oba spola uzeti su iz lokalne ne-nasljedne kolonije i pušteni da gladuju preko noći. Životinje su ubijene odsijecanjem glave i Langershanovi otočići su izolirani tehnikom izoliranja kolageneze. Adult obese hyperglycemic mice (ob/ob) of both sexes were obtained from a local non-hereditary colony and starved overnight. Animals were killed by decapitation and islets of Langershan were isolated using the collagenase isolation technique.
Suspenzija stanica je učinjena temeljno kao što je opisano u Lehrnmark, A. u Diabetologija 10, 1974, 431-438,. Ukratko otočići se disociraju u pojedinačne stanice i male grudice mućkanjem u Ca2+-Mg2+ deficitarnoj podlozi koja je dopunjena s EGTA (Etilenglikol-bis(beta-aminoetoletar) N,N,N’,N’- tetraoctene kiseline, SIGMA). Poslije toga stanice se inkubiraju na 37°C pH 7.4, preko noći u 12 ml RPMI 1640 podlozi (Podloga za kulturu tkiva od Sigma, koja sadrži L-glutamin i ne sadrži natrijum-bikarbonat) koja je dopunjena sa 10% NU-seruma TM (Collaborative Research Inc. Lexington, Ma, U.S.A.), 100 IU/ml penicilina, 60 μ/ml gentamicina i 100 μg/ml streptomicina. Kako bi se izbjegla spajanje stanica u kulturi u vrijeme inkubacije, suspenzija se blago mućka. The cell suspension was made essentially as described in Lehrnmark, A. in Diabetologia 10, 1974, 431-438. Briefly, islets are dissociated into individual cells and small clumps by shaking in a Ca2+-Mg2+ deficient medium supplemented with EGTA (Ethylene glycol-bis(beta-aminoetholeether) N,N,N',N'- tetraacetic acid, SIGMA). After that, the cells are incubated at 37°C pH 7.4 overnight in 12 ml RPMI 1640 medium (Tissue culture medium from Sigma, which contains L-glutamine and does not contain sodium bicarbonate) supplemented with 10% NU-serum TM (Collaborative Research Inc. Lexington, Ma, U.S.A.), 100 IU/ml penicillin, 60 μ/ml gentamicin, and 100 μg/ml streptomycin. In order to avoid clumping of the cells in the culture during incubation, the suspension is gently shaken.
Podloga The substrate
Temelj rabljenja podloge bio je HEPES pufer [N-(2-Hidroksietil)-piperazin-N’-(2-etansulfonska kiselina), SIGMA], pH 7.4, fiziološki balansirana sa kationima i sa Cl- kao jedinim anionom. U svim slučajevima temeljna podloga dopunjena je s 1 mg/ml albumina. The basis of the substrate used was HEPES buffer [N-(2-Hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid), SIGMA], pH 7.4, physiologically balanced with cations and with Cl- as the only anion. In all cases, the basic medium was supplemented with 1 mg/ml albumin.
Mjerenje oslobođenog inzulina Measurement of released insulin
Kinetika oslobađanja inzulina proučavana je perfuzijom beta-stanica pankreasa (pribl. 1 x 106) sa 20 mM ili 5 mM glukoze pomiješane sa Bio-Gel P-4 poliakrilamidnim petrlama (200-400, Bio-RAd Lab., Richmond, Coo, U.S.A.) u koloni od 0.5 ml. Protok je bio 0.5 ml/min a skupljene su frakcije na 1-3 minute i inzulin je analiziran radioimunološki, uporabom kristalnog inzulina štakora kao reference. Insulin release kinetics were studied by perfusion of pancreatic beta-cells (approx. 1 x 10 6 ) with 20 mM or 5 mM glucose mixed with Bio-Gel P-4 polyacrylamide beads (200-400, Bio-RAd Lab., Richmond, CO, U.S.A. ) in a 0.5 ml column. The flow rate was 0.5 ml/min and fractions were collected for 1-3 minutes and insulin was analyzed radioimmunologically, using crystalline rat insulin as a reference.
