JPH01272597A - Anti-viral compound - Google Patents

Abstract

NEW MATERIAL: A compd. which is used for treating or preventing the infection of human immunodeficiency virus(HIV), checks or destructs the interaction between gp41 and gp120 envelope proteins of HIV particles in the contact region of the proteins when brought into contact with the particles, and has an amino acid sequence contg. a sequence represented by formula I or II or a part thereof.

USE: A therapeutic or prophylactic agent for aquired immunodeficiency syndrome(AIDS).

PREPARATION: For example, a benzohydrylaminepolystyrene/divinylbenzene resin is used as a solid carrier. Amino acids with their amino groups protected are bonded to the carrier according to the amino acid sequence of peptide in the presence of a condensation agent and then amino-protecting groups are removed. This operation is repeated to synthesize a peptide resin. Peptide is separated from the resin, and all the protecting groups are removed, thus giving a compd such as represented by formula I or II.

COPYRIGHT: (C)1989,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to compounds for use in the treatment or prevention of viral infections, particularly human immunodeficiency virus (HIV) infections.

[Conventional technology]

HIV has two envelope glycoproteins, gp120 and gp41, derived from a common precursor, p160 [Barre-Sinoussi
) et al., 1984;
Haran et al., 1984; Sham et al., 1985; Levy et al., 1984; Robey et al., 1985;
n) et al., 5science, 1985;
in) et al., 1985;
Nier) et al., 1983 and Veronese (Ver.
onesc) et al., 1985].

The larger glycoprotein gp120 is on the outside, and gp4
1 is the transmembrane spanning region (tran
smembrane 5panniB region)
has.

There is strong evidence that gp120 binds CD4 on the surface of T lymphocytes, macrophages and other cells [Dalgleish et al., 198
4; Klatzman et al., 1984
and McDougal et al., 198
6 years].

Monoclonal antibodies against CD4
Preventing or limiting the infectivity and replication of V (Popovic et al., 1984; and McDougal et al., 1985). The virus binds preferentially to the surface of CD4-positive cells, and virus binding is inhibited by monoclonal antibodies against CD4 [McDougal et al., 1985
Year〕. Additionally, virus-infected cells bind and fuse with uninfected CD4-positive T cells, forming syncytia (which can be inhibited by monoclonal antibodies against CD4).
syncytia) [Dalgleish (Da
IHleish et al., 1984]. Furthermore, in radioimmunoprecipitation experiments on lysates of virus-exposed CD4-positive T cells, CD4 molecules and gp120 coprecipitate as a complex.McDougal et al.
1986a). Human cell lines that do not express the CD4 molecule are not infected, but when they are transfected with the human cD4 gene and express CD4, they are able to bind and replicate the virus [McDougal et al., 1986b. ].

It is believed that the virus enters cells by envelope protein-mediated fusion of the virus with the target cell membrane following binding of gp120 to CD4. Expression of viral envelope proteins on the surface of infected cells mediates fusion with uninfected cells, giving rise to multinucleated giant cells or syncytia [Dalgleish et al.
984; KlatzIIlan et al.
1984; McDougal et al., 1
985a; Sodrosk i
et al., 1986; and Lifson et al.
1986].

Using deletion and insertion mutations, Kowalskii
(1987) investigated the role of envelope proteins in forming syncytia in CD4-positive cells. The mutation reduces binding of the envelope protein to the CD4 molecule and prevents post-binding fusion reactions, leading to anchoring of the envelope protein at the membrane, association of the two subunits of the envelope glycocoprotein, or translation of the envelope precursor protein. It has been shown to disrupt post-proteolytic processing.

There is no disulfide bond between gp41 and gp120, as reduction of the disulfide bond does not increase the release of gp120 from infected cells (McDougall et al.
gal) et al., 1986b). Many mutations in gp1
Kowalskii (
Kowa l-5ki) et al. (1987) reported that gp12
0 makes multiple contacts with the extracellular region of gp41, and gp1
This suggests that these contacts are important for their association after gp160 and gp41 are formed from the gp160 precursor. The authors suggest that gp120 acts as a trifunctional molecule that associates with gp41 through a site in the amino terminal half and with CD4 through the carboxy-terminal region. , the interaction of the carbonyl-terminal region of p120 with CD40 varies with conformation, as denaturation of gp120 eliminates binding of CD4 to the receptor [McDougal et al., 198
6th grade b]. Kowalski et al. (19
(87) mutational studies indicate that multiple conserved regions are critical for CD4 binding interspersed with the hypertuberculosis. Collectively, these observations also suggest that the amino acid sequence of gp120, which is responsible for interacting with the CD4 molecule, is discontinuous and varies by tertiary pathways for association with it.

