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WO1998043995A1 - Nouveaux complexes anti-vih et compositions medicamenteuses - Google Patents

Nouveaux complexes anti-vih et compositions medicamenteuses Download PDF

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Publication number
WO1998043995A1
WO1998043995A1 PCT/JP1998/001366 JP9801366W WO9843995A1 WO 1998043995 A1 WO1998043995 A1 WO 1998043995A1 JP 9801366 W JP9801366 W JP 9801366W WO 9843995 A1 WO9843995 A1 WO 9843995A1
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Prior art keywords
hiv
residue
amino acid
arg
group
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PCT/JP1998/001366
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English (en)
Japanese (ja)
Inventor
Nobutaka Fujii
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Seikagaku Corporation
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Priority to AU65186/98A priority Critical patent/AU6518698A/en
Publication of WO1998043995A1 publication Critical patent/WO1998043995A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel substance having anti-HIV activity and a pharmaceutical composition containing the substance as an active ingredient. More specifically, a complex in which a reverse transcriptase inhibitor and a Z or HIV protease inhibitor are bound to a polypeptide having an anti-HIV activity by a chemical bond, an anti-HIV agent containing the substance as an active ingredient, and the like. And a pharmaceutical composition.
  • HIV Human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • AIDS-related syndrome AIDS-related syndrome
  • Drugs that inhibit each stage of HIV growth are known as anti-HIV drugs, but drugs that are actually licensed or undergoing clinical trials include reverse transcriptase inhibitors (for example, nucleoside 3′-azido-3, -deoxythymidine (hereinafter also referred to as AZT), 2,, 3,1-dexoxyinosine (hereinafter also referred to as ddl), 2 ′, 3′-deoxycytidine (hereinafter ddC) Etc.) and HIV protease inhibitors (N, 1 [1 (S) 1 benzyl-3--3- [4 a (S), 8 a (S), 3 (S)-(tert-butyl carbamoyl) ) Decahydroisoquinoline 1-2-yl] 1-2 (R) -hydroxypropyl] 1N "-quinoline-2-ylcarbamoyl) 1-L-asparagineamide (hereinafter also referred to as Ro 31-8959) )
  • these anti-HIV drugs require large doses and long-term administration, so that side effects (such as bone marrow damage) and the development of drug-resistant viruses due to reverse transcriptase or amino acid mutations in HIV protease are required. There were problems such as appearance.
  • a novel polypeptide having an antiviral activity and an anti-HIV agent containing this polypeptide as an active ingredient are known (International Publication WO92 / 043734, JP-A-5-1633).
  • the polypeptide is known to exhibit the same level of anti-HIV activity as AZT, such as reverse transcriptase inhibitors and HIV proteases. It is also known that when administered in combination with a monose inhibitor, it exhibits excellent anti-HIV activity (Japanese Patent Application Laid-Open No. 9-252440).
  • Toxic side effects such as reverse transcriptase inhibitors and HIV protease inhibitors can also be caused by administration of a combination of a polypeptide having anti-HIV activity and a reverse transcriptase inhibitor or HIV protease inhibitor, etc. It cannot be reduced and remains a problem to be solved, and anti-HIV drugs with lower toxicity are expected. Disclosure of the invention
  • the present inventors have developed a polypeptide based on a polypeptide having anti-HIV activity and having high affinity for HIV surface protein.
  • a complex in which an anti-HIV active substance such as a reverse transcriptase inhibitor or an HIV protease inhibitor has been chemically bonded to the amino (N) terminus of the polypeptide has been used as an anti-HIV agent.
  • the present inventors have found that it is possible to exhibit IV activity and reduce side effects, and thus completed the present invention.
  • the present invention relates to a polypeptide having an affinity for an HIV surface protein, preferably a polypeptide of the following formula (1), and one or more anti-HIV active substances (a reverse transcriptase inhibitor and a Z or HIV protein). And a pharmaceutical composition containing the complex as an active ingredient.
  • a polypeptide having an affinity for an HIV surface protein preferably a polypeptide of the following formula (1)
  • one or more anti-HIV active substances a reverse transcriptase inhibitor and a Z or HIV protein.
  • a pharmaceutical composition containing the complex as an active ingredient I 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3
  • 81 is ⁇ 1— (derived from the amino group of A2), or one basic amino acid selected from lysine, arginine and ordinine, or the same or selected from these amino acid residues.
  • a peptide residue having at least two different amino acid residues, or a hydrogen atom at the N- ⁇ position of the amino terminal amino acid residue of the basic amino acid residue or the peptide residue is an acyl group or substitution
  • a 2 independently represents a tyrosine, phenylalanine or tributophan residue
  • a 3 independently represents a lysine or arginine residue
  • a 4 represents an amino acid residue or a peptide residue having at least one amino acid selected from lysine and arginine;
  • Y represents a peptide residue represented by the following formula (a):
  • a 6 independently represents an alanine, nocrine, leucine, isoleucine, serine, cysteine or methionine residue
  • A7 represents a tyrosine, phenylalanine, tryptophan, alanine, palin, oral isin, isoleucine, serine, cysteine or methionine residue;
  • G 1 y represents a glycine residue.