Rezultati the results
Nakon 60 minuta inlubacije u prisustvu klukoze (8.3 mM), galanin (10-7M) je potpuno prekinuo oslobađanje inzulina iz 34+2 μU/ml After 60 minutes of incubation in the presence of glucose (8.3 mM), galanin (10-7M) completely stopped the release of insulin from 34+2 μU/ml
(P< 0.001; N= 32). Pepid iz patenta, M15, ovisno od doze, protivio se galaninom-induciranom inhibiranju oslobađanja insulina. (P< 0.001; N= 32). The patent peptide, M15, dose-dependently antagonized galanin-induced inhibition of insulin release.
Oslobađanje insulina u prisustvu glukoze, galanina i M15 na 10-6M, 10-7 ili 10-8 bilo je 28+2 μU/ml (N=16) i 10+3 μU/ml (N=16), M15 na 10-9M bio je bez učinka. Insulin release in the presence of glucose, galanin and M15 at 10-6M, 10-7 or 10-8 was 28+2 μU/ml (N=16) and 10+3 μU/ml (N=16), M15 at 10 -9M was without effect.
Kao rezultat gornjih pokusa može se zaključiti kako se peptid iz patenta, M15, protivio galanin-induciranom inhibiranju oslobađanja inzulina u otočićima. As a result of the above experiments, it can be concluded that the peptide of the patent, M15, counteracted galanin-induced inhibition of insulin release in islets.
2) Galanin inhibira oslobađanje acetilholina mjereno po metodi Fisone G., et al (proc. Natal. Acad. Sci. vol. 84. 1987. 7339-7343). Uporabom temeljno istog postupka kao što su ga citirali Fisone G.,et al iskazuje se kako peptid iz pronalaska, M15, protivi inhibirajućim učincima galanina na skopolaminom-inducirano oslobađanje acetilholina in vivo. 2) Galanin inhibits the release of acetylcholine measured by the method of Fisone G., et al (proc. Natal. Acad. Sci. vol. 84. 1987. 7339-7343). Using essentially the same procedure as cited by Fisone G., et al, it is shown that the peptide of the invention, M15, antagonizes the inhibitory effects of galanin on scopolamine-induced acetylcholine release in vivo.
Pokusi mikrodijalize Microdialysis experiments
Kirurgija i osnovna metodologija. Pokusi su obavljeni sa ženkama CD-COBS (Charls River, Como, Italy) štakora, teškim 200-260 g. Životinje su anestezirane sa ekvitenzinom (1% pentobarbitala, 4% klorohidrata). Cijev za vođenje je usađena potpuno sterotoksično u ventralni hipokampus prema slijedećim koordinatama sa atlasa Paxinos i Watson (Paxinos G.-Watsoc C. (1986) The Rat Brain in Stereotoxic Coordinates. 2nd edn. Academic Press. Sydney) : 5mm iza bregme, 4mm sa strane od srednje linije i 6.8mm ispod površine tvrde tvari. Tada se umeće stilet kako bi se vodeća cijev održala patentnom do slijedećeg dana. Istovremeno se stilet odvoji i zamijeni sondom za mikrodijalizu (CMA 10. Carnegie Medicine AB, Stockholm, Sweden) koja sadrži membranu sa dostupnom površinom 3 x 0.5mm. Sonda za mikroanalizu proteže se 3mm iza kraja vodeće cijevčice; Puferira se konstantnom brzinom od 2μl/min sa Ringerovom otopinom (NaCl, 147 mM: CaCl 2.2 mM i KCl, 4.0 μM). Koji sadrži 10 uM fizostigmin-sulfata i podesi se na pH 7.0 sa NaOH. Odbaci se početnih 40 min perfuzata. Nakon toga perfuzat se skuplja svakih 20 minuta kroz cijelo vrijeme perfulzacije od 260 min. Surgery and basic methodology. Experiments were performed with female CD-COBS (Charles River, Como, Italy) rats, weighing 200-260 g. The animals were anesthetized with equitensin (1% pentobarbital, 4% chlorohydrate). The guide tube was implanted completely stereotoxically in the ventral hippocampus according to the following coordinates from the Paxinos and Watson atlas (Paxinos G.-Watsoc C. (1986) The Rat Brain in Stereotoxic Coordinates. 2nd edn. Academic Press. Sydney): 5mm behind bregma, 4mm on the side of the midline and 6.8 mm below the surface of the hard substance. A stylet is then inserted to keep the guide tube patent until the next day. At the same time, the stylet is detached and replaced with a microdialysis probe (CMA 10. Carnegie Medicine AB, Stockholm, Sweden) which contains a membrane with an accessible surface of 3 x 0.5 mm. The probe for microanalysis extends 3 mm beyond the end of the guide tube; It is buffered at a constant rate of 2 μl/min with Ringer's solution (NaCl, 147 mM: CaCl 2.2 mM and KCl, 4.0 μM). Which contains 10 µM physostigmine sulfate and is adjusted to pH 7.0 with NaOH. Discard the initial 40 min of perfusate. After that, the perfusate is collected every 20 minutes throughout the entire perfusion time of 260 minutes.