Many attempts have been made to devise methods to treat and/or prevent HIV infection and its associated clinical manifestations.

[Problem to be solved by the invention]

The goal of antiviral therapy is to interfere with one of the key steps in the viral infection cycle. These steps include binding of the virus to host receptors, fusion to the cytoplasmic membrane, reverse transcription, integration or post-transcriptional steps;
There is release of virus or fusion of infected and uninfected cells. Since AIDS patients may have high levels of circulating antibodies, it is clear that this alone does not provide immunity once infected. To date, reverse transcriptase inhibitors such as AZT have provided favorable results in terms of prolonging the lives of AIDS patients, but drugs of this nature invariably have significant side effects.

The present inventors have unexpectedly discovered a compound that can suppress viral infection.

[Means to solve the problem]

Accordingly, one broad aspect of the invention provides compounds that can inhibit viral infection. These compounds can be peptides of varying length and amino acid composition, or non-peptide compounds such as drug molecules, which mediate the interaction between viral envelope proteins and receptors expressed on the surface of target cells. It is characterized by the fact that it can be destroyed. Disruption of the dark interactions of the viral envelope protein prevents this envelope protein from binding or associating with cellular receptors.
It inhibits the virus from entering target cells that have such receptors.

With respect to the AIDS (or HIV) virus, the present invention provides compounds for use in the treatment or prevention of human immunodeficiency virus (HIV) infection, which compounds
Upon contact with a V particle or infected cell, 8p41 of the particle or infected cell in the contact area between the proteins
and preventing or disrupting the interaction of 120 envelope proteins.

In particular, the compounds of the invention have the following amino acid sequence;
Eight to ERYLKD QQ,,.

qp120 ,,D MVE QMtfED I
l5LWD QSI, K PC,.

rib; qp41,,,IA GTK NLQARVT
^ IEK YLQD Q 81. .

qp120. ,,TVTE QAIED VW
HLFE TSIK PC,.

Preventing or disrupting the interaction of old v1 or old v2 gp41 and gp120 envelope proteins in the putative contact area defined by

Preferably, the compounds of the invention have the following amino acid sequence 2HEDIISLllIDQSLKPCVKLTPL
Or GIKQLQARILAVERYLKDQQ
or a compound having a portion thereof.

Compounds within the scope of the invention have the following peptide sequences:

++ED I 15L11 IDQsLK The amino acids of the above peptides can also be changed by substitution, addition or deletion, as long as the antiviral activity is not lost. HI
Non-peptide compounds that disrupt the interaction between V envelope protein pH41 and gp120 are also within the scope of the invention.

Due to the presence of hydrophobic residues in the gp120-gp41 putative contact region, it is possible to find drugs that can bind to this region and have the same effect as peptide binding. Hydrophobic substances are known to inhibit the action of calmodyulin [compounds such as chlorpromazine and trifluoroperazine bind to calmodyulin in the presence of calcium and inhibit its interaction with other proteins]. , Weis
ss) and Levin, 1978], therefore a similar range of substances would be expected to interfere with the gp120-gp41 interaction.

The wide variety of drugs that can be used in the present invention includes:

1. Compounds with basic functional groups that interact with acid residues in the contact region of gp120, including choline, amines,
There are compounds with a lysine or guanidine functional group.

2. Compounds with acidic functional groups that interact with basic residues in the contact region of gp41. These include compounds with carboxyl, sulfonate, sulfate or phosphate functional groups.

3. A compound that has a hydrophobic structure and binds to a hydrophobic region included in the contact region between gp41 and gp120. These compounds include phenothiazines, vimodin, heloberidol, chlordiazepoxide, phenylephrine, morphine, epinephrine, brostanic acid, phospholipids, acylglycerols, digei-I xygenin, benzamides, human rollazine, phenoxyisobutyric acid, benzothiazide,
May include steroids, probenecid, sulfinvirazone, and trioxalen.