  • D-Ordityl-proline Prolyl-D-Orditin, Prolyl-D-lysine, Prolyl-D-arginine, D-Rigid-loop phosphorus, D-Arginyl-proli And glycyl-lysine, glycyl-arginine, ornithyl-glycine, glycylornithine, lysyl-glycine, and arginyl-glycine.
  • a hydrogen atom of a side chain ⁇ -amino group of a certain D-lysine, lysine, D-orudin or ordinin indicates a peptide residue which may be substituted with an acyl group;
  • Cys indicates a cysteine residue, and the cysteine residues at positions 3 and 11 may be linked by a disulfide bond.
  • the conjugate of the present invention is characterized in that at least the above-mentioned polypeptide and an anti-HIV active substance such as AZT are bound by a chemical bond, whereby CD4 positive cells, which are targets for HIV infection in vivo, are tested. It transports an HIV active substance and releases an anti-HIV active substance in the cells to exhibit excellent anti-HIV activity.
  • a composition containing this novel complex as an active ingredient is safe. It is effective as a pharmaceutical composition such as an anti-HIV drug which is high and has few side effects.
  • the novel complex of the present invention (hereinafter sometimes referred to as the substance of the present invention) is a polypeptide which has affinity for HIV surface proteins gp120 and Z or HIV host target cell surface protein CD4, 1 or A complex in which two or more reverse transcriptase inhibitors and / or HIV protease inhibitors are bound by a chemical bond.
  • the polypeptide constituting the present invention is not particularly limited as long as it is a polypeptide having an affinity for the HIV surface proteins gp120 and Z or the HIV host target cell surface protein CD4.
  • suitable polypeptides include antibodies and polypeptides represented by the following formula (1).
  • the protein also has affinity for the cell surface protein CD4 of CD4 positive cells (host target cells), which are host targets of HIV. (Biochem. Biophys. Res. Commun., 219, 555-559 (1996), Biochem. Biophys. Acta., 1298.37-44 (1996)), which is itself represented by the following formula (1) having anti-HIV activity. Polypeptides are preferred.
  • a UiH— (derived from the amino group of A2) or one basic amino acid selected from lysine, arginine and orditin, or the same or different amino acids selected from these amino acid residues
  • a peptide residue having at least two residues, or a hydrogen atom at the N-la position of the amino terminal amino acid residue of the basic amino acid residue or the peptide residue is an acyl group or a substituted thiocarno ⁇ moyl group
  • a 2 independently represents a tyrosine, phenylalanine or tribubutane residue
  • a 3 independently represents a lysine or arginine residue
  • a 4 represents an amino acid residue or a peptide residue having one or at least two amino acids selected from lysine and arginine;
  • Y is a peptide residue represented by the following formula (a)
  • a 6 independently represents an alanine, nocrine, leucine, isoleucine, serine, cysteine or methionine residue
  • a 7 represents a tyrosine, phenylalanine, tryptophan, alanine, palin, oral isin, isoleucine, serine, cysteine or methionine residue;
  • G 1 y represents a glycine residue.
  • Cys represents a cysteine residue, and the cysteine residues at positions 3 and 11 may be linked by a disulfide bond.
  • the number indicating the position of the amino acid roughly indicates the amino acid sequence, but does not necessarily correspond to the amino acid residue in a one-to-one correspondence. are those attached to, H from the N-terminus, one OH and from the C-terminus - are NH 2 and one digit when the amino acid residue inside the other amino acid sequences present one is used, If the amino acid residue is a peptide residue according to the definition, the number of its constituent amino acid residues is used.
  • polypeptide represented by the formula 8 (1) is a polypeptide synthesized based on a horseshoe crab-derived tachyplesin family polypeptide, a horseshoe crab-derived tachyplesin I, a tachyplesin II, a tachyplesin III, a polyhumsine I or Polyphencin II (metabolism, 26, p429-439 (1989)) can be used as an alternative to the above polypeptides.
  • Having an anti-HIV activity in the present specification means having the ability to exert effects such as suppressing HIV infection, suppressing HIV proliferation, and reducing HIV.
  • the polypeptide represented by the above formula (1) (hereinafter referred to as a polypeptide chain for convenience) is a polypeptide synthesis method known per se, in particular, a liquid phase synthesis method or a solid phase synthesis method (New Protein Chemistry Laboratory Course 1 Protein VI). , Tokyo Chemical Dojin, p3-29 (1992)).
  • DNAs encoding the amino acid sequences of these polypeptides can be synthesized by introducing them into host cells by gene recombination techniques and expressing them.
  • the carboxyl group of the N-protected amino acid corresponding to the carboxyl terminal amino acid residue of the polypeptide chain for example, directly or optionally via an intervening substance such as an amino group or a hydroxyl group Has a functional group
  • the protected amino acids from position 11 to position 1 of the amino acid sequence represented by the above formula (1) are sequentially bonded in accordance with the solid phase synthesis method to obtain a protecting group-protected peptide resin.