Po završenom skupljanju uzorci se trenutno smrzavaju na čvrstom CO2 i liofiliziraju. Sadržaj acetilholina (Ach) kvantitativno se određuje specifičnim radioenzimskim procesom koji je detaljno opisan u Consolo S., et al. (J. Neurochem. 48 (1987), 1459-1465) Wu C. F., et al (Neurobiol. Aging 9, (1988), 357-361) i uključuje (a) konverziju holina u fosforoholin u prisustvu holin fosfokineze i ATP, (b) enzimsku hidrolizu acetilholina u holin i octenu kiselinu, (c)reacetiliranje dobivenog holina u [3H]acetilholin dodatkom [3]acetilkoenzima-A, 99.9 GBq/mmol i acetilkoenzima A:ChAt, i (d) odvajanje i scintilacijsko brojanje polučenog [3H]acetilholona ekstrakcijom u ketonsku fazu koja sadrži tetrafenilbor pomoću ionskoizmjenjivačke kromotografije tekućina-tekućina. After the collection is completed, the samples are instantly frozen in solid CO2 and lyophilized. The content of acetylcholine (Ach) is quantitatively determined by a specific radioenzymatic process that is described in detail in Consolo S., et al. (J. Neurochem. 48 (1987), 1459-1465) Wu C. F., et al (Neurobiol. Aging 9, (1988), 357-361) and involves (a) conversion of choline to phosphorocholine in the presence of choline phosphokinase and ATP, ( b) enzymatic hydrolysis of acetylcholine into choline and acetic acid, (c) reacetylation of the resulting choline into [3H]acetylcholine by the addition of [3]acetylcoenzyme-A, 99.9 GBq/mmol and acetylcoenzyme A:ChAt, and (d) separation and scintillation counting of the obtained [ 3H]acetylcholone by extraction into the ketone phase containing tetraphenylboron using ion-exchange liquid-liquid chromatography.
Koncentracija acetilholina u svakom uzorku računata je linearnom rigresijom na temelju radioaktivnosti standarda (linearna od 10fmola- 25 pmola acetilholina) sa nagibom koji je 7800 dpm/pmol. Koeficijent varijacije replikantnih uzoraka ili standarda acetilholina bio je približno 3%. The concentration of acetylcholine in each sample was calculated by linear regression based on the radioactivity of the standard (linear from 10 fmol - 25 pmol of acetylcholine) with a slope of 7800 dpm/pmol. The coefficient of variation of replicate samples or acetylcholine standards was approximately 3%.
Regeneriranje acetoholina in vitro kroz tri cijevi za dijalizu određeno je kako je prije opisano (Wu et al., ibid.). Prosječno regeneriranje je 17.6±0.5%. The regeneration of acetocholine in vitro through three dialysis tubes was determined as previously described (Wu et al., ibid.). Average regeneration is 17.6±0.5%.
Na kraju pokusa oslobađanja, zamjena sondi za dijalizu verificirane su histološki uporabom bojenja za Nissl supstancu. At the end of the release experiment, the replacement of the dialysis probes was verified histologically using Nissl staining.
Učinak galanina i M15 na skopolaminom-inducirano oslobađanje ACh, mjeren “in vivo” iz vetralnog hipokamusa štakora. Effect of galanin and M15 on scopolamine-induced ACh release, measured “in vivo” from rat ventral hippocampus.
