ITRM940405A1 - "HIV VIRUS DEVELOPMENT INHIBITING COMPOUNDS, THEIR PREPARATION AND USES". - Google Patents
"HIV VIRUS DEVELOPMENT INHIBITING COMPOUNDS, THEIR PREPARATION AND USES". Download PDFInfo
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- ITRM940405A1 ITRM940405A1 IT000405A ITRM940405A ITRM940405A1 IT RM940405 A1 ITRM940405 A1 IT RM940405A1 IT 000405 A IT000405 A IT 000405A IT RM940405 A ITRM940405 A IT RM940405A IT RM940405 A1 ITRM940405 A1 IT RM940405A1
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Description
DESCRIZIONE DESCRIPTION
a corredo di una domanda di brevetto di invenzione dal titolo: accompanying an invention patent application entitled:
"Composti inibitori dello sviluppo di virus HIV, loro preparazione e usi" "Compounds inhibiting the development of HIV viruses, their preparation and uses"
La presente invenzione ha per oggetto composti inibitori dello sviluppo di virus appartenenti alla famiglia dei virus della immunodeficienza umana (HIV), la loro preparazione e usi. The present invention relates to compounds inhibiting the development of viruses belonging to the human immunodeficiency virus (HIV) family, their preparation and uses.
Più in particolare l'invenzione concerne peptidi ciclici che inibiscono lo sviluppo dei virus HIV-1 , HIV-2 e di varianti naturali o ricombinanti di essi, la loro preparazione ed uso per applicazioni terapeutiche e profilattiche nella terapia dell'AIDS. More particularly, the invention relates to cyclic peptides which inhibit the development of HIV-1, HIV-2 and natural or recombinant variants thereof, their preparation and use for therapeutic and prophylactic applications in AIDS therapy.
L'infezione da HIV porta allo sviluppo della Sindrome da Immunodeficienza Acquisita (AIDS) [F. Barré-Sinoussi et al., Science, 220, 868-870, 1983; F. Clavel et al., Science, 223, 343-346, 1986], Numerosi composti capaci di interferire con la replicazione del virus sono stati sviluppati per il trattamento dell'AIDS. Tra questi sono di un certo interesse sostanze antivirali capaci di inibire la trascrizione del virus ed il suo assemblaggio [G. Skowron, Hosp. Pract., 27, Suppl. 2, 5-13, 1992; S. Scharpe et al., Biochimie, 73, 121-126, 1992; M. A. Fischi et al., N. Engl. J. Med., 317, 185-191 , 1987], vaccini che aumentano la risposta immunitaria anti-HIV [D. Birx and R. R. Redfield, Int. J. Immunoparmacol., 13, Suppi 1 , 9-18, 1991 ; R. Taylor, J. Nati. Inst. Health Res., 4, 89-93, 1992; R. R. Redfield et al., N. Eng. J. Med., 324, 1677-1684, 1991 ] oltre a sostanze dirette contro il virus [D. H. Smith et al., Science, 238, 1704-1707, 1987; A. Traunecker et al.. Nature, 331 , 84-86, 1988; K. C. Deen et al., Nature, 331 , 82-84, 1988; R. E. Hussey et al., Nature, 331 , 78-81 , 1988; R.A. Fisher et al., Nature, 331 , 76-78, 1988; G. G. Jackson et al., Lancet 9, 647-652, 1988; A. IVI. Prince et al., Proc. Nati. Acad. Sci. (USA), 85, 6944-6948, 1988; W. A. Marasco et al., J. Clin. Invest. 90, 1467-1478, 1992; V. K. Chaudhary et al., Nature, 335, 369-372, 1988; V. Ghetie et al., Proc. Nati. Acad. Sci (USA), 88, 5690-5693, 1991 ; M. A. Till et al., Science, 242, 1 166-1 168, 1988; S. H. Pincus et al., J. Immunol., 142, 3070-3075, 1989; R. W. Finberg et al., Science, 252, 1703-1705, 1991 ; J. M. Zarlìng et al., Nature, 347, 92-95, 1990] ed a combinazioni di sostanze differenti capaci di inibire sia cellule quiescenti che attivate [K.D. Bell et al.. Proc. Nati Acad. Sci.; 90, 141 1-1415, 1993]. Al momento solo Pazidotimidina è largamente adoperata per il trattamento dell'AIDS. Essa presenta comunque numerosi inconvenienti, tra cui l'elevata tossicità e lo sviluppo di virus mutanti resistenti alPazidotimidina [E. Dournon et al., Lancet ii, 1297-1302, 1988; N. Mir and C. Costello, Lancet ii, 1195-1196, 1988; B. Larder and S.D. Kemp, Science, 246, 1155-1 158, 1989]. HIV infection leads to the development of Acquired Immunodeficiency Syndrome (AIDS) [F. Barré-Sinoussi et al., Science, 220, 868-870, 1983; F. Clavel et al., Science, 223, 343-346, 1986], Numerous compounds capable of interfering with the replication of the virus have been developed for the treatment of AIDS. Among these are of some interest antiviral substances capable of inhibiting the transcription of the virus and its assembly [G. Skowron, Hosp. Pract., 27, Suppl. 2, 5-13, 1992; S. Scharpe et al., Biochimie, 73, 121-126, 1992; M. A. Fischi et al., N. Engl. J. Med., 317, 185-191, 1987], vaccines that increase the anti-HIV immune response [D. Birx and R. R. Redfield, Int. J. Immunoparmacol., 13, Suppi 1, 9-18, 1991; R. Taylor, J. Nati. Inst. Health Res., 4, 89-93, 1992; R. R. Redfield et al., N. Eng. J. Med., 324, 1677-1684, 1991] as well as substances directed against the virus [D. H. Smith et al., Science, 238, 1704-1707, 1987; A. Traunecker et al. Nature, 331, 84-86, 1988; K. C. Deen et al., Nature, 331, 82-84, 1988; R. E. Hussey et al., Nature, 331, 78-81, 1988; R.A. Fisher et al., Nature, 331, 76-78, 1988; G. G. Jackson et al., Lancet 9, 647-652, 1988; A. IVI. Prince et al., Proc. Nati. Acad. Sci. (USA), 85, 6944-6948, 1988; W. A. Marasco et al., J. Clin. Invest. 90, 1467-1478, 1992; V. K. Chaudhary et al., Nature, 335, 369-372, 1988; V. Ghetie et al., Proc. Nati. Acad. Sci (USA), 88, 5690-5693, 1991; M. A. Till et al., Science, 242, 1 166-1 168, 1988; S. H. Pincus et al., J. Immunol., 142, 3070-3075, 1989; R. W. Finberg et al., Science, 252, 1703-1705, 1991; J. M. Zarlìng et al., Nature, 347, 92-95, 1990] and to combinations of different substances capable of inhibiting both quiescent and activated cells [K.D. Bell et al .. Proc. Nati Acad. Sci .; 90, 141 1-1415, 1993]. At present, only Pazidothymidine is widely used for the treatment of AIDS. However, it has numerous drawbacks, including high toxicity and the development of mutant viruses resistant to pazidothymidine [E. Dournon et al., Lancet ii, 1297-1302, 1988; N. Mir and C. Costello, Lancet ii, 1195-1196, 1988; B. Larder and S.D. Kemp, Science, 246, 1155-1 158, 1989].
