NO176022B - Analogous Procedure for Preparation of Small Peptides That Prevent Binding to T4 Receptors and Act as Immune Organs - Google Patents
Analogous Procedure for Preparation of Small Peptides That Prevent Binding to T4 Receptors and Act as Immune Organs Download PDFInfo
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- NO176022B NO176022B NO880479A NO880479A NO176022B NO 176022 B NO176022 B NO 176022B NO 880479 A NO880479 A NO 880479A NO 880479 A NO880479 A NO 880479A NO 176022 B NO176022 B NO 176022B
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- peptide
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- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70514—CD4
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
- C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
- C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Description
Oppfinnelsen angår en analogifremgangsmåte til fremstilling av et terapeutisk aktivt peptid med formelen The invention relates to an analogue method for the preparation of a therapeutically active peptide with the formula
R<1> - R2 - R3 - R4 - R5 - R6 - R7 - Tyr - R<8> - R9 (I) hvor R<1> er en aminoterminalrest Cys eller er fraværende, R<1> - R2 - R3 - R4 - R5 - R6 - R7 - Tyr - R<8> - R9 (I) where R<1> is an amino terminal residue Cys or is absent,
R<2> er Ala eller D-Ala eller en aminoterminalrest Ala eller R<2> is Ala or D-Ala or an amino terminal residue Ala or
D-Ala eller fraværende, D-Ala or absent,
R3 er Ser eller fraværende, R3 is Ser or absent,
R<4> er Thr eller en aminoterminalrest Cys eller fraværende, R<5> er Thr, Ser, Asn eller aminoterminalrest Thr, Ser, Asn, R<6> er Thr eller Ser, R<4> is Thr or an amino terminal residue Cys or absent, R<5> is Thr, Ser, Asn or amino terminal residue Thr, Ser, Asn, R<6> is Thr or Ser,
R7 er Thr, Ser, Arg eller Asn, R7 is Thr, Ser, Arg or Asn,
R<8> er en Thr, Arg, Gly eller Ser eller karboksyterminalrest R<8> is a Thr, Arg, Gly or Ser or carboxy terminal residue
Thr, Arg, Gly, Ser eller Thr-amid eller fraværende, Thr, Arg, Gly, Ser or Thr amide or absent,
R<9> er en karboksyterminalrest Cys eller fraværende, eller et fysiologisk akseptabelt salt derav. R<9> is a carboxy terminal residue Cys or absent, or a physiologically acceptable salt thereof.
Syntetisk produserte korte peptidsekvenser hindrer binding av HTLV-III/LAV (heretter betegnet som HIV) til humane celler ved blokkering av reseptorsetene på celleflaten for således å hindre viral infektivitet av human T-celle. Peptidene, skjønt de hindrer infektivitet, induserer også antistoffproduksjon mot HIV-virusets kapselprotein (envelope protein). Følgelig har disse peptider også anvendelse som vaksiner for å hindre utvikling av Acquired Immune Deficiency Syndrome (AIDS). Monoklonale antistoffer for peptidene kan også anvendes som diagno-semidler for å identifisere HIV-viruset. Følgelig har peptider og antistoffer for peptidene anvendelse i fremstilling av testpakker for identifisering av HIV-bærere eller personer som lider av AIDS. Synthetically produced short peptide sequences prevent binding of HTLV-III/LAV (hereafter referred to as HIV) to human cells by blocking the receptor sites on the cell surface, thus preventing viral infectivity of human T cells. The peptides, although they prevent infectivity, also induce antibody production against the envelope protein of the HIV virus. Consequently, these peptides also have application as vaccines to prevent the development of Acquired Immune Deficiency Syndrome (AIDS). Monoclonal antibodies for the peptides can also be used as diagnostic agents to identify the HIV virus. Accordingly, peptides and antibodies to the peptides have applications in the manufacture of test kits for the identification of HIV carriers or persons suffering from AIDS.
Den fullstendige nukleotidsekvens for AIDS (HIV)-virus er blitt rapportert av flere forskere. (Se Lee Råtner et al., Nature 313, p. 277, januar 1985; Muesing et al., Nature, 313, p. 450, februar 1985; og Wain-Habson et al., Cell 40, pp. 9-17, januar 1985). Kapselgenet er blitt særlig forbundet med antigenisitet og infektivitet. Man vet imidlertid at kapselpartiet også har områder som er meget forskjellige. HIV-virus kapsel-glykoprotein er vist å feste seg kovalent til hjernemembranene hos mennesker, rotter og aper og til celler i immunsystemet. The complete nucleotide sequence of the AIDS (HIV) virus has been reported by several researchers. (See Lee Råtner et al., Nature 313, p. 277, January 1985; Muesing et al., Nature, 313, p. 450, February 1985; and Wain-Habson et al., Cell 40, pp. 9-17 , January 1985). The capsule gene has been particularly associated with antigenicity and infectivity. However, it is known that the capsule part also has areas that are very different. HIV virus capsular glycoprotein has been shown to attach covalently to the brain membranes of humans, rats and monkeys and to cells of the immune system.
Den forståelse at vira kan utøve celle- og vevstropisme ved festing til meget spesifikke seter på cellemembranreseptorer har oppmuntret forskere til å lete etter midler som binder seg på reseptorsetene for viruset i cellemembraner og således hindrer binding av det spesielle virus til disse celler. En demonstra-sjon på at spesifikk reseptorformidlet infektivitet av kukoppe-virus blokkeres av syntetiske peptider er tidligere blitt gitt (Epstein et al., Nature 318, pp. 663-667). The understanding that viruses can exert cell and tissue tropism by attaching to very specific sites on cell membrane receptors has encouraged researchers to look for agents that bind to the receptor sites for the virus in cell membranes and thus prevent binding of the particular virus to these cells. A demonstration that specific receptor-mediated infectivity of cowpox virus is blocked by synthetic peptides has previously been given (Epstein et al., Nature 318, pp. 663-667).
HIV-viruset er blitt vist å binde seg til et overflatemolekyl kjent som CD4- eller T4-regionen som foreligger på forskjellige celler som er mottagelige for HIV-infeksjon, innbefattet T-lymfocytter og makrofager. (Se Shaw et al., Science 226, pp. 1165-1171 for en drøftelse av tropisme av HTLV-III). The HIV virus has been shown to bind to a surface molecule known as the CD4 or T4 region present on various cells susceptible to HIV infection, including T lymphocytes and macrophages. (See Shaw et al., Science 226, pp. 1165-1171 for a discussion of tropism of HTLV-III).