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Galanin (1.56 nmola) i M15 (3.12 nmola) se injektira i. c. v. 2 min. prije skopolamina (0.3 mg/kg,s.c.). Oslobađanje ACh mjereno je u perfuzatu skupljenom za vrijeme narednih 80 min., u četiri 20 min. frakcije. Vrijednosti su izrečene kao pmoli Ach mjereni u vremenu od 20 min. frakcije koja odgovara piku skopolaminskog učina i koja odgovara drugoj 20 min. frakciji. Podaci su prosjeci opita izvršenih na dva štakora (n=2). Galanin (1.56 nmoles) and M15 (3.12 nmoles) are injected i.c. v. 2 min. before scopolamine (0.3 mg/kg, s.c.). The release of ACh was measured in the perfusate collected during the next 80 min., in four 20 min. factions. Values are expressed as pmole Ach measured over a period of 20 min. of the fraction corresponding to the peak of the scopolamine effect and corresponding to the second 20 min. faction. Data are averages of experiments performed on two rats (n=2).
Kao rezultat gornjih opita može se zaključiti kako se peptid iz izuma M15 protivi galaninom-induciranom inhibiranju skopolaminom-induciranom oslobađanju acetil-holina u hipokamusu. As a result of the above experiments, it can be concluded that the peptide of the invention M15 opposes galanin-induced inhibition of scopolamine-induced acetylcholine release in the hippocampus.
3) Učinci intratekalnog galanina u stimulaciji C-niti na fleksorni refleks opisani su u Wiesenfield-Hallin Z., et al (Brain Res. 486 (1989) 205-213). Upotrebom temeljno istog procesa kao i u citiranoj referenci, pokazano je kako peptid M15 ima antagonistički učinak na intratekalnim galaninom-inducirano olakšavanjem flekrosnog refleksa. Drugi peptid iz izuma M35 dao je u preliminarnim opitima slične rezultate. 3) The effects of intrathecal galanin in C-thread stimulation on the flexor reflex are described in Wiesenfield-Hallin Z., et al (Brain Res. 486 (1989) 205-213). Using essentially the same process as in the cited reference, peptide M15 was shown to have an antagonistic effect on intrathecal galanin-induced facilitation of the flexor reflex. Another peptide of the invention M35 gave similar results in preliminary experiments.
Elektrofiziološko proučavanje Electrophysiological study
U akutnim pokusima veličina polisinaptičkog fleksorskog refleksa podkoljenične žile, kao reakcija na aktiviranje visokih graničnih dovoda, ispitana je na decerebreriziranim, spinaliziranim, neanesteziranim štakorima zapisivanjem elektromiograma (EGM) iz zadnji biceps remoris/semidendinosus mišića. Životinje su kratko anestezirane sa Breital-omo i stavljena je kanila u dušnik. In acute experiments, the size of the popliteal artery polysynaptic flexor reflex, in response to the activation of high threshold inputs, was examined in decerebrate, spinalized, unanesthetized rats by recording the electromyogram (EGM) from the posterior biceps remoris/semidendinosus muscle. The animals were briefly anesthetized with Breital-omo and a cannula was inserted into the trachea.
Štakori su namješteni u stereotaksični okvir, decerebrizirani sa aspiracijom prednjeg i srednjeg mozga, a potom su zračeni. Leđna moždina izloži se laminoektomiji na razini teroksa i učini se presjek kod Th. Usadi se Kaundalno i.t. kateter (PE 10) u odnosu na transekciju sa svojim vrhom na lijevoj strani slabinske leđne moždine (L4-5). Fleksorski refleks izazove se pomoću stimulansa iz tijesta na listnom nervu (noge) ili njegovoj površini za intervaciju sa pojedinačnim elektro-sokovima (0,5 ms, 10mA) adekvatne jakosti kako bi se aktivirala C-vlakna (Wall and Woolf, 1984). U nekim opitima CS (kondicionalni stimulans) (1 Hz, 20 s) iste jakosti kao testirani stimulans dat u listni živac. Rats were placed in a stereotaxic frame, decerebrated with forebrain and midbrain aspiration, and then irradiated. The spinal cord is exposed to laminoectomy at the level of the thorax and a section is made at Th. It is implanted Kaundalno i.t. catheter (PE 10) in relation to the transection with its tip on the left side of the lumbar spinal cord (L4-5). The flexor reflex is elicited using a dough stimulus on the leaf nerve (legs) or its surface for intervention with individual electro-juices (0.5 ms, 10mA) of adequate strength to activate C-fibers (Wall and Woolf, 1984). In some experiments, a CS (conditioned stimulus) (1 Hz, 20 s) of the same strength as the test stimulus was given into the sciatic nerve.