The compounds according to the invention are useful for the treatment or prevention of clinical conditions associated with HIV retroviral infection, such as Kaboji's sarcoma, thrombocytopenic purpura, AIDS-related complexes, chronic neurological conditions, and patients with AIDS antibodies or AI
sero-positive for DS virus
e) is useful for patients with Related compounds of the invention are:
It is also useful in the treatment or prevention of related viral infections, such as infections with IIIV2.

According to a broader aspect of the invention, there is provided a method for preventing or preventing viral infections, comprising the use of an effective amount of a compound characterized by the ability to disrupt interactions between viral envelope proteins important for viral uptake into target cells. A method is provided comprising administering to a patient. AIDS (HIV
) For viruses, these compounds may be peptides or non-peptide compounds such as drugs that disrupt the interaction of the envelope proteins gp41 and gp120.

The compounds of the invention may be administered orally, topically or parenterally, and may be administered alone or in combination with one or more pharmaceutically acceptable carriers or excipients for the intended administration. Depending on the mode, the compounds used may be in solid, semi-solid or liquid dosage forms, such as tablets, pills, capsules, powders, liquids, suspensions, etc., preferably in unit doses suitable for single administration of precise doses. Compositions containing a compound of the invention may be administered in the form of a compound of the present invention, including conventional pharmaceutical carriers or excipients, and in addition other pharmaceutical compounds,
Such excipients, which may include pharmaceutical agents, adjuvants, etc., include other proteins such as human serum albumin or plasma preparations.

- In the shell, suitable dosage caps for the active compound are in the range - 3.0 to 120 mB per patient weight per day, preferably 6 to 90 mB per patient weight per day, most preferably 15 mB per patient weight per day. ~60mg. The desired dose is preferably presented as 2, 3, 4, 5, 6 or more sub-doses administered at appropriate intervals throughout the day. These secondary doses may be, for example, from 10 to 1500 mg of active ingredient per unit dosage form, preferably from 20 to 1 ooo*g, most preferably from 50 to
It may be taken in unit dosage form containing 700 mg.

Preferably, the dosage should be taken such that the peak plasma concentration of the active compound is from about 1 to 75 μH, preferably from about 2 to 50 μH, most preferably from about 3 to about 30 μH.

It goes without saying that the amounts mentioned herein are not absolute and may vary depending on the patient being treated, the severity of the symptoms, the method of administration, and the judgment of the prescribing physician.

For solid compositions, common non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch or magnesium stearate.

Liquid pharmaceutically acceptable compositions include, for example, a compound of the invention and an optional pharmaceutical adjuvant, such as water, saline,
They can be prepared by forming solutions or suspensions, such as by dissolving or dispersing in carriers such as aqueous dextrose, glycerol, ethanol, and the like.

If desired, the pharmaceutical composition to be taken may contain small amounts of non-toxic auxiliary substances, such as wetting agents or emulsifying agents, p) lult
It may also contain reagents such as sodium acetate or sorbitan monolaurate. Practical methods of preparing such dosage forms are known, or will be apparent to those skilled in the art, (e.g., 畦匡畦J 体岨, Hack Publishing Company,
(See Easton, Pennsylvania, 15th edition, 1975).

Patients infected with the AIDS virus can be treated with the compounds of the invention and the methods of the invention for an extended period of time, for the remainder of the patient's life. Such treatment can prevent the virus from spreading to uninfected cells. Furthermore, the viral DNA incorporated in the genetic material of the host cell (in inactive subject form) prevents it from inducing the spread of the virus particle by activation at a somewhat later stage. Virus particles released from the virus rapidly lose their viral infectivity due to disruption of the interaction between gp41 and gp120.

Humans susceptible to exposure to AIDS viruses such as hemophilia can be treated with the compounds of the invention and the methods of the invention. For example, any virus particle encountered by a human quickly becomes non-infectious.

The putative contact sequences for gp41 and gpizo were
Details are shown in Table-1.