Foruten de symptomer som oppstår fra immunsvikt, oppviser pasienter med AIDS neurofysiologiske defekter. Sentralnerve- og immunsystemene har felles et stort antall spesifikke celleoverflate-gjenkjennelsesmolekyler som tjener som reseptorer for neuropeptid-formidlet intercellulær kommunikasjon. Neuropep-tidene og deres reseptorer oppviser utstrakt evolusjonær stabilitet, idet de er godt bevart i stort sett uforandret form i encellede organismer såvel som i høyerestående dyr. Videre oppviser sentralnerve- og immunsystemene felles DC4 (T4) celleoverflate-gjenkjennelsesmolekyler som tjener som reseptorer for bindingen av HIV-kapselglykoprotein (gp 120). Da de samme godt bevarte neuropeptid-informasjonssubstanser integrerer immun- og hjernefunksjonen gjennom reseptorer som er bemerkel-sesverdig like dem for HIV, har oppfinnerne postulert at en meget lik aminosyresekvens mellom HIV-glykoproteinet gp 120 og et kort peptid tidligere identifisert i en annen sammenheng fra kapselregionen av Epstein-Barr-viruset kan indikere det kjerne-peptid som er essensielt for viral reseptorbinding. Det ble postulert at et slikt peptid kunne være nyttig for å hindre infeksjon av celler med HIV ved binding med reseptorceller og blokkering av bindingen av HIV gp 120, at slike peptider som binder seg til reseptorsetene ville gi opphav til produksjonen av antistoffer rettet på peptidsekvensen, og at disse peptider kan benyttes til å skaffe immunologisk basis for prevensjon av In addition to the symptoms arising from immunodeficiency, patients with AIDS exhibit neurophysiological defects. The central nervous and immune systems share a large number of specific cell surface recognition molecules that serve as receptors for neuropeptide-mediated intercellular communication. The neuropeptides and their receptors exhibit extensive evolutionary stability, being well conserved in largely unchanged form in unicellular organisms as well as in higher animals. Furthermore, the central nervous and immune systems share common DC4 (T4) cell surface recognition molecules that serve as receptors for the binding of HIV capsular glycoprotein (gp 120). Since the same well-conserved neuropeptide information substances integrate immune and brain function through receptors remarkably similar to those of HIV, the inventors have postulated that a very similar amino acid sequence between the HIV glycoprotein gp 120 and a short peptide previously identified in a different context from capsular region of the Epstein-Barr virus may indicate the core peptide essential for viral receptor binding. It was postulated that such a peptide could be useful in preventing infection of cells with HIV by binding to receptor cells and blocking the binding of HIV gp 120, that such peptides binding to the receptor sites would give rise to the production of antibodies directed at the peptide sequence, and that these peptides can be used to provide an immunological basis for contraception
AIDS. AIDS.
Det er en hensikt med den foreliggende oppfinnelse å skaffe en analogifremgangsmåte til fremstilling av peptider som vil tjene til å mildne symptomer på AIDS ved å hindre binding av HIV (AIDS-virus) til reseptorseter i celler av hjernemembraner og immunsystemet. It is a purpose of the present invention to provide an analogue method for the production of peptides which will serve to alleviate symptoms of AIDS by preventing the binding of HIV (AIDS virus) to receptor sites in cells of brain membranes and the immune system.
Det er også en hensikt med oppfinnelsen å skaffe, ved analogifremgangsmåten, peptider til bruk som vaksiner som kan benyttes til å gi opphav til antistoffer som vil beskytte mot utviklingen av AIDS i personer som kan bli utsatt for HIV (AIDS-virus). It is also a purpose of the invention to obtain, by the analogical method, peptides for use as vaccines which can be used to give rise to antibodies which will protect against the development of AIDS in persons who may be exposed to HIV (AIDS virus).
Det er ytterligere en hensikt med oppfinnelsen å skaffe, på samme måte, diagnostiske midler til identifisering av nærværet av antistoffer for HIV- eller HIV-kapselprotein. It is a further object of the invention to provide, in the same way, diagnostic means for identifying the presence of antibodies to HIV or HIV capsular protein.
Disse hensikter oppnås ved en analogifremgangsmåte som angitt i patentkravet. These purposes are achieved by an analogous method as stated in the patent claim.
Detaljert beskrivelse av oppfinnelsen. Detailed description of the invention.
Et oktapeptid i HIV-kapselglykoproteinet (gp 120) ble identifisert ved datamaskinassistert analyse. Dette peptid, kalt "peptid T" på grunn av sitt høye threonin-innhold, er vist å hindre binding av gp 120 til hjernemembranene. Peptidet har sekvensen Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr. Senere analyse avdekket en klasse beslektede pentapeptider med lignende bindingsegenskaper. An octapeptide in the HIV capsular glycoprotein (gp 120) was identified by computer-assisted analysis. This peptide, called "peptide T" because of its high threonine content, has been shown to prevent binding of gp 120 to the brain membranes. The peptide has the sequence Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr. Later analysis revealed a class of related pentapeptides with similar binding properties.
I henhold til en første side ved den foreliggende oppfinnelse er der skaffet et peptid med formelen (I): hvor Ra er en aminoterminalrest Ala- eller D-Ala og R<b> er en karboksyterminalrest -Thr eller -Thr-amid eller et derivat derav med en ytterligere Cys-rest ved én eller begge amino- og karboksyterminalene, eller et peptid med formelen (II): According to a first aspect of the present invention, a peptide of the formula (I) has been obtained: where Ra is an amino-terminal residue Ala- or D-Ala and R<b> is a carboxy-terminal residue -Thr or -Thr-amide or a derivative thereof with an additional Cys residue at one or both of the amino and carboxy termini, or a peptide of the formula (II):
hvor R"<1> er en aminoterminalrest Thr-, Ser- eller Asn-, where R"<1> is an amino terminal residue Thr-, Ser- or Asn-,
R2 er Thr eller Ser, R 2 is Thr or Ser,
R3 er Thr, Ser, Asn eller Arg, R 3 is Thr, Ser, Asn or Arg,
R<4> er Tyr, R<4> is Taurus,
og R<5> er fortrinnsvis en karboksyterminalrest -Thr, -Arg eller - Gly eller et Thr-amid. Skjønt de foretrukne aminosyrer ved R5 • begge er angitt, er det kjent at aminosyren i denne posisjon kan variere meget. Det er faktisk mulig å avslutte peptidet med R<4 >(tyrosin) som karboksyterminal-aminosyren, idet R<5> er fraværende. Slike peptider beholder bindingsegenskapene av de grupper som her er omtalt. Serin og threonin synes å være ut-skiftbare for de biologiske formål som her er omtalt. De aktive forbindelser ifølge oppfinnelsen kan eksistere som fysiologisk akseptable salter av peptidene. and R<5> is preferably a carboxy terminal residue -Thr, -Arg or -Gly or a Thr amide. Although the preferred amino acids at R5 • are both indicated, it is known that the amino acid in this position can vary greatly. It is actually possible to terminate the peptide with R<4 >(tyrosine) as the carboxy-terminal amino acid, R<5> being absent. Such peptides retain the binding properties of the groups discussed here. Serine and threonine seem to be interchangeable for the biological purposes discussed here. The active compounds according to the invention can exist as physiologically acceptable salts of the peptides.
De mest foretrukne peptider, såvel som peptid T ovenfor, er de følgende oktapeptider med formelen (I): The most preferred peptides, as well as peptide T above, are the following octapeptides of formula (I):
og og de følgende'pentapeptider med formelen (II): and and the following pentapeptides of the formula (II):
eventuelt med et amidderivat ved karboksyterminalen. optionally with an amide derivative at the carboxy terminus.
Forbindelsene fremstilt ved analogifremgangsmåten ifølge oppfinnelsen kan fordelaktig modifiseres ved de fremgangsmåter som er kjent for å fremme passasje av molekyler gjennom blod/hjerne-barrieren. Acetylering har vist seg å være spesielt nyttig for å fremme bindingsaktiviteten av peptidet. De terminale amino- og karboksyseter er særlig foretrukne seter for modifikasjon. The compounds produced by the analogue method according to the invention can advantageously be modified by the methods known to promote the passage of molecules through the blood/brain barrier. Acetylation has been shown to be particularly useful in promoting the binding activity of the peptide. The terminal amino and carboxyl sites are particularly preferred sites for modification.
Peptidene kan også modifiseres i en begrensende struktur som skaffer øket stabilitet og oral tilgjengelighet. The peptides can also be modified in a limiting structure that provides increased stability and oral availability.