Fleksorski refleks zabilježen je kao EMG aktivnost pomoću elektroda tipa igle od nerđajućeg čelika ubačenih u ipsilateralne mišiće podkoljenici žile. Broj akcijskih potencijala izazvanih za vrijeme refleksa integriran je tijekom 2s. Integrirani refleks zapisan je Guold pisaču model 2400 S. Za vrijeme pokusa pračena je brzina srca i rektalna temperatura štakora te održavani u normalnim granicama. Ispravna lokacija i.t. katetera potvrđena je iza svakog pokusa laminoektomijom. The flexor reflex was recorded as EMG activity using stainless steel needle-type electrodes inserted into the ipsilateral calf muscles of the vessel. The number of action potentials evoked during the reflex was integrated over 2s. The integrated reflex was recorded on a Goold model 2400 S printer. During the experiment, the heart rate and rectal temperature of the rats were monitored and maintained within normal limits. The correct location of the i.t. of the catheter was confirmed after each trial by laminoectomy.
Galanin (Bachem, Bubendorff, Switerland) i Somatostatin (Fering, Malm. Sweden) otopljeni su u 0.9%-no) otopini soli. Svi peptidi injektirani su i.t. u vol. 10 μl i potom još 10 μl otopine soli u propuhani kateter. Galanin (Bachem, Bubendorff, Switzerland) and Somatostatin (Fering, Malm. Sweden) were dissolved in 0.9% saline solution. All peptides were injected i.t. in volume 10 μl and then another 10 μl of salt solution into the inflated catheter.
Prikupljanje podataka Data collection
Odgovarajući temeljni iznos refleksa utvrđen je min. 20 min. prije svake i.t. injekcije ili živaca CS. Učinak i.t. peptida ili živaca CS na fleksorski refleks izražen je kao postotak promjene u iznosu refleksa uporedan sa temeljnom linijom koja je naznačena kao 100%. Za ispitivanje interakcije galanina s drugim peptidima ili listnim CS, davan je 15 minuta prije injekcije drugih peptida kako je olakšavajući učinak galanina presta ili istovremeno sa listnim CS. The corresponding basic amount of the reflex is determined min. 20 min. before each i.t. injection or nerve CS. The effect of i.t. of peptides or CS nerves on the flexor reflex is expressed as a percentage change in the amount of the reflex compared to the baseline, which is indicated as 100%. To test the interaction of galanin with other peptides or leaf CS, it was administered 15 minutes before the injection of other peptides until the facilitative effect of galanin has ceased or simultaneously with the leaf CS.
Mjeren je antagonistički učinak intraketalnog (i.t.) M15 na i.t. golaninom-inducirano olakšanje fleksorskog refleksa. Maksimalan olakšavajući efekt 30 pmola galanina bio je 167+42.8% preko razine refleksa temeljne linije. Antagonizam M-15 injektiranog 5 do 10 minuta poslije galanina, izračunat je kao postotak smanjenja maksimalnog olakšavajućeg učina galanina. Podaci su izraženi kao prosjek S.E.M. The antagonistic effect of intraketal (i.t.) M15 on i.t. golanin-induced facilitation of the flexor reflex. The maximal facilitatory effect of 30 pmol galanin was 167+42.8% over the baseline reflex level. The antagonism of M-15 injected 5 to 10 minutes after galanin was calculated as the percentage reduction in the maximal facilitatory effect of galanin. Data are expressed as mean S.E.M.
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Opiti su ponovljeni upotrebom peptida iz izuma, M35, i postignuti su slični rezultati. The experiments were repeated using the peptide of the invention, M35, and similar results were obtained.
Kao rezultat gornjih opita može se zaključiti kako se peptidi iz izuma suprostavljaju intraketalnim galaninom induciranom olakšavanju fleksorskog refleksa na način koji zavisi od doze. As a result of the above experiments, it can be concluded that the peptides from the invention counteract intraketal galanin-induced facilitation of the flexor reflex in a dose-dependent manner.