  • the insoluble resin is not particularly limited as long as it can be covalently bonded directly to the carboxyl group of the N-protected amino acid at the carboxyl (C) terminal, or optionally through an intervening substance, and can be subsequently removed. Is not done.
  • examples of such an insoluble resin include a resin that releases an amino acid or a eptidic acid after elimination of the insoluble resin, an alkoxy resin (A1 ko-resin or the like), an amino acid amide after elimination, An amide-type resin (Rinkami de resin or the like) that releases a peptide amide may be used.
  • resins that release amino acids or eptidic acids include chloromethyl resin, oxymethyl resin, PAM (4- (hydroxymethyl) phenylacetamidomethyl) resin and Alko-resin.
  • the amide resin include p-alkoxybenzyl alcohol (Wang) resin, hydroxymethyl phenoxy acetic acid (HMPA) resin, dialkoxy benzyl alcohol type resin, and trialkoxy diphenyl methyl alcohol type resin.
  • BHA benzhydrylamine
  • MBHA p_methylbenzhydrylamine
  • PAL pepti de ami de 1 i nker resin and R i n k ami de resin
  • a protected amino acid is an amino acid in which a functional group is protected with a protecting group by a known method, and various protected amino acids are commercially available.
  • the protecting group is preferably selected from those known per se according to the peptide synthesis conditions.
  • the coupling of the protected amino acids can be carried out according to a conventional condensation method.
  • the DCC (dicyclohexylcarbodiimide) method the DCC-HOBt (1-hydroxybenzotriazole) method
  • the DIPCI N, N-diisopropyl carpoimide
  • BOP benzotriazolyl-N-hydroxytrisdimethylaminophosphonium hexafluorophosphoride
  • HBTU 2,3,3-tetramethylperoniumhexafluoro
  • symmetric anhydride method etc.
  • These condensation reactions are usually performed in an organic solvent such as dichloromethane, dimethylformamide (DMF), N-methylpyrrolidone (NMP) or a mixture thereof.
  • the desired protecting group-protected peptide resin or the peptide released from the protecting group-protected polypeptide resin and the substituted isothiocyanate compound are slightly diluted.
  • a ⁇ terminal ⁇ -monosubstituted thiocarbamoylated polypeptide of the substance of the present invention can be obtained.
  • the carboxyl terminus of the amino acid residue at position 13 can be free (A 5 in formula (1) corresponds to - ⁇ ), or the acid amide (formula (In (1), A 5 is equivalent to —NH 2 ).
  • the polypeptide having the protecting group and bonded to the insoluble resin is hereinafter also referred to as a protecting group-protected polypeptide resin.
  • each symbol represents an amino acid residue or a substituted amino acid residue in internationally recognized three-letter display, and each symbol represents the following amino acid residue or substituted amino acid residue.
  • each symbol represents the following amino acid residue or substituted amino acid residue.
  • D- before an amino acid label, it means that all are "L”.
  • amino acid used as a protecting group for the side chain functional group of the amino acid examples include the following.
  • B u ' tertiary butyl
  • B oc tertiary butyloxy
  • Examples of a method for obtaining only a polypeptide chain from the protective group-protected polypeptide resin include a known method and a method for removing a protective group and an insoluble resin and forming a disulfide bond in one step described below.
  • TFA trifluoroacetic acid
  • dichloromethane HC 1Z dioxane
  • piperidine / DMF or piperidine Zin ZNMP is used, and is appropriately selected depending on the type of the protecting group.
  • Protected group-protected polypeptide resins include hydrogen fluoride, TFMSA (trifluoromethanesulfonic acid), TMSOTf (trimethylsilino retriflate), TMSB r (trimethylsilyl bromide), TMSC1 (trimethylsilino chloride) ) Or by treating with TFA, etc., the resin and the protecting group can be simultaneously eliminated.
  • the above elimination reagent is appropriately selected depending on the synthesis strategy (Boc method or Fmoc method), the insoluble resin to be used, and the type of protecting group.
  • the obtained polypeptide chain forms a disulfide bond (1-S—S—) via a mercapto group between the cysteines at positions 3 and 11 or, optionally, at positions ⁇ and 6 ,.
  • These disulfide bonds can be formed by a method known per se, for example, mild air oxidation or the like.
  • an oxidizing agent such as oxygen perfluorophosphate in the atmosphere (for example, potassium perfluoride) is used.
  • the protecting group and the resin are eliminated, and the polypeptide formed by forming a disulfide bond can be further isolated and purified by a known method. Most effective.
  • a reverse transcriptase inhibitor and a NO or HIV protease inhibitor which are anti-HIV active substances, are chemically bound to the protecting group-protected polypeptide resin.
  • Reverse transcriptase inhibitors are substances that inhibit the activity of HIV reverse transcriptase, and are classified into nucleoside-based inhibitors and non-nucleoside-based inhibitors.
  • the nucleoside-based inhibitor includes a nucleoside comprising a pyrimidine base, a purine base, an imidazole base or a triazole base, and a furanose having at least one hydroxyl group or an acyclo-form thereof.