De følgende forkortelser er heretter brukt: The following abbreviations are used hereafter:
Med mindre noe annet er angitt, foreligger aminosyrene selvsagt i den naturlige form av L-stereoisomerer. Unless otherwise stated, the amino acids naturally exist in the natural form of L-stereoisomers.
En sammenligning av aminosyresekvenser for 12 pentapeptider er vist i tabell 1. Skjønt den opprinnelige datamaskinundersøkelse historisk viste at peptid T (inneholdes i ARV-isolatet) var den relevante molekyldel, ble det etter hvert som ytterligere virussekvenser ble tilgjengelige klart at den relevante bio-aktive sekvens kunne være et kortere pentapeptid omfattende, nominelt, peptid T t4-8] eller sekvensen TTNYT. I isolater som ble sammenlignet (tabell 1), ble betydelige homologier oppdaget bare i denne kortere region. Mesteparten av forandringene er interomsetninger av serin (S) og threonin (T), to nært beslektede aminosyrer. Tyrosinet i posisjon 7 på peptid T er et invariant trekk i alle disse konstruerte stykker som indikerer at det kan være obligatorisk for bioaktivitet. Substitusjoner som finner sted ved posisjon 5 omfatter T, G, R eller S. Posisjon 4 og 6 ble først begrenset (med ett unntak) til S, T og N, som alle er aminosyrer inneholdende uladede polare grupper med nært beslektede steriske egenskaper. En vurdering av generell sekvensoverensstemmelse blant 5 forskjellige AIDS-virusisolater viser at regionen rundt og omfattende peptid-T-sekvensen er et meget variabelt område. Slik variabili-tet kan antyde spesialisering gjennom sterk selektiv variasjon av den eller de funksjoner som kan defineres ved dette locus. På samme måte som opiatpeptidene synes disse peptid-T-analoger å eksistere i multiple former som minner om met- og leu-enkefalin. Disse pentapeptidsekvenser representert i disse forskjellige AIDS-virusisolater er biologisk aktive og i stand til å inter-reagere som agonister av CD4-reseptoren - tidligere kjent stort sett som en overflate-"markør" av T-hjelpeceller. A comparison of amino acid sequences for 12 pentapeptides is shown in Table 1. Although the original computer investigation historically showed that peptide T (contained in the ARV isolate) was the relevant molecular moiety, as additional virus sequences became available it became clear that the relevant bio-active sequence could be a shorter pentapeptide comprising, nominally, peptide T t4-8] or the sequence TTNYT. In isolates compared (Table 1), significant homologies were detected only in this shorter region. Most of the changes are interconversions of serine (S) and threonine (T), two closely related amino acids. The tyrosine at position 7 of peptide T is an invariant feature in all these engineered fragments indicating that it may be mandatory for bioactivity. Substitutions occurring at position 5 include T, G, R or S. Positions 4 and 6 were initially restricted (with one exception) to S, T and N, all of which are amino acids containing uncharged polar groups with closely related steric properties. An assessment of general sequence agreement among 5 different AIDS virus isolates shows that the region surrounding and including the peptide T sequence is a highly variable area. Such variability may suggest specialization through strong selective variation of the function(s) that can be defined at this locus. Like the opiate peptides, these peptide T analogs appear to exist in multiple forms reminiscent of met- and leu-enkephalin. These pentapeptide sequences represented in these different AIDS virus isolates are biologically active and capable of inter-reacting as agonists of the CD4 receptor - previously known largely as a surface "marker" of T helper cells.
Peptidet med syv aminosyrer Cys-Thr-Thr-Asn-Tyr-Thr-Cys er også aktivt. Tilsetning av cysteiner til en kjerne virker ikke ugunstig inn på aktivitet. The peptide of seven amino acids Cys-Thr-Thr-Asn-Tyr-Thr-Cys is also active. Addition of cysteines to a core does not adversely affect activity.
Peptidene ble syntetisert på bestilling (custom synthesized) av The peptides were custom synthesized by
Peninsula Laboratories under en konfidensiell avtale mellom oppfinnerne og produsenten. Merrifields metode for fastfase-peptidsyntese ble benyttet. (Se US-PS 3.531.258 her innlemmet som referanse.) De syntetiserte peptider er særlig foretrukket. Skjønt peptid T og pentapeptidet som er en del derav kunne isoleres fra viruset, var de peptider som ble fremstilt i henhold til Merrifields metode frie for virus- og celleavfall. Følgelig finner der ikke sted noen uheldige reaksjoner med forurensende materialer når de syntetiserte peptider benyttes. Peninsula Laboratories under a confidentiality agreement between the inventors and the manufacturer. Merrifield's method for solid phase peptide synthesis was used. (See US-PS 3,531,258 herein incorporated by reference.) The synthesized peptides are particularly preferred. Although peptide T and the pentapeptide which is part of it could be isolated from the virus, the peptides produced according to Merrifield's method were free of virus and cell debris. Consequently, no adverse reactions with polluting materials take place when the synthesized peptides are used.
Peptidene ifølge oppfinnelsen kan fremstilles ved vanlige fremgangsmåter for peptidsyntese. Både fastfase- og væskefase-metoder kan benyttes. Det er funnet at Merrifields fastfase-metode er særlig passende. I denne fremgangsmåte blir peptidet syntetisert på en trinnvis måte mens karboksyenden av kjeden er kovalent festet til den uoppløselige bærer. Under de mellomlig-gende syntetiske trinn forblir peptidet i den faste fase og kan derfor lett manipuleres. Den faste bærer er en klormetylert styrendivinylbenzen-kopolymer. The peptides according to the invention can be prepared by usual methods for peptide synthesis. Both solid-phase and liquid-phase methods can be used. Merrifield's solid phase method has been found to be particularly suitable. In this method, the peptide is synthesized in a stepwise manner while the carboxy end of the chain is covalently attached to the insoluble support. During the intermediate synthetic steps, the peptide remains in the solid phase and can therefore be easily manipulated. The solid support is a chloromethylated styrenedivinylbenzene copolymer.
En N-beskyttet form av karboksyterminalaminosyren, f.eks. en t-butoksykarbonyl-beskyttet (Boe-) aminosyre omsettes med klor-metylresten av den klormetylerte styrendivinylbenzen-kopolymer-harpiks for å produsere et beskyttet aminoacyl-derivat av harpiksen, hvor aminosyren er koblet til harpiks-benzylesteren. Denne blir de-beskyttet og omsatt med en beskyttet form av den neste ønskede aminosyre, for således å produsere et beskyttet dipeptid som er festet til harpiksen. Aminosyren vil generelt benyttes i aktivert form, f.eks. ved bruk av et karbodiimid eller aktiv ester. Denne sekvens gjentas og peptidkjeden vokser med en rest ad gangen ved kondensasjon ved aminoenden med de ønskede N-beskyttede aminosyrer til det ønskede peptid er blitt bygget opp på harpiksen. Peptidharpiksen behandles deretter med en vannfri hydrofluorsyre for å kløyve esteren som binder det oppbygde peptid til harpiksen for å frigjøre det ønskede peptid. Funksjonelle sidekjedegrupper av aminosyrer som må blokkeres under den syntetiske fremgangsmåte ved bruk av vanlige metoder kan også fjernes på samme tid. Syntese av et peptid med en aminogruppe på dets karboksyterminal kan utføres på vanlig måte An N-protected form of the carboxy-terminal amino acid, e.g. a t-butoxycarbonyl protected (Boe) amino acid is reacted with the chloromethyl residue of the chloromethylated styrenedivinylbenzene copolymer resin to produce a protected aminoacyl derivative of the resin, where the amino acid is linked to the resin benzyl ester. This is deprotected and reacted with a protected form of the next desired amino acid, thus producing a protected dipeptide which is attached to the resin. The amino acid will generally be used in activated form, e.g. using a carbodiimide or active ester. This sequence is repeated and the peptide chain grows one residue at a time by condensation at the amino end with the desired N-protected amino acids until the desired peptide has been built up on the resin. The peptide resin is then treated with an anhydrous hydrofluoric acid to cleave the ester that binds the assembled peptide to the resin to release the desired peptide. Functional side chain groups of amino acids that must be blocked during the synthetic process using conventional methods can also be removed at the same time. Synthesis of a peptide with an amino group at its carboxy terminus can be carried out in a conventional manner
ved bruk av en 4-metylbenzhydrylamin-harpiks. using a 4-methylbenzhydrylamine resin.