4) Učinjeno je proučavanje spajanja liganda. Proučavano je potiskivanje 125J-galanina galaninom, galaninskim fragmentom (1-13), fragmentom Tvari P (4-11), galaninskim fragmentom (1-13) sa fragmentom Tvari P (4-11) u ekvimolarnim koncentracijama i peptidima iz izuma M15, M35 i M34,A. Dokazano je kako se peptidi iz izuma specifično spaja za galaninski receptor. 4) Ligand docking studies were done. Suppression of 125J-galanin by galanin, galanin fragment (1-13), substance P fragment (4-11), galanin fragment (1-13) with substance P fragment (4-11) was studied in equimolar concentrations and peptides from the invention M15, M35 and M34,A. It has been proven that the peptides from the invention bind specifically to the galanin receptor.
Dobivanje 125J-monojoda-Tyr26-porcinskog galanina Obtaining 125J-monoiodo-Tyr26-porcine galanin
Sintetički porcinski galanin (1-29) jodira se pomoću postupka sa kloraminom-T tako da se dobije 125J-monojodin-Tyrpocinski galanin (specifična aktivnost 1800-2000 Ci/mmol), kao što je opisano u Land T., et al u: Methods in Neurosciences Ed. M. Conn. 5 1991, 225-234), i upotrebljen je u proučavanju spajanja liganda koja su izvedena u ravnoteži. Synthetic porcine galanin (1-29) is iodinated using the chloramine-T procedure to give 125J-monoiodine-Tyrpocine galanin (specific activity 1800-2000 Ci/mmol), as described in Land T., et al in: Methods in Neurosciences Ed. M. Conn. 5 1991, 225-234), and was used in the study of ligand binding performed at equilibrium.
Dobivanje membranske frakcije Rin m 5F stanica Obtaining the membrane fraction of Rin m 5F cells
Uspostava i stanična kultura Rin m 5F (stanična linija β-tumora pankreasa štakora) opisana je prije (Gazdov et al. Proc. Natl. Acad. Sci. USA, 77 (1980) 3519-3523). Ukratko, stanice su kulivirane u RPMI-1640 (Gibco) podlozi koja sadrži 19% (v/v) seruma fetusa krave, 2,06 mM L-glutamina, 100 IU/ml penicilina i 100 mg/ml streptomocina u 10% CO2-90% zraka na 37oC. The establishment and cell culture of Rin m 5F (a rat pancreatic β-tumor cell line) has been described previously (Gazdov et al. Proc. Natl. Acad. Sci. USA, 77 (1980) 3519-3523). Briefly, cells were cultured in RPMI-1640 (Gibco) medium containing 19% (v/v) fetal bovine serum, 2.06 mM L-glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin in 10% CO2- 90% air at 37oC.
Propuštana su svakih 5 dana, odvojena su od površine boce upotrebom 0.25% tripsina koji sadrži 1 mM EDTA i centrifugiraju se pri 2000 x g tijekom 5 min. na 4oC. Pilula se izloži 15 min. hipoosmodskom 5 mM HEPES puferu (pH 7.4). Suspenzija se centrifugira na 20000 x g 15 min., dobivena pilula se resuspendira bacitracinu koji sadržava (1 mg/ml) 5 mM HEPES puferiranog Krebs-Ringerove otopine (137 mM NaCl, 2.68 nM KCl, 1.8 mM CaCl2 g/l glukoze), pH 7.4 i rabi se trenutno u optima za povezivanje ravnoteže. They were passed every 5 days, separated from the bottle surface using 0.25% trypsin containing 1 mM EDTA and centrifuged at 2000 x g for 5 min. at 4oC. The pill is exposed for 15 min. hypoosmotic 5 mM HEPES buffer (pH 7.4). The suspension is centrifuged at 20,000 x g for 15 min., the resulting pellet is resuspended in bacitracin containing (1 mg/ml) 5 mM HEPES buffered Krebs-Ringer solution (137 mM NaCl, 2.68 nM KCl, 1.8 mM CaCl2 g/l glucose), pH 7.4 and is currently used in optima for connecting balance.