Nuove molecole sono state proposte come alternativa per il controllo dello sviluppo del virus HIV. Queste molecole, analoghe della ciclosporina [Domanda di Brevetto PCT WO 93/14780] sono in generale dotate di elevata tossicità, ma soprattutto presentano un'attività immunosoppressiva, controindicata nei pazienti affetti da AIDS; inoltre la loro efficacia non è stata ancora accertata. New molecules have been proposed as an alternative for controlling the development of the HIV virus. These molecules, analogous to cyclosporine [PCT Patent Application WO 93/14780] are generally highly toxic, but above all they have an immunosuppressive activity, contraindicated in AIDS patients; moreover, their effectiveness has not yet been ascertained.
Da qui la necessità di sviluppare nuove molecole che possano essere adoperate per la preparazione di composizioni farmaceutiche adatte alla profilassi e terapia dell'AIDS e che non presentino gli inconvenienti delle molecole fino ad ora sviluppate. Hence the need to develop new molecules which can be used for the preparation of pharmaceutical compositions suitable for AIDS prophylaxis and therapy and which do not have the drawbacks of the molecules developed up to now.
Gli autori della presente invenzione hanno sviluppato nuove molecole non note dai documenti di tecnica anteriore con un'attività antivirale, in particolare per il virus HIV. Le abbreviazioni, quando non specificate, si riferiscono ai venti amminoacidi naturali. The authors of the present invention have developed new molecules not known from prior art documents with antiviral activity, in particular for the HIV virus. Abbreviations, when not specified, refer to the twenty natural amino acids.
I composti ciclici della presente invenzione hanno formula generale: The cyclic compounds of the present invention have the general formula:
in cui: A1 e A2 rappresentano qualsiasi imminoacido e possono essere uguali o diversi tra loro; A3 e A4 rappresentano qualsiasi amminoacido idrofobico e possono essere uguali o diversi tra loro; A5 rappresenta un braccio spaziatore di lunghezza compresa tra circa 12 Å e 21 Å, legato covalentemente alle estremità N-terminale di A1 e C-terminale di A4. where: A1 and A2 represent any imino acid and can be the same or different from each other; A3 and A4 represent any hydrophobic amino acid and can be the same or different from each other; A5 represents a spacer arm approximately 12 Å to 21 Å in length, covalently bonded to the N-terminal ends of A1 and C-terminal of A4.
Forme preferite di attuazione dell'invenzione comprendono composti ciclici in cui A1 e A2 rappresentano un qualsiasi imminoacido idrofobico N-sostituito con un gruppo alchilico contenente da 1 a 4 atomi di carbonio. Preferibilmente A1 e A2 sono compresi nel gruppo di: acido azetidinico (Azt), prolina (Pro), acido pipecolico (Pipi, sarcosina (Sar); A3 e A4 sono compresi nel gruppo di: fenilalanina (Phe), tirosina (Tyr), triptofano (Trp), β-1-naftilalanina (1-Nal), β-2-naftilalanina (2-Nal), cicloesilalanina (Cha), cicloesilglicina (Chg); A5 è un braccio spaziatore comprendente un peptide contenente da 3 a 5 residui amminoacidici a carattere prevalentemente idrofobico. Sono preferiti bracci spaziatori peptidìci compresi nel gruppo: Aib-Aib-Val di lunghezza di circa 12 Å, in cui Aib rappresenta l'acido α-amminoisobutirrico; Pro-Pro-Phe-Phe, di lunghezza di circa 16.5 Å, Leu-lle-lle-Leu-Val; Aib-Aib-lle-D-Ala-Val, di lunghezza di circa 21 Å; Cys(aOMe)-SSCys(aBoc)-Val, di lunghezza di circa 15.3 A, in cui OMe rappresenta il metilestere e Boc il tert-butilossicarbonile. Preferred embodiments of the invention comprise cyclic compounds in which A1 and A2 represent any N-substituted hydrophobic imino acid with an alkyl group containing from 1 to 4 carbon atoms. Preferably A1 and A2 are included in the group of: azetidine acid (Azt), proline (Pro), pipecolic acid (Pipi, sarcosine (Sar); A3 and A4 are included in the group of: phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), β-1-naphthylalanine (1-Nal), β-2-naphthylalanine (2-Nal), cyclohexylalanine (Cha), cyclohexylglycine (Chg); A5 is a spacer arm comprising a peptide containing 3 to 5 amino acid residues with a predominantly hydrophobic character. Preferred peptide spacer arms included in the group: Aib-Aib-Val with a length of about 12 Å, in which Aib represents the α-aminoisobutyric acid; Pro-Pro-Phe-Phe, with a length of about 16.5 Å, Leu-lle-lle-Leu-Val; Aib-Aib-lle-D-Ala-Val, about 21 Å long; Cys (aOMe) -SSCys (aBoc) -Val, about 15.3 long A, where OMe represents the methyl ester and Boc the tert-butyloxycarbonyl.
Secondo una forma preferita di attuazione dell'invenzione A1 rappresenta Pro, A2 rappresenta Pro, A3 rappresenta Tyr, A4 rappresenta Phe, A5 rappresenta Leu-lle-lle-Leu-Val di lunghezza calcolata di circa 21 Å. According to a preferred embodiment of the invention A1 represents Pro, A2 represents Pro, A3 represents Tyr, A4 represents Phe, A5 represents Leu-ille-ille-Leu-Val of calculated length of about 21 Å.
Secondo una forma alternativa preferita di attuazione dell'invenzione A1 rappresenta Pro, A2 rappresenta Pro, A3 rappresenta Phe, A4 rappresenta Phe, A5 rappresenta Aib-Aib-lle-D-Ala-Val di lunghezza calcolata di circa 21 Å. According to a preferred alternative embodiment of the invention A1 represents Pro, A2 represents Pro, A3 represents Phe, A4 represents Phe, A5 represents Aib-Aib-11le-D-Ala-Val of calculated length of about 21 Å.
Secondo una forma alternativa preferita di attuazione dell'invenzione A1 rappresenta Pro, A2 rappresenta Pro, A3 rappresenta Phe, A4 rappresenta Phe, A5 rappresenta Cys(aOMe)-SSCys(aBoc)-Val- di lunghezza calcolata di circa 15.3 A. According to a preferred alternative embodiment of the invention A1 represents Pro, A2 represents Pro, A3 represents Phe, A4 represents Phe, A5 represents Cys (aOMe) -SSCys (aBoc) -Val- of calculated length of about 15.3 A.