  • AZT CAS REGISTRY NUMBERS: 30516-87-1: zidovudine
  • dd I CAS REGISTRY NUMBERS: 69655-05-6: didanosine
  • d dC CAS REGISTRY NUMBERS: 7481-89- 2: zalci tabin e
  • 2 ', 3'—didehydro 1', 3, 1-dideoxythymidine CAS REGISTRY NUMBERS: 3056-17 -5: d 4 T: stavudine
  • 3'-thia-2 ', 3' didoxycytidine CAS REGISTRY NUMBERS: 134678-17-4: 3 TC: l 5 Lamivudine
  • 2 '-3-Fluoro dd C 3' Fluorotymidine
  • non-nucleoside inhibitors examples include, for example, tetrahydroimidazo-benzodiazepine one-one or one-thione (T IBO) derivative (specifically,
  • nucleoside reverse transcriptase inhibitors are preferred, among the nucleoside-based HIV transcriptase inhibitors, it is preferably AZT, ddl, ddC, 14 or 3 fingers already administered to humans in the clinic, more preferably the polypeptide chain and AZT whose antiviral activity is particularly synergistically enhanced when chemically combined with the substance of the present invention.
  • nucleoside reverse transcriptase inhibitors are incorporated into DNA when HIV synthesizes DNA from RNA by reverse transcription.As a result, unnatural nucleosides or nucleoside analogs are inhibited to inhibit DNA synthesis. Nigs are preferred.
  • the nucleoside analog refers to a non-nucleoside compound having a steric structure similar to that of a nucleoside.
  • these reverse transcriptase inhibitors can be commercially available or those prepared according to known synthesis methods.
  • an inhibitor which is a substance which inhibits the activity of HIV protease and which is a mimic compound of the substrate transition state of the protease is preferable.
  • the substrate transition state mimic refers to a substance that can bind to the substrate binding site of the enzyme and has a similar tertiary structure to the substrate in the enzyme-substrate complex.
  • Ro 3 1—8 9 5 9 (CAS REGISTRY NUMBERS: 127779-20-8: sa quinavir), A—7703 (CAS REGISTRY NUMBERS: 134878-17-4), A—8 0 9 8 7 (CAS REGISTRY NUMBERS: 144141-97-9), KN I—93 (CAS REGISTR Y NUMBERS: 138258-64-7), KN I—102 (CAS REGISTRY NUMBERS: 139694-6 5- 8), KN I- 1 7 4, KN I-2 2 7 (CAS REGISTRY NUMBERS: 147384-69-8) .KN I-2 7 2 (CAS REGISTRY NUMBERS: 147318-81-8), L- 7 3 5 5 2 7 (CAS REGISTRY NUMBERS: 150378-17-9: indinavir), SC-5 2 1 5 1 (CAS REGISTRY NUMBERS: 143224-34-4: Tel inavir).
  • Ro31-8959, L1-735527 and KN-272 having high antiviral activity are preferred, but there is no particular limitation.
  • these HIV protease inhibitors commercially available ones or those prepared according to known synthetic methods can be used.
  • Ro 31 -8959 for example, the preparation method described in J. Med. Chem. 36, P2300-2310 (1993) can be mentioned.
  • the substance of the present invention is not particularly limited as long as the force at which the polypeptide chain and the anti-HIV active substance are chemically bonded is not particularly limited as long as the bond is a chemically formed bond.
  • the ester bond is formed by transporting the bound anti-HIV active substance into a target cell in a living body, followed by intracellular esterase or the like.
  • the anti-HIV active substance can be released near the action point of the anti-HIV active substance.
  • the ester bond is a bond having such a stability that the ester bond is not easily cleaved during transportation, and is not limited to a preferable force.
  • the position of the polypeptide chain that binds to the anti-HIV active substance is not particularly limited, but the polypeptide chain itself, HIV surface protein gp120, and CD4 positive which is a host target cell
  • An amino terminal site capable of maintaining affinity with cell surface protein CD4 of the cell is preferred.
  • the ⁇ -amino group or ⁇ -amino group of the amino acid at the amino terminal, or Tam was used to prepare the antibody.
  • MAP system multiple antigen peptide peptide system
  • JP Tam 'Peptides synthesi s, structures, and applications "(B.
  • Dendrimer using radially branched lysine Is formed at the amino terminus of the polypeptide chain by the method of Tam described above, and a plurality of anti-HIV active substances are bound to a plurality of amino groups present at the terminus and side chains to obtain one molecule of the polypeptide chain.
  • the substance used for forming the dendritic structure is most preferably the above-described lysine, but may be prepared by a basic amino acid such as ornithine or by synthesis in addition to lysine. Even amino acids and the like can be used as long as they are substances containing two or more amino groups. .
  • the binding site of the anti-HIV active substance that binds to the amino group of the polypeptide chain should be appropriately selected depending on the type of the active substance to be bound, but when a carboxyl group or a hydroxyl group is present in the substance. Is preferably a chemical bond formed through these functional groups, particularly preferably a hydroxyl group. Further, even if the position of the functional group is a site having a central role in anti-HIV activity, a chemical bond is cleaved in the target cell, so that the functional group in the molecule serving as the binding site is The position is not particularly limited.