Forbindelsene ifølge oppfinnelsen ble funnet å effektivt blokkere cellers reseptorseter og å hindre celleinfektivitet med HIV (AIDS-virus) i ape, rotte og humane hjernemembraner og celler i immunsystemet. The compounds of the invention were found to effectively block cell receptor sites and to prevent cell infectivity with HIV (AIDS virus) in monkey, rat and human brain membranes and cells of the immune system.
Forbindelsene fremstilt ved analogifremgangsmåten ifølge oppfinnelsen kan f.eks. anvendes i en farmasøytisk blanding som omfatter en slik forbindelse i tilslutning til en farmasøytisk aksep-tabel bærer eller eksipiens tilpasset til bruk i human eller veterinær medisin. Slike blandinger kan frembys for bruk på vanlig måte i blanding med én eller flere fysiologisk akseptable bærere eller eksipienser. Blandingene kan valgfritt videre inneholde ett eller flere andre terapeutiske midler som om ønskelig kan være et forskjellig antivirusmiddel. The compounds produced by the analog method according to the invention can e.g. is used in a pharmaceutical mixture comprising such a compound in addition to a pharmaceutically acceptable carrier or excipient adapted for use in human or veterinary medicine. Such mixtures can be presented for use in the usual way in admixture with one or more physiologically acceptable carriers or excipients. The mixtures can optionally further contain one or more other therapeutic agents which, if desired, can be a different antiviral agent.
Peptidene i henhold til oppfinnelsen kan således formuleres for oral tilførsel, tilførsel via kinn eller hulrom (buccal), parenteral, topisk eller rektal tilførsel. The peptides according to the invention can thus be formulated for oral administration, administration via the cheek or cavity (buccal), parenteral, topical or rectal administration.
Nærmere bestemt kan peptidene fremstilt ved analogifremgangsmåten i henhold til oppfinnelsen formuleres for injisering eller infusjon og kan gis i enhetdoseform i ampuller eller i multi-dose-beholdere med et tilsatt preservativ. Blandingene kan anta slike former som suspensjoner, oppløsninger eller emulsjoner i oljeholdige eller vandige vehikler, og kan inneholde formu-leringsmidler såsom suspensjonsmidler, stabiliseringsmidler og/eller dispergeringsmidler. Alternativt kan den aktive ingre-diens foreligge i pulverform for sammensetning med en egnet vehikel, f.eks. sterilt, pyrogenfritt vann før bruk. More specifically, the peptides produced by the analog method according to the invention can be formulated for injection or infusion and can be given in unit dose form in ampoules or in multi-dose containers with an added preservative. The mixtures can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulating agents such as suspending agents, stabilizing agents and/or dispersing agents. Alternatively, the active ingredient can be in powder form for composition with a suitable vehicle, e.g. sterile, pyrogen-free water before use.
Slike farmasøytiske blandinger kan også inneholde andre aktive ingredienser såsom antimikrobielle midler eller preservativer. Such pharmaceutical mixtures may also contain other active ingredients such as antimicrobial agents or preservatives.
Blandingene kan inneholde 0,001-99% av det aktive materiale. The mixtures can contain 0.001-99% of the active material.
For tilførsel ved injeksjon eller infusjon, vil den daglige dose som anvendes for behandling av et voksent menneske på ca. 70 kg kroppsvekt ligge i området fra 0,2 mg til 10 mg, fortrinnsvis fra 0,5 til 5 mg, som kan gis i 1-4 doser, f.eks., avhengig av tilførselsveien og pasientens tilstand. For administration by injection or infusion, the daily dose used to treat an adult person of approx. 70 kg body weight be in the range from 0.2 mg to 10 mg, preferably from 0.5 to 5 mg, which can be given in 1-4 doses, for example, depending on the route of administration and the condition of the patient.
Det ble postulert at affinitetskonstantene ligner på dem for morfin. På grunnlag av denne affinitet, ble doseringer på 0,33-0,0003 mg/kg pr. dag foreslått. Dette har vist seg å være effektivt. En blodkonsentrasjon på 10~*> - 10"^ molar blodkonsentrasjon er foreslått. Hos aper oppnår man ved 3 mg/kg pr. dag en serumkonsentrasjon på 150 x 10"^ M. Denne konsentrasjon er 15 ganger større enn nødvendig for oppnåelse av en konsentrasjon på 10-<8> M. Primater krever generelt 10 ganger så stor dose som den som benyttes i mennesker. It was postulated that the affinity constants are similar to those for morphine. On the basis of this affinity, dosages of 0.33-0.0003 mg/kg per day suggested. This has proven to be effective. A blood concentration of 10~*> - 10"^ molar blood concentration has been proposed. In monkeys, at 3 mg/kg per day, a serum concentration of 150 x 10"^ M is achieved. This concentration is 15 times greater than necessary to achieve a concentration of 10-<8> M. Primates generally require 10 times the dose used in humans.
Vaksinepreparater inneholdende et peptid i henhold til oppfinnelsen skaffer beskyttelse mot infeksjon av AIDS-virus. Vaksinen vil inneholde en virksom immunogen mengde peptid, f.eks. lug-20 mg/kg regnet på vertens vekt, eventuelt konjugert til et protein såsom humanserumalbumin, i en passende vehikel, f.eks. sterilt vann, saline eller buffret saline. Hjelpmidler kan anvendes, f.eks. aluminiumhydroksid-gel. Tilførselen kan være ved injeksjon, f.eks. intramuskulært, intreperitonealt, subkutanøst eller intravenøst. Tilførselen kan finne sted en gang eller ved en rekke forskjellige tidspunkter, f.eks. ved 1-4 ukers mellomrom. Vaccine preparations containing a peptide according to the invention provide protection against infection by the AIDS virus. The vaccine will contain an effective immunogenic amount of peptide, e.g. lug-20 mg/kg based on the host's weight, optionally conjugated to a protein such as human serum albumin, in a suitable vehicle, e.g. sterile water, saline or buffered saline. Aids can be used, e.g. aluminum hydroxide gel. The supply can be by injection, e.g. intramuscularly, intraperitoneally, subcutaneously or intravenously. The supply can take place once or at a number of different times, e.g. at 1-4 week intervals.
Antigensekvenser fra krabbe samt proteiner fra andre virvelløse dyr kan også settes til peptidene fremstilt ved analogifremgangsmåten ifølge oppfinnelsen for å fremme antigenisitet. Antigen sequences from crab as well as proteins from other invertebrates can also be added to the peptides produced by the analog method according to the invention to promote antigenicity.