Secondo una forma alternativa preferita di attuazione dell'invenzione A1 rappresenta Pro, A2 rappresenta Pro, A3 rappresenta Phe, A4 rappresenta Phe, A5 rappresenta Pro-Pro-Phe-Phe di lunghezza calcolata di circa 16.5 Å. According to a preferred alternative embodiment of the invention A1 represents Pro, A2 represents Pro, A3 represents Phe, A4 represents Phe, A5 represents Pro-Pro-Phe-Phe of calculated length of about 16.5 Å.
Secondo una forma alternativa preferita di attuazione dell'invenzione A1 rappresenta Pro, A2 rappresenta Pro, A3 rappresenta Phe, A4 rappresenta Phe, A5 rappresenta Aib-Aib-Val di lunghezza calcolata di circa 12 Å. According to a preferred alternative embodiment of the invention A1 represents Pro, A2 represents Pro, A3 represents Phe, A4 represents Phe, A5 represents Aib-Aib-Val of calculated length of about 12 Å.
Costituisce ulteriore oggetto dell'invenzione l'uso dei composti dell'invenzione per la preparazione di un medicamento farmaceuticamente accettabile per la terapia di infezioni da HIV. A further object of the invention is the use of the compounds of the invention for the preparation of a pharmaceutically acceptable medicament for the therapy of HIV infections.
Gli autori hanno trovato che il segmento A1-A2-A3-A4 rappresenta la parte attiva della molecola, quando opportunamente strutturata attraverso la struttura ciclica ottenuta con il braccio spaziatore A5. The authors found that the A1-A2-A3-A4 segment represents the active part of the molecule, when suitably structured through the cyclic structure obtained with the A5 spacer arm.
Il termine "qualsiasi imminoacido" qui adoperato si riferisce agli isomeri L e D sia di imminoacidi naturali sia di quelli "non proteici", comunemente adoperati nella chimica dei peptidi per la preparazione di analoghi sintetici di peptidi naturali. Esempi di imminoacidi "non proteici" sono tioprolina, deidroprolina, idrossiprolina, acido pipecolico, acido azetidinico, sarcosina. The term "any imino acid" used herein refers to the L and D isomers of both natural and "non-protein" imino acids, commonly used in peptide chemistry for the preparation of synthetic analogues of natural peptides. Examples of "non-protein" imino acids are thioproline, dehydroproline, hydroxyproline, pipecolic acid, azetidine acid, sarcosine.
Il termine "qualsiasi amminoacido idrofobico" qui adoperato si riferisce agli isomeri L e D sia di amminoacidi naturali sia di quelli "non proteici" comunemente adoperati nella chimica dei peptidi per la preparazione di analoghi sintetici di peptidi naturali. Gli amminoacidi idrofobici naturali sono glieina, alanina, vaiina, leucina, isoleucina, metionina, fenilalanina, tirosina, triptofano, prolina, istidina, asparagina, glutammina. Esempi di amminoacidi idrofobici "non proteici" sono norleucina, norvalina, alloisoleucina, omofenilalanina, acido α-amminobutirrico, acido α-amminocaproico, acido α-amminovalerico, cicloesilglicina, cicloesilalanina, acido amminofenilbutirrico, fenilalanine mono- e di- sostituite nelle posizioni orto, meta e para dell'anello aromatico con uno o più alogeni, β-2- e 3-tienilalanina, β-2- e 3-furanilalanina, β-2-, 3- e 4-piridilalanina, β-(1 - o 2-naftil)alanina, derivati O-alchilati della serina, omoserina, treonina, allotreonina e tirosina, cisteina S-alchilata, omocisteina S-alchilata, lisina ε-alchilata, ornitina δ-alchilata. The term "any hydrophobic amino acid" used herein refers to the L and D isomers of both natural and "non-protein" amino acids commonly used in peptide chemistry for the preparation of synthetic analogues of natural peptides. The natural hydrophobic amino acids are glieine, alanine, goina, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, proline, histidine, asparagine, glutamine. Examples of "non-protein" hydrophobic amino acids are norleucine, norvaline, alloisoleucine, homophenylalanine, α-aminobutyric acid, α-aminocaproic acid, α-aminovaleric acid, cyclohexylglycine, cyclohexylalanine, aminophenyl-substituted monobutyric acid, phenyl-substituted phenyl, phenyl meta and para of the aromatic ring with one or more halogens, β-2- and 3-thienylalanine, β-2- and 3-furanylalanine, β-2-, 3- and 4-pyridylalanine, β- (1 - or 2 -naphthyl) alanine, O-alkylated derivatives of serine, homoserine, threonine, allotreonine and tyrosine, S-alkylated cysteine, S-alkylated homocysteine, ε-alkylated lysine, δ-alkylated ornithine.
Il termine "braccio spaziatore” qui adoperato si riferisce a quella parte di una struttura chimica che definisce il più piccolo numero di legami chimici consecutivi che possono essere tracciati da una estremità all'altra di detta struttura chimica. I componenti atomici del braccio spaziatore corrispondono a qualsiasi tipo di atomo capace di formare almeno due legami chimici. The term "spacer arm" used here refers to that part of a chemical structure which defines the smallest number of consecutive chemical bonds that can be traced from one end of said chemical structure to the other. The atomic components of the spacer arm correspond to any type of atom capable of forming at least two chemical bonds.
Il termine "lunghezza" si riferisce alla misura predittiva ottenuta sommando le lunghezze di legame tra gli atomi compresi nel braccio spaziatore. Le lunghezze di legame tra due atomi è ben nota dalla letteratura [CRC Handbook of Chemistry and Physics, 65 edizione, R.C. Weist, CRC press, Ine. Boca Raton, Florida USA pp. F-166-170 (1984)]. The term "length" refers to the predictive measure obtained by adding the bond lengths between the atoms included in the spacer arm. The bond lengths between two atoms are well known from the literature [CRC Handbook of Chemistry and Physics, 65 edition, R.C. Weist, CRC press, Ine. Boca Raton, Florida USA pp. F-166-170 (1984)].
Il braccio spaziatore così definito gioca un ruolo importante nel determinare la struttura tridimensionale e quindi la potenza biologica della porzione responsabile dell’attività inibitoria dello sviluppo del virus HIV e dei retrovirus relativi, oltre che a determinarne la rigidità conformazionale idonea per una maggiore resistenza alle proteasi piasmatiche. The spacer arm thus defined plays an important role in determining the three-dimensional structure and therefore the biological power of the portion responsible for the inhibitory activity of the development of HIV and related retroviruses, as well as determining its conformational rigidity suitable for greater resistance to proteases. piasmatics.