  • the substance of the present invention when the substance of the present invention is obtained by binding an anti-HIV active substance to a polypeptide chain via a carboxyl group in the substance, the amino group at the amino terminal of the protecting group-protected polypeptide resin is reacted with the amino group. It is possible to form an amide bond between the carboxyl groups of the HIV active substance and to make the bond directly, and to form a chemical bond that is easily cleaved in vivo through an appropriate spacer substance. It is also possible to form.
  • the substance of the present invention is obtained by binding an anti-HIV active substance to a polypeptide chain via a hydroxyl group in the substance, the functional group at the amino terminal of the protecting group-protected polypeptide resin and the anti-HIV activity It can be attached through a multifunctional spacer having a functional group that can be chemically attached to both hydroxyl groups of the substance.
  • a multifunctional supplier has one carboxyl group capable of binding to an amino group at the amino terminal of the polypeptide chain to be used, and further contains water in the anti-HIV active substance. It is preferable to have a carboxyl group different from the above which can form an ester bond with an acid group, and specific examples include an aromatic compound having two or more carboxyl groups or an aliphatic compound. Aliphatic compounds are preferred because they form a chain structure.
  • an amide bond is formed between one of the carboxyl groups and the amino group at the amino end of the polypeptide chain, and the other carboxyl group is formed.
  • an ester bond is formed with the hydroxyl group in the anti-HIV active substance, a polypeptide chain and the anti-HIV active substance can be bound.
  • an aliphatic dicarboxylic acid having 4 to 12 carbon atoms is a multifunctional spacer.
  • an aliphatic dicarboxylic acid having 4 to 8 carbon atoms is more preferable.
  • Succinic acid or glutaric acid is particularly preferred from the viewpoint of stability and ease of handling.
  • the dicarboxylic acid may have another functional group as long as it does not prevent the binding between the polypeptide chain and the anti-HIV active substance.
  • a method of bonding the protecting group-protected polypeptide resin and AZT to a dicarboxylic acid for example, succinic acid or glutaric acid
  • AZT and a polyfunctional spacer are combined.
  • AZT is reacted with succinic anhydride or glucuric anhydride in the presence of dimethylaminopyridine, and after a conventional treatment, the binding of AZT, in which succinic acid or glutaric acid is ester-bonded to AZT, to a multifunctional spacer. Get the body.
  • the AZT-polyfunctional spacer conjugate is added to the ⁇ -amino group or ⁇ -amino group of the amino acid at the N-terminal of the protecting group-protected polypeptide resin, or dendrimer-like to the amino acid.
  • Peptide resin is also referred to as AZT complex).
  • the protecting group-protected polypeptide resin spacer-AZT complex obtained as described above can be prepared in a single step, for example, by the following method to remove the insoluble resin and the protecting group and form a disulfide bond.
  • the substance of the present invention can be obtained. That is, for example, 10% or less, preferably less than 5%, and more preferably 0.5 to 1.5% (v / v) of aniso is added to the protecting group-protected polypeptide resin-spacer AZT complex.
  • TMSC1 trimethylsilyl chloride
  • TFA trimethylsilyl chloride
  • the reaction is carried out for 0 minutes at 0 to 80 ° C, preferably at 10 to 40 ° C, more preferably at 15 to 30 ° C, followed by preferably 10 ° C or less, more preferably ice.
  • 100 equivalents or more, preferably 200 to 400 equivalents of DMSO is added under cooling, and 20 minutes or more, preferably 30 to 120 minutes, more preferably 60 to 100 minutes.
  • TMSC1-1DMSO / TFA system The system for removing the protecting group and the resin and forming a disulfide bond is hereinafter referred to as TMSC1-1DMSO / TFA system.
  • the thus-obtained substance of the present invention can be isolated and purified by a known method, but the method by reversed-phase high-performance liquid chromatography is most effective.
  • the active ingredient of the pharmaceutical composition of the present invention is the above-mentioned substance of the present invention or a pharmacologically acceptable salt thereof.
  • Pharmaceutically acceptable salts include, for example, inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.), organic carboxylic acids (acetic acid, propionic acid, maleic acid, succinic acid, malic acid, Salts with citric acid, tartaric acid, salicylic acid, etc., acidic sugars (eg, glucuronic acid, galacturonic acid, dalconic acid, ascorbic acid, etc.), or organic sulfonic acids (eg, methanesulfonic acid, p-toluenesulfonic acid, etc.).
  • inorganic acids hydroochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.
  • organic carboxylic acids acetic acid, propionic acid, maleic acid, succinic acid, malic acid, Salts with citric acid, tartaric acid, salicylic acid, etc.
  • acidic sugars
  • the substance of the present invention can be administered as it is, it may be a pharmaceutical composition further containing a pharmacologically acceptable carrier selected according to the administration method and administration form of the drug.
  • This pharmaceutical composition is administered orally or parenterally according to the treatment method and the like, and is injected, injectable, externally applied, suppository (for example, powder) using an appropriate drug carrier according to the administration method.
  • suppository for example, powder
  • Granules, liquid for injection or oral use, tablets, pessaries, ointments, creams, aerosols, etc. It can be a formulation.