Nok en anvendelse av peptidet angår testpakker for påvisning av AIDS-viruset og antistoffer for AIDS-viruset inneholdende et peptid fremstilt i henhold til oppfinnelsen som en kilde til antigen, eller et monoklonalt antistoff frembragt ved et peptid fremstilt i henhold til oppfinnelsen. F.eks. kan et slikt peptid benyttes i en testpakke for å påvise AIDS-infeksjon og diagnos-tisere AIDS og pre-AIDS-betingelser ved bruk av det som test-reagensen i en enzymkoblet immunosorbent-analyse (ELISA) , eller en enzym-immunpunktsanalyse (enzyme immunodot assay). Slike testpakker kan innbefatte en uoppløselig porøs overflate Another application of the peptide concerns test kits for detecting the AIDS virus and antibodies for the AIDS virus containing a peptide produced according to the invention as a source of antigen, or a monoclonal antibody produced by a peptide produced according to the invention. E.g. can such a peptide be used in a test kit to detect AIDS infection and diagnose AIDS and pre-AIDS conditions by using it as the test reagent in an enzyme-linked immunosorbent assay (ELISA), or an enzyme-immunospot assay (enzyme immunodot assay). Such test packs may include an insoluble porous surface
eller fast substrat, til hvilket det antigene peptid eller monoklonale antistoff er blitt forabsorbert eller kovalent bundet, idet en slik overflate eller et substrat fortrinnsvis er i form av mikrotiterplater eller -brønner, testsera, heteroanti-sera som spesielt binder seg til og metter det antigen eller antistoff som absorberes på overflaten eller bæreren, forskjellige fortynningsmidler og buffere, merkede konjugater for påvisning av spesielt bundne antistoffer eller andre signalgene-rerende reagenser såsom enzymsubstrater, kofaktorer og kromo-gener. or solid substrate, to which the antigenic peptide or monoclonal antibody has been preabsorbed or covalently bound, such a surface or a substrate preferably being in the form of microtiter plates or wells, test sera, heteroantisera which specifically bind to and saturate the antigen or antibody absorbed on the surface or support, various diluents and buffers, labeled conjugates for the detection of specifically bound antibodies or other signal-generating reagents such as enzyme substrates, cofactors and chromogens.
Peptidet kan benyttes som et immunogen for å frembringe monoklonale antistoffer som spesielt binder seg til det aktuelle parti av kapselsekvensen i AIDS-viruset ved bruk av vanlige teknikker. The peptide can be used as an immunogen to produce monoclonal antibodies that specifically bind to the relevant part of the capsule sequence in the AIDS virus using common techniques.
EKSPERIMENTELLE METODER OG DATA EXPERIMENTAL METHODS AND DATA
Radioaktiv merking av gp 120, preparering av hjernemembraner, binding og tverrbinding av gp 120 til reseptor og immunoutfelling av T4- antigen Radioactive labeling of gp 120, preparation of brain membranes, binding and cross-linking of gp 120 to receptor and immunoprecipitation of T4 antigen
HTLV-IIIb av HIV ble formert i H9-celler, og gp 120 ble isolert ved immunoaffinitetskromatografi og preparativ NaDodSO^PAGE. HTLV-IIIb of HIV was propagated in H9 cells, and gp 120 was isolated by immunoaffinity chromatography and preparative NaDodSO^PAGE.
125 125
Renset gp 120 ble merket med I ved kloramin-T-metoden. Purified gp 120 was labeled with I by the chloramine-T method.
Frisk hippokampus fra menneske, ape og rotte ble hurtig homoge-nisert (Polytron, Brinkmann Instruments) i 100 volumer iskald 50 mM Hepes (pH 7,4). Membranene som ble samlet opp ved sentri-fugering (15 000 x g) ble vasket i det opprinnelige buffervolum og ble brukt friske eller lagret ved -70°C. Før bruk ble hjernemembranene og meget høyt rensede T-celler (ref. 16; gave fra Larry Wahl) preinkubert i 15-30 minutter i fosfatbuffret saline (PBS). Membraner som ble oppnådd fra 20 mg (opprinnelig våtvekt) hjerne (ca. 100 ug protein) ble inkubert med 28 000 cpm av <125>I-gp 120 i 1 time ved 37°C i 200 1 (endelig volum) av 50 mM Hepes inneholdende 0,1% bovinserumalbumin og peptidaseinhibito-rene bacitracin (0,005%), aprotinin (0,005%), leupeptin (0,001%) og chymostatin (0,001%). Inkubasjonene ble hurtig vakuumfiltrert og tellet for å bestemme det reseptorbundne materiale. Fresh hippocampus from humans, monkeys and rats was quickly homogenized (Polytron, Brinkmann Instruments) in 100 volumes of ice-cold 50 mM Hepes (pH 7.4). The membranes collected by centrifugation (15,000 x g) were washed in the original buffer volume and used fresh or stored at -70°C. Before use, the brain membranes and highly purified T cells (ref. 16; gift from Larry Wahl) were preincubated for 15-30 minutes in phosphate-buffered saline (PBS). Membranes obtained from 20 mg (initial wet weight) brain (approximately 100 µg protein) were incubated with 28,000 cpm of <125>I-gp 120 for 1 h at 37°C in 200 L (final volume) of 50 mM Hepes containing 0.1% bovine serum albumin and the peptidase inhibitors bacitracin (0.005%), aprotinin (0.005%), leupeptin (0.001%) and chymostatin (0.001%). The incubations were rapidly vacuum filtered and counted to determine the receptor bound material.
Immunoutfelling Immunoprecipitation
Immunoutfellinger ble fremstilt ved inkubasjon (over natten ved 4°C) av 0,5% Triton X-100/PBS oppløseliggjort, laktoperoksidase-/glukoseoksidase/l-joderte hjernemembraner eller intakte T-celler som indikerte mAbs ved 10 jig pr. reaksjon. En fastfase-immunoabsorbant (immunoperler, Bio-Rad) ble brukt til å utfelle immunkomplekser før deres reoppløsning ved NaDodS04/PAGE. Kontrollinkubasjoner inneholdt ikke noe primært mAb eller en underklassekontroll mAb (OKT8). Immunoprecipitates were prepared by incubation (overnight at 4°C) of 0.5% Triton X-100/PBS solubilized, lactoperoxidase/glucose oxidase/I-iodinated brain membranes or intact T cells indicating mAbs at 10 µg per reaction. A solid-phase immunoabsorbent (immunobeads, Bio-Rad) was used to precipitate immune complexes prior to their reresolution by NaDodSO 4 /PAGE. Control incubations contained no primary mAb or a subclass control mAb (OKT8).
Kjemisk neuroanatomi og datamaskinassistert densitometri Kryostatkuttede 25-jjm seksjoner av nylig frosset human-, ape- og rottehjerne ble tinet-montert og tørket på gelatinbelagte objektglass, og reseptorer ble synliggjort som beskrevet (18). Inkubasjoner med eller uten antistoffer (10 ug/ml) mot T4, T4A, T8 og T11 ble utført over natten ved 0°C i RPMI-medium, tverrbundet på o deres antigener og synliggjort, med <125>I-merket geit-antimus-antistoff. Inkubasjoner av på-objektglassmonterte vevseksjoner for å o merke antigenreseptoren med <125>I-gp 120 ble utført i 5 ml's objektglassbærere med (1 *iM) eller uten umerket gp 120 eller mAb OKT4A (10 ug/ml) (Ortho Diagnostics). Chemical neuroanatomy and computer-assisted densitometry Cryostat-cut 25-μm sections of fresh-frozen human, monkey, and rat brain were thaw-mounted and dried on gelatin-coated slides, and receptors were visualized as described (18). Incubations with or without antibodies (10 µg/ml) against T4, T4A, T8 and T11 were performed overnight at 0°C in RPMI medium, cross-linked on o their antigens and visualized, with <125>I-labeled goat anti-mouse -antibody. Incubations of slide-mounted tissue sections to o label the antigen receptor with <125>I-gp 120 were performed in 5 ml slide carriers with (1 *iM) or without unlabeled gp 120 or mAb OKT4A (10 µg/ml) (Ortho Diagnostics).