Le molecole oggetto della presente invenzione possono essere egualmente sintetizzate con le varie tecniche riportate nella letteratura; si veda per esempio Schroeder et al. "The Peptides" voi 1 , Academic Press, 1965; Bodanszky et al. "Peptide Synthesis" Interscience Publischer, 1966; Barany & Merrifield, "The peptides; Analysis, Synthesis, Biology", 2, Capitolo 1 , Academic Press, 1980. Queste tecniche includono sintesi peptidica in fase solida, sintesi peptidica in soluzione, metodiche sintetiche di chimica organica, oppure una qualunque combinazione di esse. Lo schema di sintesi prescelto dipenderà dalla composizione della particolare molecola. Di preferenza, essendo le molecole rivendicate interamente su base peptidica, sono utilizzate metodiche sintetiche basate su appropriate combinazioni di tecniche in fase solida e di metodi classici in soluzione, che comportano bassi costi produttivi, in particolare su scala industriale. Nel dettaglio tali metodiche consistono nella: The molecules object of the present invention can be equally synthesized with the various techniques reported in the literature; see for example Schroeder et al. "The Peptides" vol 1, Academic Press, 1965; Bodanszky et al. "Peptide Synthesis" Interscience Publischer, 1966; Barany & Merrifield, "The peptides; Analysis, Synthesis, Biology", 2, Chapter 1, Academic Press, 1980. These techniques include solid phase peptide synthesis, solution peptide synthesis, synthetic organic chemistry methods, or any combination of they. The synthesis scheme chosen will depend on the composition of the particular molecule. Preferably, since the claimed molecules are entirely on a peptide basis, synthetic methods are used based on appropriate combinations of solid phase techniques and classical methods in solution, which involve low production costs, in particular on an industrial scale. In detail, these methods consist of:
i) Sintesi in soluzione di frammenti della catena peptidica attraverso l'accoppiamento successivo di amminoacidi N-protetti, opportunamente attivati, ad un amminoacido o ad una catena peptidica C-protetta, con isolamento degli intermedi, successiva deprotezione selettiva delle estremità N- e C-terminali di detti frammenti ed accoppiamento di essi fino all'ottenimento del peptide desiderato. Infine si esegue la deprotezione selettiva dei gruppi coinvolti nella ciclizzazione, come ad esempio delle estremità N e C terminali e si procede quindi alla loro condensazione. Infine, ove necessario, si deproteggono le catene laterali. i) Solution synthesis of fragments of the peptide chain through the subsequent coupling of N-protected amino acids, suitably activated, to an amino acid or to a C-protected peptide chain, with isolation of the intermediates, subsequent selective deprotection of the N- and C ends -terminals of said fragments and coupling of them until the desired peptide is obtained. Finally, the selective deprotection of the groups involved in the cyclization, such as for example of the N and C terminal ends, is carried out and their condensation is then carried out. Finally, where necessary, the side chains are deprotected.
ii) Sintesi in fase solida della catena peptidica dall'estremità C-terminale verso quella N-terminale su di un supporto polimerico insolubile. Il peptide viene rimosso dalla resina per idrolisi con acido fluoridrico anidro o con acido trifluoroacetico in presenza degli opportuni scavengers. ii) Solid phase synthesis of the peptide chain from the C-terminus towards the N-terminus on an insoluble polymeric support. The peptide is removed from the resin by hydrolysis with anhydrous hydrofluoric acid or trifluoroacetic acid in the presence of the appropriate scavengers.
Nel caso dei peptìdi in cui la ciclizzazione è ottenuta attraverso formazione del ponte disolfuro tra due residui cisteinici, protetti permanentemente alle funzioni N- e C-terminali, la reazione di ossidazione può essère condotta con metodi convenzionali sia in soluzione che in fase solida dopo rimozione dei gruppi protettori delle catene laterali dei residui cisteinici. In the case of peptides in which cyclization is obtained through the formation of the disulfide bridge between two cysteine residues, permanently protected at the N- and C-terminal functions, the oxidation reaction can be carried out with conventional methods both in solution and in solid phase after removal of the protecting groups of the side chains of the cysteine residues.
Per le molecole oggetto della presente invenzione in cui la struttura ciclica è realizzata attraverso la formazione di legami peptidici, la ciclizzazione può sia essere effettuata in soluzione diluita delle estremità N- e C-terminali, preventivamente liberate dei gruppi protettori, oppure direttamente in fase solida mediante l'uso delle metodiche ben note della sintesi peptidica in fase solida. For the molecules object of the present invention in which the cyclic structure is achieved through the formation of peptide bonds, the cyclization can either be carried out in a dilute solution of the N- and C-terminal ends, previously released from the protecting groups, or directly in the solid phase through the use of the well known methods of solid phase peptide synthesis.
L'efficacia delle molecole della presente invenzione di inibire lo sviluppo del virus HIV-1 è stata valutata in vitro in due gruppi di esperimenti. Il primo riguarda il rilascio della proteina virale p55gag da parte di cellule di insetto infettate con Baculovirus ricombinante ÌD. Gheysen et al., Celi, voi. 59, 103-1 12, 1989; A. Gallina et al., Proc. Biotech, 94, AIDS, pag. 89; T. Fitzon et al., Ibid . ; Gederblom et al., Micron. Microsc., 19, 41-60, 1988; Katsumoto et al., Intervirology, 27, 148-153, 1987]; il secondo riguarda l'effetto in coltura dei composti oggetto della presente invenzione nella produzione dell' HIV-1 su linfociti T [Karpas et al. Lancet, 697-699, 1985; Karpas et al., Proc. Acad. Nati. Sci., 89, 8351-8355, 1992]. The efficacy of the molecules of the present invention to inhibit the development of the HIV-1 virus was evaluated in vitro in two groups of experiments. The first concerns the release of the viral protein p55gag by insect cells infected with recombinant Baculovirus ÌD. Gheysen et al., Celi, vol. 59, 103-1 12, 1989; A. Gallina et al., Proc. Biotech, 94, AIDS, p. 89; T. Fitzon et al., Ibid. ; Gederblom et al., Micron. Microsc., 19, 41-60, 1988; Katsumoto et al., Intervirology, 27, 148-153, 1987]; the second relates to the effect in culture of the compounds object of the present invention in the production of HIV-1 on T lymphocytes [Karpas et al. Lancet, 697-699, 1985; Karpas et al., Proc. Acad. Born. Sci., 89, 8351-8355, 1992].
Le molecole oggetto della presente invenzione, a differenza delle molecole note dallo stato della tecnica, non presentano attività immunosoppressiva, come accertato in vitro sulla proliferazione di linfociti umani di sangue periferico e sono strutturalmente molto diverse da composti proposti per la terapia e profilassi dell'AIDS come la ciclosporina o l'FK506. The molecules object of the present invention, unlike the molecules known from the state of the art, do not exhibit immunosuppressive activity, as ascertained in vitro on the proliferation of human peripheral blood lymphocytes and are structurally very different from compounds proposed for the therapy and prophylaxis of AIDS. such as cyclosporine or FK506.