Potiskivanje 125J-galanina sa galaninom i galaninskim receptorskim ligandima Suppression of 125J-galanin by galanin and galanin receptor ligands
Opiti potiskivanja činjeni su u završnom volumenu od 400 μl bacitracia koja ima (1 mg/ml) HEPES-puferirane (5 mM) Krebs-Ringerove otopine, 0.05% (mas./v) BSA (pH 7.4) uz 0.1-0.2 125J-galanina, membranskog proizvoda i povećavajućih koncentracija (10-12 12-10-6 M) nemarkiranog porcinskog galanina ili drugih galaninskih receptorskih liganda. Probe se inkubiraju 30 min. na 37oC. Inkubacija okončava dodavanjem 10 ml ledeno-hladnog HEPES-puferirane Krebs-Ringerovoe otopine, i potom brzim filtriranjem preko Whatman CF/C filtera, prethodno prekritih tijekom 5-6 sati sa 0.3% (v/v) u polietilenaminskoj otopini. Filtri se isplahnjuju sa 10 ml otopine za testiranje. Radioaktivnost zadržana na filterima određena je u Packard gama brojaču. Suppression experiments were performed in a final volume of 400 μl of bacitrac containing (1 mg/ml) HEPES-buffered (5 mM) Krebs-Ringer solution, 0.05% (wt/v) BSA (pH 7.4) with 0.1-0.2 125J- of galanin, a membrane product and increasing concentrations (10-12 12-10-6 M) of unlabeled porcine galanin or other galanin receptor ligands. Samples are incubated for 30 min. at 37oC. Incubation is terminated by adding 10 ml of ice-cold HEPES-buffered Krebs-Ringer solution, and then by rapid filtration through Whatman CF/C filters, previously covered for 5-6 hours with 0.3% (v/v) in polyethyleneamine solution. The filters are rinsed with 10 ml of the test solution. The radioactivity retained on the filters was determined in a Packard gamma counter.
Specifično spajanje definira se kao spajanje koje se može potisnuti sa 1 μm galanina (ili adekvatnom koncentracijom drugog potiskivača). Specific splicing is defined as splicing that can be suppressed by 1 μ m galanin (or an adequate concentration of another suppressor).
Iznos IC50 potisnutih liganda računa se na temelju kompjutor-generiranih LC50 iznosa kao što je opisano u Land, t., et al. (ibid). The amount of IC50 suppressed ligands is calculated based on computer-generated LC50 values as described in Land, t., et al. (ibid).
Pripasiranje opitnih podataka izvedeno je na Macintosh SE uz model najmanjih nelinearnih kvadrata uporabom programa “KaleidaGraph”. Fitting of the experimental data was performed on a Macintosh SE with the nonlinear least squares model using the "KaleidaGraph" program.
Potiskivanjem 125J-galanina iz membrane Rin m SF sa galaninskim receptorskim ligandima: By suppressing 125J-galanin from the Rin m SF membrane with galanin receptor ligands:
[image] [image]
Kao što je očito na osnovu gornjih opita, peptidi iz izuma su ligandi galaninskih receptora. As is apparent from the above experiments, the peptides of the invention are ligands of galanin receptors.
Tako farmakološki opiti 1), 2), 3), dokazuju kako su peptidi iz izuma galaninski antagonisti, a opit 4) dokazuje kako su liganidi galaninski receptori, za razliku od drugih spojeva za koje je dokazano kako antagoniziraju specifičan efekt galanina u specifičnom tkivu na temelju interakcije sa reakcijskom fazom van galaninskog receptora – koja je uključena u biološkom djelovanju galanina u datom staničnom tipu. Takovi antagonizam nije specifičan za učinak galanina i nije vidljiv na galaninskom receptoru. Također nije primjenjiv na nekoliko tkiva gdje galanin djeluje, dok su antagonisti prema izumu bona fide antagonisti u farmakološkom značaju i pokazuju svoj učinak na receptorskom mijestu na vanjštini stanice konkurirajući s endogenim ligandom. Thus, pharmacological tests 1), 2), 3) prove that the peptides from the invention are galanin antagonists, and test 4) proves that the ligands are galanin receptors, unlike other compounds that have been proven to antagonize the specific effect of galanin in a specific tissue on based on the interaction with the reaction phase outside the galanin receptor - which is involved in the biological action of galanin in a given cell type. Such antagonism is not specific for the effect of galanin and is not visible at the galanin receptor. It is also not applicable to several tissues where galanin acts, while the antagonists according to the invention are bona fide antagonists in pharmacological significance and exert their effect at the receptor site on the outside of the cell by competing with the endogenous ligand.