Separasjon av T- lymfocytt- subsett Separation of T lymphocyte subsets
Subsett av T-celler ble oppnådd ved behandling av Percoll densitetsrensede perifere blod T-celler med spesielle monoklonale antistoffer (T4 eller T8) ved 10 ug/ml. De behandlede celler ble deretter helt ut (21) på en Petri-skål av plast som var belagt med geit-antimus-immunoglobulin [F(ab')2^ (Sero Lab, Eastbury, MA) i 30 minutter ved 4°C. De ikke-vedheftende celler ble deretter fjernet, vasket og analysert for reaktivitet ved strømningscytometri. De separerte T4- og T8-cellepopulasjoner har <5% forurensning av andre T-cellesubsett. Celler ble deretter dyrket med fytohemagglutinin (1 ug/ml) i 72 timer og utsatt for HIV som beskrevet nedenunder. Infiserte celler ble fenotypisk karakterisert når cytotoksisitetsanalyser ble utført. Subsets of T cells were obtained by treating Percoll density-purified peripheral blood T cells with specific monoclonal antibodies (T4 or T8) at 10 µg/ml. The treated cells were then plated (21) onto a plastic Petri dish coated with goat anti-mouse immunoglobulin [F(ab') 2 ^ (Sero Lab, Eastbury, MA) for 30 minutes at 4°C. The non-adherent cells were then removed, washed and analyzed for reactivity by flow cytometry. The separated T4 and T8 cell populations have <5% contamination by other T cell subsets. Cells were then cultured with phytohemagglutinin (1 µg/ml) for 72 hours and exposed to HIV as described below. Infected cells were phenotypically characterized when cytotoxicity assays were performed.
Virusinfeksi on Viral infection
Det HTLV-lll-virus som ble benyttet til infeksjon ble isolert fra en interleukin 2 (IL-2)-avhengig dyrket T-cellelinje etablert fra nytt materiale fra AIDS-pasienter og ført inn i HuT 78, en ettergivende (permissive) IL-2-uavhengig cellelinje. The HTLV-lll virus used for infection was isolated from an interleukin 2 (IL-2)-dependent cultured T-cell line established from fresh material from AIDS patients and introduced into HuT 78, a permissive IL- 2-independent cell line.
Beskrivelse av tegningen Description of the drawing
1 25 1 25
Fig. 1A viser en tverrbinding av I-gp 120 til hjernemembraner og T-celler (a) <125>I-gp 120 kun; (b) ape; (c) rotte; (d) human hjerne og (e) humane T-celler. Fig. 1B og 1C viser immunoutf elling av 1 ^Si^e^e^e henholdsvis ape-hjernemembraner og humane T-celler; (f,i) ingen primær antistoffkontroll; (g,j) OKT4 Mab; (h,k) 0KT8 Mab. Fig. 2a viser en fortrengning av spesifikk <125>j_gp 120-binding til friske hippokampale membraner fra rotte. Hver bestemmelse ble utført i triplikat; resultatene av et eksperiment som ble utført tre ganger med lignende resultater er vist. Spesifikk binding fortrengbar med 10 ug/ml OKT4 og 4A lå i området 27-85% av den samlede binding som var 2201 ± 74 cpm i det forsøk som er vist. Fig. 2b viser at den virale infektivitet blokkeres ved peptid T og dets syntetiske analoger. Hver bestemmelse ble utført i duplikat. Resultatene representerer et enkelt eksperiment som ble gjentatt tre ganger med lignende resultater. Fig. 1A shows a cross-linking of I-gp 120 to brain membranes and T cells (a) <125>I-gp 120 only; (b) monkey; (c) rat; (d) human brain and (e) human T cells. Figures 1B and 1C show immunoprecipitation of 1 ^Si^e^e^e monkey brain membranes and human T cells, respectively; (f,i) no primary antibody control; (g,j) OCT4 Mab; (h,k) 0KT8 Mab. Fig. 2a shows a displacement of specific <125>j_gp 120 binding to healthy rat hippocampal membranes. Each determination was performed in triplicate; the results of an experiment performed three times with similar results are shown. Specific binding displaceable by 10 µg/ml OKT4 and 4A was in the range 27-85% of the total binding which was 2201 ± 74 cpm in the experiment shown. Fig. 2b shows that the viral infectivity is blocked by peptide T and its synthetic analogues. Each determination was performed in duplicate. The results represent a single experiment that was repeated three times with similar results.
Eksempel 1 Example 1
Et enkelt radioaktivt merket tverrbundet produkt på ca. 180 Kd fås etter spesifikk binding av <125>j_gp 120 til membraner fra enten ekornape, rotte eller humane hjernemembraner som ikke kan skjeldnes fra dem hos humane T-celler (fig. 1A). Dette resultat antyder at gp 120 kan kobles til et protein på ca. 60 Kd; ureagert ^<25>j_gp 120 løper ved siden av ikke-membrankontrollen (bane a). A single radioactively labeled cross-linked product of approx. 180 Kd is obtained after specific binding of <125>j_gp 120 to membranes from either squirrel monkey, rat or human brain membranes which is indistinguishable from that of human T cells (Fig. 1A). This result suggests that gp 120 can be linked to a protein of approx. 60 Kd; unreacted ^<25>j_gp 120 runs alongside the non-membrane control (lane a).
Immunoutfelling av radiojoderte humane hjernemembraner med 0KT4 og OKT8 (10 jig/ml) (fig. 1B) viser at hj ernemembraner inneholder et T4-antigen på ca. 60 Kd som ikke kan skjeldnes fra det som identifiseres på humane T-lymfocytter (fig. 1C); i motsetning til dette immunoutfeller 0KT8 et protein med lav molekylvekt (ca. 30 Kd) fra T-lymfocytter (fig. 1C) som er fraværende i hjernemembraner (fig. 1B), hvilket tyder på at hjerne-T4 ikke fås fra lymfocytter som finnes på stedet. Lignende resultater observeres hos ape og rotte (ikke vist) hippokampale membraner. Disse resultater viser at T4-antigenet tjener som virusresepto-ren og er et godt bevart 60 Kd molekyl som deles med immun- og sentralnervesystemene. Immunoprecipitation of radioiodinated human brain membranes with 0KT4 and OKT8 (10 µg/ml) (Fig. 1B) shows that brain membranes contain a T4 antigen of approx. 60 Kd indistinguishable from that identified on human T lymphocytes (Fig. 1C); in contrast, 0KT8 immunoprecipitates a low molecular weight protein (about 30 Kd) from T lymphocytes (Fig. 1C) that is absent from brain membranes (Fig. 1B), suggesting that brain T4 is not derived from lymphocytes present On-site. Similar results are observed in monkey and rat (not shown) hippocampal membranes. These results show that the T4 antigen serves as the virus receptor and is a well-conserved 60 Kd molecule that is shared with the immune and central nervous systems.