La dose di un peptide di questa invenzione può variare da 0.05 mg/Kg a 250 mg/Kg di peso corporeo del paziente per giorno, in funzione del paziente, della gravità dell'infezione virale e del peptide scelto. La dose opportuna, che può essere facilmente determinata, dovrebbe prevedere fino ad un massimo di 4 dosi giornaliere variabili da 1 a 100 mg di composto attivo per dose. Le dosi possono essere utilizzate in combinazione con composizioni e metodi per il trattamento dell'AIDS. I composti oggetto della presente invenzione sono adatti alla somministrazione a fini terapeutici all'uomo attraverso la via parenterale, subcutanea, topica o nasale, raggiungendo effetti farmacologici in accordo con le proprietà sopra descritte. Per la via orale si possono preparare soluzioni o sospensioni acquose o oleose, emulsioni, sciroppi, elisir o liofilizzati. Applicazioni topiche possono essere effettuate mediante preparazioni sotto forma di gel acquoso, sospensione oleosa o emulsione. Le dosi di principio attivo nelle composizioni suddette possono essere comprese tra 0.1 e 10 mg/kg di peso corporeo. I composti dell'invenzione possono essere somministrati anche nella forma di un "depot injection" o "implant preparation", che possono essere formulati in maniera tale da permettere un rilascio sostanziale di ingrediente attivo. L'ingrediente attivo può essere compresso in pasticche o piccoli cilindri e impiantato subcutaneamente o intramuscolo come "depot injections" o impianti. Gli impianti possono impiegare materiali inerti come polimeri biodegradabili o siliconi sintetici. The dose of a peptide of this invention can vary from 0.05 mg / kg to 250 mg / kg of the patient's body weight per day, depending on the patient, the severity of the viral infection and the selected peptide. The appropriate dose, which can be easily determined, should include up to a maximum of 4 daily doses ranging from 1 to 100 mg of active compound per dose. Doses can be used in conjunction with compositions and methods for treating AIDS. The compounds object of the present invention are suitable for administration to humans for therapeutic purposes through the parenteral, subcutaneous, topical or nasal route, achieving pharmacological effects in accordance with the properties described above. For the oral route, aqueous or oily solutions or suspensions, emulsions, syrups, elixirs or lyophilisates can be prepared. Topical applications can be carried out by means of preparations in the form of aqueous gel, oily suspension or emulsion. The doses of active principle in the above compositions can be comprised between 0.1 and 10 mg / kg of body weight. The compounds of the invention can also be administered in the form of a "depot injection" or "implant preparation", which can be formulated in such a way as to allow a substantial release of active ingredient. The active ingredient can be compressed into tablets or small cylinders and implanted subcutaneously or intramuscularly as "depot injections" or implants. The implants can use inert materials such as biodegradable polymers or synthetic silicones.
I seguenti esempi illustrano ulteriormente l'invenzione e non devono essere interpretati in maniera limitativa dell'ambito di protezione della presente domanda di brevetto. The following examples further illustrate the invention and must not be interpreted as limiting the scope of protection of the present patent application.
ESEMPIO 1 Preparazione del composto di struttura ciclo(Pro-Pro-Tyr-Phe-Leu-lle-lle-Leu-Val) EXAMPLE 1 Preparation of the cycle structure compound (Pro-Pro-Tyr-Phe-Leu-lle-lle-Leu-Val)
Per la sintesi si utilizza un sintetizzatore di peptidi automatico partendo da 0.714 g di Boc-Tyr(OBz)-OCH2-PAM resina (0.70 meq/g) pari a 0.5 mmoli di gruppi amminici. Sono quindi fatti reagire nell’ordine i seguenti amminoacidi: Boc-Pro-OH, Boc-Pro-OH, Boc-Val-OH, Boc-Leu-OH, Boc-lle-OH, Boc-lle-OH, Boc-Leu-OH, Boc-Phe-OH. La resina secca viene posta in un reattore di teflon con 1 mi di anisolo; la miscela viene portata a -50°C e vi vengono distillati 10 mi di acido fluoridrico, quindi viene mantenuta sotto agitazione per 1 h in un bagno di ghiaccio. L'acido fluoridrico viene rimosso con corrente di azoto, il prodotto viene essiccato sotto vuoto per 2 ore, lavato con etere etilico (2 x 15 mi) ed estratto con acido acetico al 50% (3 x 15 mi), filtrato su un imbuto con setto poroso per allontanare la resina esaurita. La soluzione risultante è diluita con acqua e liofilizzata. Il peptide è infine purificato per cromatografia in fase inversa e caratterizzato mediante HPLC analitica con colonna Vydac C18 0.46 x 25 cm con un gradiente lineare in acetonitrile contenente lo 0.1 % (v/v) di acido trifluoroacetico (fase B) contro acido trifluoroacetico acquoso 0.1 % (v/v) (Fase A), dal 5 al 70% in B in 35 min ad un flusso di 1 ml/min, con rivelazione UV a 210 nm. Tempo di ritenzione (Rt) = 24.0 min; purezza cromatografica >99%. La ciclizzazione viene effettuata in soluzione diluita (circa 1 mM) di N ,N-dimetilacetammide utilizzando benzotriazolo-1 -il-oxi-Tris-pirrolidino-fosfonioesafluorofosfato (pyBop) come agente attivante. Dopo circa 24 h il prodotto viene purificato ed analizzato come descritto precedentemente. Tempo di ritenzione Rt = 26.0 min. An automatic peptide synthesizer is used for the synthesis starting from 0.714 g of Boc-Tyr (OBz) -OCH2-PAM resin (0.70 meq / g) equal to 0.5 mmoles of amino groups. The following amino acids are then reacted in order: Boc-Pro-OH, Boc-Pro-OH, Boc-Val-OH, Boc-Leu-OH, Boc-lle-OH, Boc-lle-OH, Boc-Leu -OH, Boc-Phe-OH. The dry resin is placed in a Teflon reactor with 1 ml of anisole; the mixture is brought to -50 ° C and 10 ml of hydrofluoric acid are distilled into it, then it is kept under stirring for 1 h in an ice bath. Hydrofluoric acid is removed with nitrogen stream, the product is dried under vacuum for 2 hours, washed with ethyl ether (2 x 15 ml) and extracted with 50% acetic acid (3 x 15 ml), filtered on a funnel with porous septum to remove the spent resin. The resulting solution is diluted with water and lyophilized. The peptide is finally purified by reverse phase chromatography and characterized by analytical HPLC with a 0.46 x 25 cm Vydac C18 column with a linear gradient in acetonitrile containing 0.1% (v / v) of trifluoroacetic acid (phase B) against 0.1 aqueous trifluoroacetic acid. % (v / v) (Phase A), from 5 to 70% in B in 35 min at a flow of 1 ml / min, with UV detection at 210 nm. Retention time (Rt) = 24.0 min; chromatographic purity> 99%. Cyclization is carried out in a dilute solution (about 1 mM) of N, N-dimethylacetamide using benzotriazole-1 -yl-oxi-Tris-pyrrolidino-phosphoniumhexafluorophosphate (pyBop) as activating agent. After about 24 hours the product is purified and analyzed as previously described. Retention time Rt = 26.0 min.