  • the pharmaceutical composition of the present invention is directly administered to a living body, for example, as an injection, 10 mg to 5 g per 1 kg of human body weight per day can be administered as a drug component.
  • the anti-HIV activity of the pharmaceutical composition of the present invention is determined by transporting an anti-HIV active substance into a target cell by a polypeptide chain having affinity for at least an HIV surface protein, and a polypeptide chain on the target cell.
  • the anti-HIV agent By breaking the chemical bond with the anti-HIV active substance, the anti-HIV agent with a different mechanism of action, the polypeptide chain and the anti-HIV active substance, ie, the steps of adsorption, fusion and entry of HIV to CD4-positive cells It is thought to be caused by the combination of a polypeptide that is thought to suppress the activity and an anti-HIV agent that inhibits the enzyme activity of HIV reverse transcriptase or HIV protease.
  • FIG. 1 is a BIAcore analysis diagram showing the affinity of the substances 12 (AZT-Suc-T22) and 22 (AZT-Glu-T22) of the present invention for gp120 and CD4.
  • Fmo c As p (But) — OH, Fmo c — G lu (B u ') — OH, Fm oc — H is (Bo c) _0H, Fmo c — Ser (Bu l ) — 0H, Fmo c- Th r (B u ') -OH, Fmo c— Lys (Bo c) one 0H, Fmo c— A rg (Pmc) — To each 6 mg of OH, add TMSC 1 (42 ⁇ 1), DMSO (762 ⁇ 1), and TFA (5.2 ml), and treat at 4 ° C to 5, 1 0, 3 0, 6 0, 9 and sampled by 1 0 0 beta 1 after 0 minutes ⁇ 2 0- Me CN (1: 1) with diluted lml, by injection a part of the solution to the H PLC Fmoc amino acids, side chain protected Fmoc amino acids were quantified.
  • H—Leu—A1 koresin (0.60 mmo1 / g, 2 ⁇ mo1) in which Leu is bound to an alkoxyl-type resin that releases amino acids or eptidic acid by cleavage, or amino acids by cleavage H-Arg (Pmc) with Arg (Pmc) bound to an amide-type resin that releases amide or peptide amide
  • An internal standard for Rink am ideresin (0.32 mmo1 / g, 2 no1) Boc—Gly—OH (3 no 1), TMS C 1 (7 ⁇ 1, 750 eq) and TFA (870 (1) were added, and the mixture was stirred at room temperature for 1 hour.
  • Preparation of the protecting group-protected polypeptide resin having the polypeptide chain of the formula (A) was performed manually using the Fmoc-type solid phase synthesis method.
  • Fmoc-Arg (Pmc) -A1 koresin 0.52 mmo1 / g in terms of Arg
  • the cells were treated with 20% piperidine ZDMF for 15 minutes to remove the Fmoc group.
  • DIPCI-HOBt / DMF was used for the condensation of Fmoc amino acids.
  • Fmoc amino acids, condensing reagents, and the like were each used in 2.5 equivalents to the resin.
  • the substance synthesized in this manner is referred to as a protecting group-protected polypeptide resin 1 for convenience.
  • a protecting group-protected polypeptide resin that generates an amide form of a polypeptide chain in which the 13-position of the above formula (A) is NH 2 is used as a resin for peptide synthesis, as Fmoc-Arg (Pmc).
  • Fmoc-Arg Pmc
  • a protecting group-protected polypeptide resin having a polypeptide chain represented by the following formula (B) is used as a resin for synthesizing a peptide, which releases an amino acid amide or a peptide amide by a cleavage treatment.
  • Fmoc-PAL (peptideamid) The compound was synthesized by the Fmoc-type solid phase synthesis method as described above using delinker) resin (0.38 mmo 1 / g in terms of amino group). The substance synthesized in this manner is described as a protecting group-protected polypeptide resin 2 for convenience. 1 1 2 3 4 5 6 7 7 7 7 7 8
  • AZT (534.8 mg, 2 mmo 1) was reacted with glutaric anhydride (228.2 mg, 2 mmo 1) under the same reaction conditions and the same reaction procedure as in the preparation of AZT succinate in Preparation Example 2. After the same treatment, the desired crystals of AZT glutaric acid ester were obtained (yield: 80.0%).
  • Bondasphere 5 C18—100 people (3.9 x 50 mm, manufactured by Nippon Millipore) has a retention time of 14.8 minutes in analytical HPLC, and amino acid analysis of hydrolyzate with 6N HCl (in Nikko) Cy s not determined (theoretical values are theoretical) (1), Tyr
  • polypeptide amide of the above formula (B) (protecting group-protected polypeptide)
  • synthetic polypeptide the substance obtained by this method is referred to as a synthetic polypeptide
  • synthetic polypeptide (T13 31) having the polypeptide chain of the above formula (A) is referred to as synthetic polypeptide 1
  • synthetic polypeptide (T22) prepared by the same method as described above using the protecting group-protected polypeptide resin 2 having the above polypeptide chain will be referred to as synthetic polypeptide 2.