Pravljenje membrane iz hipotalamusa štakora Making a membrane from the rat hypothalamus
Odraslim mužjacima štakora (Sprague-Dawley, 180-200 g) odsiječe se glava, hipotalamusi se hitro podvrgnu disekciji i homogeniziraju (10% mas./vol.) u 0.05 M TRIS-Cl puferu, pH 7.4. Potom se deseterostruko razblaži i centrifugira pri 1000 x g 10 minuta. Supernatant se centrifugira pri 10000 x g 45 min. i pilule se resuspendiraju u 5 mM Hepes puferirane Krebs-Ringerove otopine (137 mM NaCl, 2.68 mM KCl, 1.8 CaCl2 1 g/l glukoze, pH 7.4 i tako se dobije finalna koncentracija proteina 1.0 – 15 mg/ml. Adult male rats (Sprague-Dawley, 180-200 g) are decapitated, the hypothalamus are quickly dissected and homogenized (10% wt./vol.) in 0.05 M TRIS-Cl buffer, pH 7.4. It is then diluted tenfold and centrifuged at 1000 x g for 10 minutes. The supernatant is centrifuged at 10,000 x g for 45 min. and the pellets are resuspended in 5 mM Hepes-buffered Krebs-Ringer solution (137 mM NaCl, 2.68 mM KCl, 1.8 CaCl2 1 g/l glucose, pH 7.4 and thus a final protein concentration of 1.0 – 15 mg/ml is obtained.
Proučavanje spajanja liganda na hipotalamusu štakora Study of ligand binding on the rat hypothalamus
Pokusi potiskivanja obavljeni su u završnom volumenu 400 μl Hepes-puferirane (5 mM) Krebs-Ringerove otopine, 0.05% (ms./v.) BSA (pH 7.4) dopunjenog s bacitracijom (1 mg/ml) u prisustvu 0.1 – 0.2 nM 125J-galanina, membranskog preparata iz hipotalimusa štakora i rastućih koncentracija (10-12-10-6 M) nemarkiranog galanina ili drugih galaninskih receptorskih liganada. Uzorci se inkubiraju 30 min. na 37oC. Inkubacija okončava dodavanjem 10 ml ledeno-hladne Hepes-puferirane Krebs-Ringerove otopine, a potom brzim filtriranjem preko Whatman GF/C filtera, prethodno prevučenih sa polietilenaminskom otopinom, 5-6 sati u 0.3% (vol/vol). Filteri se ispiru sa 10 ml otopine za testiranje. Radioaktivnost koja je zadržana na filterima određuje se na Packard gama brojaču. Specifično spajanje se definira kao ono koje se može zamjeniti sa 1 μM galanina. Galanin štakora i svinje doveli su do krivih potiskivanja koja se ne mogu razlučiti sa membranama iz hipotalamusa štakora. IC50 vrijednost liganda izračunate su komponiranjem opitnih podataka na Macintosh SE pomoću princima najmanjih nelinearnih kvadrata iz programa “Kaleida-Graph”. Suppression experiments were performed in a final volume of 400 μl of Hepes-buffered (5 mM) Krebs-Ringer solution, 0.05% (w/v) BSA (pH 7.4) supplemented with bacitrate (1 mg/ml) in the presence of 0.1 – 0.2 nM 125J-galanin, a membrane preparation from rat hypothalamus and increasing concentrations (10-12-10-6 M) of unlabeled galanin or other galanin receptor ligands. The samples are incubated for 30 min. at 37oC. Incubation ends by adding 10 ml of ice-cold Hepes-buffered Krebs-Ringer solution, and then by rapid filtration through Whatman GF/C filters, previously coated with polyethyleneamine solution, for 5-6 hours in 0.3% (vol/vol). The filters are washed with 10 ml of the test solution. The radioactivity retained on the filters is determined on a Packard gamma counter. Specific binding is defined as that which can be replaced by 1 μM galanin. Rat and pig galanin produced suppression curves indistinguishable from rat hypothalamic membranes. The IC50 values of the ligands were calculated by plotting the experimental data on a Macintosh SE using non-linear least squares from the "Kaleida-Graph" program.
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