ESEMPIO 2 Preparazione del composto di struttura ciclo(Pro-Pro-Phe-Phe-Aib-Aib-lle-D-Ala-Val) EXAMPLE 2 Preparation of the cycle structure compound (Pro-Pro-Phe-Phe-Aib-Aib-lle-D-Ala-Val)
Per la sintesi si utilizza un sintetizzatore di peptidi automatico partendo da 0.714 g di Boc-Phe-OCH2-PAM resina (0.70 meq/g) pari a 0.5 mmoli di gruppi amminici. Sono quindi fatti reagire nell'ordine i seguenti amminoacidi: Boc-Pro-OH, Boc-Pro-OH, Boc-Val-OH, Boc-D-Ala-OH, Boc-lle-OH, Boc-Aib-OH, Boc-Aib-OH, Boc-Phe-OH. Dopo distacco dalla resina e successiva purificazione, la ciclizzazione viene effettuata come riportato nell'esempio 1 . Rt = 28.5 min. For the synthesis an automatic peptide synthesizer is used starting from 0.714 g of Boc-Phe-OCH2-PAM resin (0.70 meq / g) equal to 0.5 mmoles of amino groups. The following amino acids are then reacted in order: Boc-Pro-OH, Boc-Pro-OH, Boc-Val-OH, Boc-D-Ala-OH, Boc-lle-OH, Boc-Aib-OH, Boc -Aib-OH, Boc-Phe-OH. After detachment from the resin and subsequent purification, the cyclization is carried out as reported in Example 1. Rt = 28.5 min.
ESEMPIO 3 Preparazione del composto di struttura ciclo(Boc-Cvs-Val-Pro-Pro-Phe-Phe-Cvs-OMe) EXAMPLE 3 Preparation of the cycle structure compound (Boc-Cvs-Val-Pro-Pro-Phe-Phe-Cvs-OMe)
Per la sintesi si adopera la stessa procedura descritta in letteratura [G. Zanotti et al. Biopolymers, 33, 1083-1091 , 1993] For the synthesis the same procedure described in the literature is used [G. Zanotti et al. Biopolymers, 33, 1083-1091, 1993]
ESEMPIO 4 Preparazione del composto di struttura ciclo(Pro-Pro-Phe-Phe)2EXAMPLE 4 Preparation of the cycle structure compound (Pro-Pro-Phe-Phe) 2
Per la sintesi si utilizza la stessa procedura descritta nell'esempio 1 partendo da 0.714 g di Boc-Phe-OCH2-PAM resina (0.70 meq/g) pari a 0.5 mmoli di gruppi amminici. Sono quindi fatti reagire nell'ordine i seguenti amminoacidi: Boc-Pro-OH, Boc-Pro-OH, Boc-Phe-OH, Boc-Phe-OH, Boc-Pro-OH, Boc-Pro-OH, Boc-Phe-OH. For the synthesis the same procedure described in Example 1 is used starting from 0.714 g of Boc-Phe-OCH2-PAM resin (0.70 meq / g) equal to 0.5 mmoles of amino groups. The following amino acids are then reacted in order: Boc-Pro-OH, Boc-Pro-OH, Boc-Phe-OH, Boc-Phe-OH, Boc-Pro-OH, Boc-Pro-OH, Boc-Phe -OH.
Dopo distacco dalla resina e successiva purificazione, la ciclizzazione viene effettuata come riportato nell'esempio 1. Rt = 27.5 min. After detachment from the resin and subsequent purification, the cyclization is carried out as reported in example 1. Rt = 27.5 min.
ESEMPIO 5 Preparazione del composto di struttura ciclo(Pro-Pro-Phe-Phe-Aib-Aib-Val) EXAMPLE 5 Preparation of the cycle structure compound (Pro-Pro-Phe-Phe-Aib-Aib-Val)
Per la sintesi si utilizza la stessa procedura descritta nell'esempio 1 partendo da 0.714 g di Boc-Phe-OCH2-PAM resina 10.70 meq/g) pari a 0.5 mmoli di gruppi amminici. Vengono quindi fatti reagire nell'ordine i seguenti amminoacidi: Boc-Pro-OH, Boc-Pro-OH, Boc-Val-OH, Boc-Aib-OH, Boc-Aib-OH, Boc-Phe-OH. For the synthesis the same procedure described in example 1 is used starting from 0.714 g of Boc-Phe-OCH2-PAM resin 10.70 meq / g) equal to 0.5 mmoles of amino groups. The following amino acids are then reacted in order: Boc-Pro-OH, Boc-Pro-OH, Boc-Val-OH, Boc-Aib-OH, Boc-Aib-OH, Boc-Phe-OH.
Dopo distacco dalla resina e successiva purificazione, la ciclizzazione viene effettuata come riportato nell'esempio 1 . Rt = 27.0 min. After detachment from the resin and subsequent purification, the cyclization is carried out as reported in Example 1. Rt = 27.0 min.
ESEMPIO 6 Valutazione dell'attività anti-HIV EXAMPLE 6 Evaluation of anti-HIV activity
à. Impiego di cellule di insetto Sf9 infettate con Baculovirus ricombinante. to. Use of Sf9 insect cells infected with recombinant Baculovirus.
Si procede preliminarmente al clonaggio del gene gag di HIV-1 in Baculovirus e selezione del Baculovirus ricombinante. Il plasmide pHXB2 contenente l'intero genoma di HIV-1 è stato digerito con Sacl ed Hindi ed il frammento 1852 coppie di basi, corrispondente all'intero gene gag, è stato clonato nel vettore pBS ( /-). Il plasmide risultante è stato quindi ritagliato con EcoRI e BamHl ed il frammento estratto ligato al vettore pVL1392, (pVL1392-GAG) in cui il gene gag si trova ad essere inserito a valle del promotore del gene della poliedrina. Preliminary cloning of the HIV-1 gag gene in Baculovirus and selection of the recombinant Baculovirus. The pHXB2 plasmid containing the entire HIV-1 genome was digested with Sacl and Hindi and the 1852 base pair fragment, corresponding to the entire gag gene, was cloned into the pBS (/ -) vector. The resulting plasmid was then cut with EcoRI and BamHl and the extracted fragment ligated to the vector pVL1392, (pVL1392-GAG) in which the gag gene is inserted downstream of the promoter of the polyhedrine gene.