  • substance 1 of the present invention substance 1 of the present invention
  • substance 1 of the present invention (AZT-Suc-T131) having the polypeptide chain of the above formula (A) will be referred to as substance 11 of the present invention
  • substance 1 of the present invention (AZT-Sue-T22) having the polypeptide chain of (B) is referred to as the substance 12 of the present invention.
  • substance 2 of the present invention substance 2 of the present invention
  • substance 2 of the present invention (AZT-G1U-T131) having the polypeptide chain of the above formula (A) is referred to as substance 21 of the present invention
  • substance 22 of the present invention substance 22 of the present invention
  • the protecting amino acid is further protected on the ⁇ -amino group of the N-terminal amino acid of the protecting group-protected polypeptide resin 1 or 2 prepared in Preparation Example 1 using a similar amino acid condensation method (DIPCI-HOBt method, etc.).
  • Fmo c — Lys Fmo c was bound.
  • the removal of Fmoc group and the introduction of Fmoc-Lys (Fmoc) were repeated twice, and Lys having a dendritic structure in which seven Lys were introduced into the amino terminal of the polypeptide.
  • An introduced protecting group-protected polypeptide resin was prepared.
  • the resin prepared from the protecting group-protected polypeptide 1 obtained in Preparation Example 1 is referred to as Lys-introduced protecting group-protected polypeptide resin 1
  • the resin prepared from the protecting group-protected polypeptide 2 is referred to as Lys Described as introduced protection group-protected polypeptide resin 2.
  • the F moc group of the Lys-introduced protecting group-protected polypeptide resin 1 was removed, and the AZT succinate (2.5 eq) prepared in Preparation Example 2 was prepared in the same manner as in Preparation Example 5 by DI PC I- Condensed by the HOBt method.
  • the Lys-introduced protecting group-protected polypeptide resin-AZT succinate complex thus obtained was subjected to deprotection, deresinization and disulfide bond formation in the same manner as in the preparation of synthetic polypeptide 1 in Preparation Example 4.
  • a dendrimer-type substance of the present invention into which eight target AZTs were introduced was obtained.
  • the substance of the present invention according to the present preparation method will be referred to as “dendrimer type substance 1 of the present invention” for convenience.
  • the AZT glutaric acid ester prepared in Preparation method 3 is bonded to the Lys-introduced protecting group-protected polypeptide resin 1, and the same deprotection, deresinization, disulfide bond formation, isolation, Purification was performed to obtain the target substance of the present invention.
  • the substance of the present invention is referred to as “dendrimer type present substance 2”.
  • the AZT succinate prepared in Preparation Example 2 was bonded to the Lys-introduced protecting group-protected polypeptide resin 2.
  • the obtained Lys-introduced protecting group-protected polypeptide-AZT succinate complex resin (10 Omg) was added to m-cresol (3001), 1,2-ethanedithiol (300/1), Add thioanisole (30031), TFA (4.8 ml) and distilled water (300 ⁇ 1) and stir at room temperature for 2 hours to deprotect and deresin. After treating and air-drying according to the procedure for preparing synthetic polypeptide 1 or 2, the crude peptide derivative obtained was 1 N
  • dendrimer-type substance 3 of the present invention the substance of the present invention according to the present preparation method is referred to as dendrimer-type substance 3 of the present invention for convenience.
  • two disulfide isomers were obtained, they are represented as isomers 1 and 2, respectively.
  • Dendrimer-type substance 3 of the present invention 3 two kinds of disulfide isomers were obtained.
  • the AZT glutaric acid ester prepared in Preparation Example 3 was bonded to the Lys-introduced protecting group-protected polypeptide resin 2, and the same deprotection, resin removal, disulfide bond formation, isolation, Purification was performed to obtain the target substance of the present invention.
  • the substance of the present invention is referred to as “dendrimer type present substance 4”.
  • HIV human immunodeficiency virus
  • the antiviral activity against HIV of Invention Substance 1 and AZT was tested and evaluated according to the following method. That is, HIV-infected MT-4 cells (2.5 ⁇ 10 4 cells, Zwell, multiplicity of infection (MOI): 0.001) are added to a 96-well microtiter plate immediately after infection, together with various concentrations of the test substance.
  • MOI multiplicity of infection
  • C0 2 incubator one, they were cultured for 5 days at 37 ° C, the number of viable cells the MTT method (Pauwel et. Al., J. Virol. M ethods 20, 309-321 (1988)) was measured in.
  • Antiviral activity is expressed as the concentration that inhibits cell death by 50% by HIV infection (E Cso: 50 : effective concentration).
  • E Cso 50 : effective concentration
  • virus-uninfected cells were cultured with the test substance at various concentrations in the same manner as described above. Cytotoxicity is expressed as 50% cytotoxic concentration (C Cso: 503 ⁇ 4 cytotoxic concentration) by the test substance. Also CC 5 . And EC 5 .
  • the approximate ratio (CC 5; ZEC 5 ) was expressed as the selection coefficient (SI).
  • Table 3 shows the results of the anti-HIV activity measurement.