Den oppdagelse at Epstein-Barr og HTLV-III/LAV deler en nesten identisk oktapeptidsekvens forårsaket syntesen og undersøkelsen av "peptid T". Fig. 2 viser at den høye affinitet (0,1 nM-området) og saturabilitet (fig. 2a) av <125>I-gp 120-bindingen til nylig preparerte rotte-hjernemembraner. Spesifisitet (fig. 2b) er vist ved blokkade med OKT4 og OKT4A, men ikke 0KT3 (0,1 )Ag/ml). Peptid T og to av dets syntetiske analoger (men ikke den irrelevante oktapeptid-substans P [1-8]) hindret i betydelig grad <1>2<5>I-gp 120-binding i 0,1 nM-området (fig. 2c). Substitu-sjon av et D-threoninamid i posisjon 8 førte til minst et 100 ganger større tap av reseptorbindingaktivitet. Den klassiske [D-Ala]-substitusjon for [L-Ala ] resulterer i en konsekvent mer virkningsfull, øyensynlig mer peptidaseresistent analog enn peptid T; amidering av C-terminalthreoninet gir også konsekvent en noe større virkningsgrad (fig. 3). The discovery that Epstein-Barr and HTLV-III/LAV share an almost identical octapeptide sequence led to the synthesis and investigation of "peptide T". Fig. 2 shows that the high affinity (0.1 nM range) and saturability (Fig. 2a) of <125>I-gp 120 binding to freshly prepared rat brain membranes. Specificity (Fig. 2b) is shown by blockade with OKT4 and OKT4A, but not 0KT3 (0.1 )Ag/ml). Peptide T and two of its synthetic analogs (but not the irrelevant octapeptide substance P [1-8]) significantly inhibited <1>2<5>I-gp 120 binding in the 0.1 nM range (Fig. 2c). Substitution of a D-threoninamide in position 8 led to at least a 100-fold greater loss of receptor binding activity. The classical [D-Ala] substitution for [L-Ala] results in a consistently more potent, apparently more peptidase-resistant analog than peptide T; amidation of the C-terminal threonine also consistently gives a somewhat greater efficiency (Fig. 3).
Når de syntetiske peptider ble testet for deres evne til å blokkere virusinfeksjon på humane T-celler, kjente ikke eksperi-mentatorene til resultatene av bindingsanalysene. Ved 10"<7>M kan de tre peptider som er aktive i bindingsanalysen redusere påvi-selige nivåer av revers transkriptase-aktivitet med nesten 9 ganger. Den mindre aktive bindingsfortrenger [D-Thr]-peptid T oppviste på samme måte sterkt redusert blokkering av virusinfeksjon og krevde konsentrasjoner 100 ganger høyere for å oppnå betydelig inhibering. Således er ikke bare rangordningen for virkningsfullhet av de fire peptider (D- [Ala] j-peptid T-amid > D-[Ala] j-peptid T > peptid T > D-[Thr] 8-peptid T-amid) , men også deres absolutte konsentrasjoner med hensyn til å inhi-bere reseptorbinding og virusinfektivitet nøye korrelert (fig. 3) . When the synthetic peptides were tested for their ability to block viral infection of human T cells, the experimenters were unaware of the results of the binding assays. At 10"<7>M, the three peptides active in the binding assay can reduce detectable levels of reverse transcriptase activity by nearly 9-fold. The less active binding displacer [D-Thr]-peptide T similarly showed greatly reduced blocking of virus infection and required concentrations 100-fold higher to achieve significant inhibition.Thus, not only is the ranking of efficacy of the four peptides (D- [Ala] j-peptide T-amide > D-[Ala] j-peptide T > peptide T > D-[Thr] 8-peptide T-amide), but also their absolute concentrations with regard to inhibiting receptor binding and virus infectivity closely correlated (fig. 3).
Eksempel 2 Example 2
Et protein på tilnærmet 60 Kd som er likt om ikke identisk med humane T-celler T4-antigen forelå i øyensynlig bevart molekylær form på membraner fremstilt fra human hjerne; videre kan det radioaktivt merkede HIV-kapselglykoprotein (^<2>^I-gp 120) være kovalent tverrbundet til et molekyl som foreligger i tre pattedyrhjerner, hvis størrelse og immunoutfellingsegenskaper ikke kunne skjeldnes fra T4-antigenets. Ved bruk av en fremgangsmåte for synliggjøring av antistoffbundne reseptorer på skiver av hjerne, ble det neuroanatomiske fordelingsmønster for hjerne-T4 som er tettest over den kortikale neuropil og analogt organisert i alle tre pattedyrhjerner presentert. Videre antydet igjen radioaktivt merket HIV-viruskapsel-glykoprotein bundet i et identisk mønster på tilgrensende hjerneseksjoner at T4 var HIV-reseptoren. A protein of approximately 60 Kd that is similar if not identical to human T-cell T4 antigen was present in apparently conserved molecular form on membranes prepared from human brain; furthermore, the radioactively labeled HIV capsular glycoprotein (^<2>^I-gp 120) may be covalently cross-linked to a molecule present in three mammalian brains, whose size and immunoprecipitation properties could not be distinguished from those of the T4 antigen. Using a method for visualizing antibody-bound receptors on brain slices, the neuroanatomical distribution pattern of brain T4, which is densest over the cortical neuropil and analogously organized in all three mammalian brains, was presented. Furthermore, radiolabeled HIV viral capsular glycoprotein bound in an identical pattern on adjacent brain sections again suggested that T4 was the HIV receptor.
Eksempel 3 Example 3
Kjemisk neuroanatomi, datamaskinassistert densitometri. Kryostat-kuttede 25 mikrometer seksjoner av nylig frosset human-, ape- og rottehjerne ble tine-montert og tørket på gelbelagte objektglass og reseptorer synliggjort som beskrevet av Herkenham og Pert, J. Neurosci., 2, pp. 1129-1149 (1982). Inkubasjoner med eller uten antistoffer (10 *ig/ml) mot T4, T4Ak T8 og T11 ble utført over natten ved 0 °C i RPMI, tverrbundet på deres antigener og synliggjort med <125>j_geit-antimus-antistoff. Inkubasjoner av på-objektglass monterte vevsseksjoner for å merke antigen/reseptoren med 12^I_9P 120 ble utført i 5 ml's objektglassbeholdere med (10"<*> M) eller uten umerket gp 120 eller Mab OKT4A (10 ug/ml) (Ortho Diagnostics) som beskrevet ovenfor for membraner. Chemical neuroanatomy, computer-assisted densitometry. Cryostat-cut 25 micrometer sections of freshly frozen human, monkey and rat brain were thaw-mounted and dried on gel-coated slides and receptors visualized as described by Herkenham and Pert, J. Neurosci., 2, pp. 1129-1149 (1982) . Incubations with or without antibodies (10 µg/ml) against T4, T4Ak T8 and T11 were performed overnight at 0°C in RPMI, cross-linked to their antigens and visualized with <125>j_goat anti-mouse antibody. Incubations of slide-mounted tissue sections to label the antigen/receptor with 12^I_9P 120 were carried out in 5 ml slide containers with (10"<*> M) or without unlabeled gp 120 or Mab OKT4A (10 ug/ml) (Ortho Diagnostics ) as described above for membranes.