Per ottenere il virus ricombinante è stato utilizzato un protocollo standard come descritto da Summers & Smith (Manual of methods for Baculovirus vectors and insect celi culture procedures. Text. Agricoltural experimental station, Bullettin, 15551. 3 mg di plasmide pVL1392-gag sono stati mescolati a 100 ng di DNA di Autoorapha californica (AcRP23 lacZ) in presenza di lipofectina ed aggiunti alle cellule Sf9 per 6 ore a 28°C. Il supernatante viene quindi eliminato e sostituito con terreno fresco. Il virus ricombinante viene quindi isolato attraverso un esperimento di "plaque assay". Le cellule Sf9 vengono piastrate in sei pozzetti ad una densità di 1.5x10® e quindi infettate con diverse diluizioni del supernatante ottenuto dalla cotransfezione. Dopo una incubazione di 2 ore il supernatante viene eliminato e le cellule ricoperte con una soluzione contenente il terreno ed agarosio. Le placche ricombinanti riconosciute con microscopio ottico vengono quindi isolate e ripropagate nelle cellule Sf9 e purificate attraverso due ulteriori cicli di "plaque assay". A standard protocol was used to obtain the recombinant virus as described by Summers & Smith (Manual of methods for Baculovirus vectors and insect celi culture procedures. Text. Agricoltural experimental station, Bullettin, 15551. 3 mg of plasmid pVL1392-gag were mixed to 100 ng of DNA of Autoorapha californica (AcRP23 lacZ) in the presence of lipofectin and added to Sf9 cells for 6 hours at 28 ° C. The supernatant is then eliminated and replaced with fresh medium. The recombinant virus is then isolated through an experiment of "plaque assay". Sf9 cells are plated in six wells at a density of 1.5x10® and then infected with different dilutions of the supernatant obtained from cotransfection. After a 2 hour incubation the supernatant is removed and the cells covered with a solution containing the medium and agarose. The recombinant plaques recognized with an optical microscope are then isolated and repropagated in the cells. the Sf9 and purified through two further cycles of "plaque assay".
La presenza del gene eterologo nel virus ricombinante viene saggiata con PCR. Dopo tale verifica viene preparato uno stock virale ad alto titolo per valutare l'effetto dei peptidi di cui agli esempi 1-5 sulla produzione di Virus-Like Particles (VLPS) [D. Gheysen et al., Celi, 59, 103-112,1989; A. Gallina et al,. Proc. Biotech 94, AIDS, pag. 89; T. Fitzon et al., ibid.]. Fiasche di formato T25 con 4 mL di terreno senza siero con cellule ad una concentrazione di 2x10® vengono infettate con lo stock virale ad una molteplicità di infezione di 10-20; dopo due ore viene rimosso il supernatante e sostituito con terreno con siero, Trascorse 20 ore dall'infezione si aggiungono in cinque esperimenti paralleli i peptidi di cui agli esempi 1-5 a due diverse concentrazioni (1 e 10 mg/mL). Dopo 72 ore si raccolgono i supernatanti, si concentrano 10 volte e si caricano su un gradiente preformato di saccarosio (dal 20 al 60%). Il gradiente viene ultracentrifugato con rotore SW per 18 ore a 35Krpm a 20°C. A fine corsa si suddivide il gradiente in frazioni, che vengono esaminate in Western blot. Le cellule infettate in assenza del peptide producono le particelle che sedimentano tra il 40 e 45% di saccarosio, mentre la presenza del peptide inibisce per tutti i composti saggiati la formazione di VLPS ad una concentrazione di 1 mg/mL, The presence of the heterologous gene in the recombinant virus is assayed by PCR. After this verification, a high titer viral stock is prepared to evaluate the effect of the peptides referred to in examples 1-5 on the production of Virus-Like Particles (VLPS) [D. Gheysen et al., Cell, 59, 103-112, 1989; A. Gallina et al ,. Proc. Biotech 94, AIDS, p. 89; T. Fitzon et al., Ibid.]. T25 format flasks with 4 mL of serum-free medium with cells at a concentration of 2x10® are infected with the viral stock at an infection multiplicity of 10-20; after two hours the supernatant is removed and replaced with medium with serum. After 20 hours from the infection, the peptides indicated in examples 1-5 are added in five parallel experiments at two different concentrations (1 and 10 mg / mL). After 72 hours the supernatants are collected, concentrated 10 times and loaded onto a preformed sucrose gradient (20 to 60%). The gradient is ultracentrifuged with a SW rotor for 18 hours at 35Krpm at 20 ° C. At the end of the stroke the gradient is divided into fractions, which are examined in Western blot. The infected cells in the absence of the peptide produce particles that settle between 40 and 45% of sucrose, while the presence of the peptide inhibits the formation of VLPS for all the compounds tested at a concentration of 1 mg / mL,
E' stata successivamente verificata l'inibizione mediata dai cinque peptidi (esempi 1-5) con citofluorimetro (FACscan, Becton Dickinson, San Josè, CA). Cellule Sf9 infettate con virus selvatico, con virus p55 o con virus p55 in presenza del peptide vengono incubate per 30 min a 4°C con un anticorpo anti p24 6BW (Sorin Biomedica, Saluggia, Italy) coniugato con FITC, a varie concentrazioni. Circa il 50% delle cellule infettate con p55 appaiono fluorescenti, mentre l'aggiunta del peptide porta, in tutti i cinque esperimenti, la fluorescenza a livello del controllo con le cellule Sf9 selvatiche. The inhibition mediated by the five peptides (examples 1-5) was subsequently verified with a flow cytometer (FACscan, Becton Dickinson, San Josè, CA). Sf9 cells infected with wild virus, with p55 virus or with p55 virus in the presence of the peptide are incubated for 30 min at 4 ° C with an anti p24 6BW antibody (Sorin Biomedica, Saluggia, Italy) conjugated with FITC, at various concentrations. Approximately 50% of the cells infected with p55 appear fluorescent, while the addition of the peptide brings, in all five experiments, the fluorescence to the level of the control with wild Sf9 cells.
b. Impiego di linfociti periferici (PBL) infettati con HIV-1. b. Use of HIV-1 infected peripheral lymphocytes (PBLs).