  • the anti-HIV activity of the substance 11 of the present invention was twice as high as the anti-HIV activity of AZT (EC 5. Comparison of values), the selection coefficient was more than twice, and the anti-HIV activity was clearly enhanced. It also has higher anti-HIV activity than synthetic polypeptide 1, which is a known anti-HIV active polypeptide. Furthermore, the dendrimer type substance 1 of the present invention showed the highest anti-HIV activity.
  • Table 3 Measurement of anti-HIV activity by MTT method
  • HIV infection inhibitory activity of synthetic polypeptides 1 and 2 obtained in Preparation Example 4 above, substances 1 to 22 of the present invention obtained in Preparation Examples 4, 5, and 6, and dendrimer-type substance 1 of the present invention, and AZ-cho. was tested and evaluated according to the following methods. That is, PHA (Phaseolus vulgaris agglutinin) activated P BMC (perip heral blood mononuclear cells) using T cell line tropic HIV-1 (NL 4-3) and macrophage tropic HIV-1 (JR-CSF) ) was measured by a p24 antigen expression suppression test by an ELISA method using an antibody against the HIV-1 p24 antigen.
  • PHA Phaseolus vulgaris agglutinin
  • P BMC peripheral blood mononuclear cells
  • T cell line tropic HIV-1 NL 4-3
  • macrophage tropic HIV-1 JR-CSF
  • dendrimer type substance of the present invention 1 88 7.3
  • a pharmaceutical composition having high anti-HIV activity can be provided by effectively inducing an anti-HIV active substance on target cells by affinity for CD4 positive cell surface protein and HIV surface protein.

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Abstract

La présente invention concerne des compositions médicamenteuses comprenant des complexes dans lesquels au moins un inhibiteur de transcriptase inverse et /ou inhibiteur de protéase de VIH sont chimiquement liés à des polypeptides présentant une affinité envers la protéine de surface gp120 du VIH et/ou la protéine de surface CD4 de la cellule hôte cible du VIH et pouvant par conséquent, en raison cette affinité pour la protéine de surface CD4 et la protéine de surface du VIH, amener les substances à activé anti-VIH vers les cellules cibles et déployer une activité anti-VIH élevée.
PCT/JP1998/001366 1997-03-28 1998-03-26 Nouveaux complexes anti-vih et compositions medicamenteuses WO1998043995A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6440946B1 (en) 1999-02-25 2002-08-27 Takeda Chemical Industries, Ltd. Multiple-agents-binding compound, production and use thereof
CN115057911A (zh) * 2022-05-20 2022-09-16 广州朗圣药业有限公司 一种hiv抑制剂

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02152932A (ja) * 1988-08-26 1990-06-12 Tosoh Corp 抗ウイルス薬及びその製法
JPH03206888A (ja) * 1989-11-13 1991-09-10 Green Cross Corp:The Ｈｉｖ抗原に対し特異性を持つマウス―ヒトキメラ抗体
JPH03504556A (ja) * 1987-05-29 1991-10-09 タノツクス・バイオシステムズ・インコーポレーテツド Ｈｉｖ‐１を中和するモノクローナル抗体
WO1992004374A1 (fr) * 1990-09-11 1992-03-19 Seikagaku Kogyo Co., Ltd. Nouveau polypeptide et medicament anti-vih prepare a partir de ce polypeptide
JPH05163298A (ja) * 1991-05-02 1993-06-29 Seikagaku Kogyo Co Ltd 新規ポリペプチドおよびこれを用いる抗ｈｉｖ剤
JPH08504837A (ja) * 1993-10-14 1996-05-28 生化学工業株式会社 新規ポリペプチドおよびこれから調製される抗ｈｉｖ剤
JPH0925240A (ja) * 1995-07-11 1997-01-28 Seikagaku Kogyo Co Ltd 医薬組成物

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03504556A (ja) * 1987-05-29 1991-10-09 タノツクス・バイオシステムズ・インコーポレーテツド Ｈｉｖ‐１を中和するモノクローナル抗体
JPH02152932A (ja) * 1988-08-26 1990-06-12 Tosoh Corp 抗ウイルス薬及びその製法
JPH03206888A (ja) * 1989-11-13 1991-09-10 Green Cross Corp:The Ｈｉｖ抗原に対し特異性を持つマウス―ヒトキメラ抗体
WO1992004374A1 (fr) * 1990-09-11 1992-03-19 Seikagaku Kogyo Co., Ltd. Nouveau polypeptide et medicament anti-vih prepare a partir de ce polypeptide
JPH05163298A (ja) * 1991-05-02 1993-06-29 Seikagaku Kogyo Co Ltd 新規ポリペプチドおよびこれを用いる抗ｈｉｖ剤
JPH08504837A (ja) * 1993-10-14 1996-05-28 生化学工業株式会社 新規ポリペプチドおよびこれから調製される抗ｈｉｖ剤
JPH0925240A (ja) * 1995-07-11 1997-01-28 Seikagaku Kogyo Co Ltd 医薬組成物

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6440946B1 (en) 1999-02-25 2002-08-27 Takeda Chemical Industries, Ltd. Multiple-agents-binding compound, production and use thereof
CN115057911A (zh) * 2022-05-20 2022-09-16 广州朗圣药业有限公司 一种hiv抑制剂

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