Datamaskinassistert overføring av autoradiografisk filmopasitet til kvantitative fargebilder ble utført. Ko-eksponering av standard kjente økninger av radioaktivitet med seksjoner av apehjernen genererte et lineært plott ( 4 = > 0,99) av log O.D. som funksjon av cpm, hvorfra den relative konsentrasjon av radioaktivitet kan ekstrapoleres på meningsfull måte. Celle-farging av hjerneseksjoner med thionin ble utført ved klassiske metoder og synliggjøring av reseptorer som lå oppå farget vev. Computer-assisted transfer of autoradiographic film opacity to quantitative color images was performed. Co-exposure of standard known increases of radioactivity with monkey brain sections generated a linear plot ( 4 = > 0.99) of log O.D. as a function of cpm, from which the relative concentration of radioactivity can be meaningfully extrapolated. Cell-staining of brain sections with thionin was carried out by classical methods and visualization of receptors that lay on top of stained tissue.
Eksempel 4 Example 4
Eksperimenter ble utført for å bestemme fordelingen av T4-antigen på en rostral til caudal serie eller koronale seksjoner av ekornape-hjerne. Disse eksperimenter viser at der er påvise-lige nivåer av T4-monoklonalt antistoff-binding til cytoarkitek-tonisk meningsfulle områder i hjernestammen (f.eks. substantia nigra), men det slående mønster av kortikal anrikning er tydelig på hvert nivå i neuroaksen. 0KT8, et T-lymfocytt-styrt monoklonalt antistoff fra den samme underklasse som 0KT4, oppviser intet observerbart mønster. Generelt inneholder de superfisielle lagene inne i hjernebarken de tetteste konsentrasjoner av T4-antigenet; den frontale og perilimbiske hjernebark som ligger oppå amydala er spesielt reseptorrik gjennom alle de dype lag. Den hippokampale formasjon har den tetteste konsentrasjon av reseptorer i hjernen hos ape, rotte og menneske. Mørkfeltmikro-skopi av ekornape-seksjoner dyppet i fotografisk emulsjon avslørte at båndet med den tetteste reseptormerking ligger inne i de molekylære lag av den takkede gyrus og hippokampus proper (som inneholder meget få neuroner). Således synes reseptorene å være riktig fordelt over neuropilet (de neuronale forlengelser av dendritter og aksoner) eller de kan være lokalisert til et spesifikt subsett av ikke-fargede astrogliale celler. Experiments were performed to determine the distribution of T4 antigen on a rostral to caudal series or coronal sections of squirrel monkey brain. These experiments show that there are detectable levels of T4 monoclonal antibody binding to cytoarchitectonically meaningful areas of the brainstem (eg, substantia nigra), but the striking pattern of cortical enrichment is evident at every level of the neuroaxis. 0KT8, a T-lymphocyte-directed monoclonal antibody from the same subclass as 0KT4, shows no observable pattern. In general, the superficial layers within the cerebral cortex contain the densest concentrations of the T4 antigen; the frontal and perilimbic cerebral cortex that lies on top of the amydala is particularly rich in receptors through all the deep layers. The hippocampal formation has the densest concentration of receptors in the brains of monkeys, rats and humans. Dark-field microscopy of squirrel monkey sections dipped in photographic emulsion revealed that the band with the densest receptor labeling lies within the molecular layers of the dentate gyrus and hippocampus proper (which contains very few neurons). Thus, the receptors appear to be properly distributed across the neuropil (the neuronal extensions of dendrites and axons) or they may be localized to a specific subset of unstained astroglial cells.
Bevis for spesifisiteten av den kjemiske neuroanatomi og resultater som viser at T4 og viruskapsel-gjenkjennelsesmoleky-let ikke kan skjeldnes fra hverandre er blitt bestemt. Koronale seksjoner av rottehjerne viste et meget likt hjernebark/hippokampus-rikt mønster for reseptorfordeling, hva enten 0KT4 eller <125>j_gp 120 ble brukt for synliggjøring. Videre var dette mønster ikke åpenbart når inkubasjon fant sted i nærvær av umerket gp 120 (1 m), OKT4A (10 ug/ml) eller OKT4 (10 ^g/ml). Andre muse-Mabs rettet mot andre humane T-celleoverflateanti-gener innbefattet OKT8 og 0KT11 ga intet påviselig mønster på rottehjerne når det ble synliggjort ved <125>i-geit-antimus-IgG sekundært antistoff, på samme måte som der ikke var noen reproduserbar påviselig antigen/reseptor med sekundært antistoff alene. Evidence for the specificity of the chemical neuroanatomy and results showing that T4 and the viral capsid recognition molecule are indistinguishable from each other have been determined. Coronal sections of rat brain showed a very similar cerebral cortex/hippocampus-rich pattern of receptor distribution, whether 0KT4 or <125>j_gp 120 was used for visualization. Furthermore, this pattern was not evident when incubation took place in the presence of unlabeled gp 120 (1 m), OKT4A (10 µg/ml) or OKT4 (10 µg/ml). Other mouse Mabs directed against other human T-cell surface antigens including OKT8 and 0KT11 produced no detectable pattern on rat brain when visualized by <125>i goat anti-mouse IgG secondary antibody, just as there was no reproducible detectable antigen/receptor with secondary antibody alone.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US86991986A | 1986-06-03 | 1986-06-03 | |
| US87858686A | 1986-06-26 | 1986-06-26 | |
| US4814887A | 1987-05-11 | 1987-05-11 | |
| PCT/US1987/001270 WO1987007613A1 (en) | 1986-06-03 | 1987-05-27 | Small peptides which inhibit binding to t-4 receptors and act as immunogens |
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| Publication Number | Publication Date |
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| NO880479D0 NO880479D0 (en) | 1988-02-03 |
| NO880479L NO880479L (en) | 1988-02-03 |
| NO176022B true NO176022B (en) | 1994-10-10 |
| NO176022C NO176022C (en) | 1995-01-18 |
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| NO880479A NO176022C (en) | 1986-06-03 | 1988-02-03 | Analogous Procedure for Preparation of Small Peptides That Prevent Binding to T4 Receptors and Act as Immune Organs |
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| KR (1) | KR930008448B1 (en) |
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| NO (1) | NO176022C (en) |
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| CN101088557A (en) | 2006-06-12 | 2007-12-19 | 天津市扶素生物技术有限公司 | Medicine composition for preventing and treating HIV infection and its application |
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1987
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1988
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| MX172337B (en) | 1993-12-14 |
| IL82719A (en) | 1992-11-15 |
| AU604719B2 (en) | 1991-01-03 |
| NO880479D0 (en) | 1988-02-03 |
| DK53288A (en) | 1988-02-02 |
| NO880479L (en) | 1988-02-03 |
| AU7540887A (en) | 1988-01-11 |
| FI885630A0 (en) | 1988-12-02 |
| JP2680011B2 (en) | 1997-11-19 |
| HUT48907A (en) | 1989-07-28 |
| NZ220485A (en) | 1989-08-29 |
| IL82719A0 (en) | 1987-11-30 |
| JPH01502659A (en) | 1989-09-14 |
| FI885630A (en) | 1988-12-02 |
| FI94352C (en) | 1995-08-25 |
| DK173667B1 (en) | 2001-05-28 |
| FI94352B (en) | 1995-05-15 |
| CA1341066C (en) | 2000-08-01 |
| WO1987007613A1 (en) | 1987-12-17 |
| ES2061497T3 (en) | 1994-12-16 |
| PT84992A (en) | 1987-07-01 |
| KR930008448B1 (en) | 1993-09-04 |
| KR880701247A (en) | 1988-07-26 |
| DE3787927T2 (en) | 1994-03-03 |
| NO176022C (en) | 1995-01-18 |
| DE3787927D1 (en) | 1993-12-02 |
| IE871388L (en) | 1987-12-03 |
| PT84992B (en) | 1990-03-08 |
| DK53288D0 (en) | 1988-02-02 |
| IE61725B1 (en) | 1994-11-30 |
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