PBL sono stati preparati a partire da donatori sani attraverso gradiente Ficoll/Hypaque. Le cellule sono state seminate in una fiasca contenente terreno RPMM 64Q contenente siero fetale bovino e 10% IL-2. Al quarto giorno le cellule sono state raccolte, lavate ed esposte ad un supernatante contenente un isolato clinico di HIV-1 per 4-5 ore, lavate nuovamente, suddivise e coltivate in piastra a 24 pozzetti ad una densità di 1x10® per mL. In cinque esperimenti paralleli, le cellule sono state esposte a varie concentrazioni (1-10 mg) di peptide di cui agli esempi 1-5, operando in duplicato. La produzione dei precursori gag è stata esaminata in immunofluorescenza al citofluorimetro, come in precedenza descritto. Inoltre i diversi supernatanti sono stati esaminati per la capacità di produrre l'antigene p24, utilizzando un test preparato internamente. PBLs were prepared from healthy donors via Ficoll / Hypaque gradient. Cells were seeded in a flask containing RPMM 64Q medium containing fetal bovine serum and 10% IL-2. On the fourth day the cells were harvested, washed and exposed to a supernatant containing a clinical isolate of HIV-1 for 4-5 hours, washed again, divided and cultured in a 24-well plate at a density of 1x10® per mL. In five parallel experiments, the cells were exposed to various concentrations (1-10 mg) of the peptide of Examples 1-5, operating in duplicate. The production of the gag precursors was examined in immunofluorescence with the flow cytometer, as previously described. In addition, the different supernatants were tested for their ability to produce the p24 antigen, using an internally prepared test.
Il test al citofluorimetro ha dimostrato che i peptidi descritti negli esempi 1-5 determinano, come visto per le cellule di insetto infettate con Baculovirus gag ricombinante, un'aumento della fluorescenza sulla membrana ed una parallela diminuzione dei valori di p24 nel supernatantè di coltura. Analogamente si è osservato lo stesso fenomeno utilizzando il farmaco ciclosporina. The cytofluorimeter test showed that the peptides described in Examples 1-5 cause, as seen for the insect cells infected with recombinant Baculovirus gag, an increase in fluorescence on the membrane and a parallel decrease in the p24 values in the culture supernatant. Similarly, the same phenomenon was observed using the drug cyclosporine.
Inoltre, allo scopo di determinare se i peptidi descritti negli esempi 1-5 determinano un abbassamento della capacità infettiva di HIV-1 , sono state utilizzate cellule MOLT-4 con alta espressione del recettore cellulare CD4 per condurre il saggio della formazione dei sincizi. Le cellule MOLT-4 sono state infettate con un ceppo fortemente citotossico di H1V-1 denominato NDK. Quando le cellule infettate con NDK vengono coltivate con cellule MOLT-4 non infettate con un rapporto 1 :100 si formano cellule giganti entro le 24 ore. Il saggio di inibizione mediato dal farmaco è stato condotto come descritto precedentemente [Karpas et al. Lancet, 697-699, 1985]; le cellule bersaglio vengono preincubate a varie concentrazioni prima dell'infezione con le cellule MOLT-4 recanti HIV-1 . Furthermore, in order to determine whether the peptides described in examples 1-5 cause a lowering of the infectious capacity of HIV-1, MOLT-4 cells with high expression of the cellular receptor CD4 were used to conduct the assay for the formation of syncytia. MOLT-4 cells were infected with a strongly cytotoxic strain of H1V-1 called NDK. When NDK-infected cells are cultured with uninfected MOLT-4 cells at a 1: 100 ratio, giant cells are formed within 24 hours. The drug-mediated inhibition assay was conducted as previously described [Karpas et al. Lancet, 697-699, 1985]; target cells are preincubated at various concentrations prior to infection with HIV-1 bearing MOLT-4 cells.
I risultati di questo esperimento hanno dimostrato che l'aggiunta di uno qualsiasi dei peptidi descritti negli esempi 1-5 ad una concentrazione di 20 mg/mL vi era un ridottissimo numero di cellule giganti ( < 1 %) rispetto alle cellule non trattate. The results of this experiment showed that the addition of any of the peptides described in Examples 1-5 at a concentration of 20 mg / mL there was a very small number of giant cells (<1%) compared to the untreated cells.
ESEMPIO 7 Valutazione dell'attività immunosoppressiva EXAMPLE 7 Evaluation of the immunosuppressive activity
Cellule mononucleari da sangue venoso eparinizzato sono state preparate mediante centrifugazione su Ficoll-Hypaque e coltivate come 2 x 10<5 >cellule/piastra in 0.1 mL di RPMI contenente 10% siero fetale bovino, 100 U/mL penicillina e 100 mg/mL streptomicina. Mononuclear cells from heparinized venous blood were prepared by centrifugation on Ficoll-Hypaque and cultured as 2 x 10 <5> cells / plate in 0.1 mL of RPMI containing 10% fetal bovine serum, 100 U / mL penicillin and 100 mg / mL streptomycin .
1-10 mg di peptidi disciolti in DMSO oppure in Tween 80 in etanolo, diluiti nel mezzo di coltura con 0.05 mL di fitoemoagglutinina (2pg/piastra) sono stati aggiunti a ciascuna piastra contenente 2 x 10<5 >cellule in 0.1 mL di mezzo di coltura. Come controllo è stata adoperata una appropriata concentrazione di solvente e di soluzioni di ciclosporina a varie concentrazioni. Le cellule sono state coltivate per 72 h a 37°C in atmosfera umida e dopo 48 h è stato aggiunto 1 μCi [<6-3>H]timidina in 0.05 mL. Nessuna variazione significativa nell'incorporazione di timidina è osservata in presenza dei peptidi descritti negli esempi 1-5. 1-10 mg of peptides dissolved in DMSO or Tween 80 in ethanol, diluted in the culture medium with 0.05 mL of phytohemagglutinin (2pg / plate) were added to each plate containing 2 x 10 <5> cells in 0.1 mL of medium of culture. An appropriate concentration of solvent and cyclosporine solutions at various concentrations was used as a control. The cells were cultured for 72 h at 37 ° C in a humid atmosphere and after 48 h 1 μCi [<6-3> H] thymidine in 0.05 mL was added. No significant change in thymidine incorporation is observed in the presence of the peptides described in Examples 1-5.
La presente invenzione è stata descritta, a titolo illustrativo ma non limitativo, secondo sue forme preferite di realizzazione, ma è da intendersi che variazioni e/o modifiche potranno essere apportate dagli esperti nel ramo senza per questo uscire dal relativo ambito di protezione. The present invention has been described, for illustrative but not limitative purposes, according to its preferred embodiments, but it is to be understood that variations and / or modifications may be made by those skilled in the art without thereby departing from the relative scope of protection.
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| ITRM940405A IT1272295B (en) | 1994-06-22 | 1994-06-22 | "HIV VIRUS DEVELOPMENT INHIBITING COMPOUNDS, THEIR PREPARATION AND USES". |
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| ITRM940405A IT1272295B (en) | 1994-06-22 | 1994-06-22 | "HIV VIRUS DEVELOPMENT INHIBITING COMPOUNDS, THEIR PREPARATION AND USES". |
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| ITRM940405A0 ITRM940405A0 (en) | 1994-06-22 |
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| IT1272295B IT1272295B (en) | 1997-06-16 |
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| ITRM940405A0 (en) | 1994-06-22 |
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