Table 1 Amino acid sequences of envelope proteins are Mayer (M
yer) et al., 1988, isolate PV22, H
IV 2 is taken from isolate ROD and SIVMAC from SIVMM142. Correspondingly charged residues are underlined and amino acid residue substitutions found in other isolates are indicated in parentheses. The single letter code for amino acids is as follows:

A is alanine, C is cysteine, D is aspartic acid,
E is glutamic acid, F is phenylalanine, G is glycine, H is histidine, ■ is inleucine, K is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, ■ is valine, W is tryptophan and Y is 1,000 rosin.

The contact sequence is characterized by a set of complementary repeating charged residues, especially glutamic acid (E), aspartic acid (D), arginine (R) and lysine (K) interspersed between other amino acids. These four harmoniously charged residues, extending over 16 amino acids, are conserved between strains IIIV1 and IIIV2. The spacing or number of amino acids between each pair of residues (4.5
．． 3 residue intervals) are conserved. This pattern is also observed for the closely related simian immunodeficiency virus (see Table 19), where the corresponding gp41 and gp120 contact sequences are arranged in an α-helical configuration.
A corresponding amphipathic helix is created between a pair of charged residues and an associated hydrophobic residue. The space is 3.6 residues per turn of the helix or 3. . Preliminary computer graphic analysis of the proposed contact configuration, which does not correspond exactly to three residues per turn of the helix, shows that the structures are highly complementary to the contact sequences identified by the present invention. is novel and specific, defines a single primary contact sequence,
The core of this sequence is contained within the 16 residue pairs of the sequence and can accommodate as many as 3 out of 4 harmonious charge pair interactions.

The confirmation of the contact area described herein is surprising;
Firstly, we learned that a peptide corresponding to a portion of the gp120 sequence has antiviral activity (see discussion below), and secondly, the antiviral sequence is related to any other sequences found in envelope proteins. This was due to the question of whether or not to do so. Examination of the gp41 sequence surprisingly revealed that it has a structure complementary to the antiviral peptide sequence.

Twiteno residues 571-591 in gp41()IIV1
) (7) The contact sequence is part of the sequence identified by Wag et al. (1986) and the major epitope and other residues (residues 579-601) and is Viraemic patients are those who have antibodies in this region.

The corresponding region in gp120 (D” to C19) contains the important THi cell epitope identified by Cease et al. (1987). This T cell epitope was created by computer algorithms and Kaiser et al. (1984) proposed a role for amphipathic helices in various peptides and proteins, including calcitonin. T and B cell epitopes are associated with contact regions. Moreover, since the epitopes are recognized by the immune surveillance system, they are likely to be exposed, and therefore the corresponding synthetic peptides have a region corresponding to the CD4 binding region. Kowalski, which is not expected to have antiviral activity unless it is derived from
, 1987, and the gp1 described herein.
It is clear that the 20 putative contact sequences originate from regions distant from the CD4-binding region. 0 The potent and T-cell proteins identified in gp41 and gp120 overlap with the contact regions described in this document. Epitopes have been proposed as potential key regions in vaccine development. On the other hand, the identification of contact areas performed in the present invention suggests that these cannot be achieved with intact virus particles. Masking of B and Tm cell epitopes in the contact area of infectious particles explains why patients sick with the virus cannot be protected from disease progression if the patient has circulating antibodies. Since the loss of gp120 from an infectious particle alone, rendering it non-infectious, generates an immune response against the major epitope in gp41, the antibodies generated will recognize the non-infectious particle and therefore provide protective immunity. cannot be provided.

The invention will now be illustrated by way of example only in the following non-limiting examples.

〔Example〕

Peptides can be prepared in a variety of ways, including solution or solid phase methods. A variety of manual, semi-automatic and automated synthesis equipment are commercially available and can be used according to known protocols. The peptides referred to herein are derived from automated applied biosystems.
Synthesize using the Merrifield method on a biosystem synthesizer (Holladhes and Merrifield, 1975).
.

Peptides were collected on benzhydrylamine polystyrene/divinylbenzene resin. Benzyl side chain protection and BOC
α-amine protection was used. All residues were added by the direct dicyclohexylcarbodiimide method, except that glutamine, asparagine and arginine were coupled as hydroxybenzotriazole esters. Dinitrobenzo and formyl protecting groups for histidine and tryptophan, respectively, are 20% (
Volume/Volume) 2-Mercaptoethanol was removed by treatment of the peptide resin amide with 1M ethanolamine/dimethylene formamide. Stewart and Yo wanted to remove the collected peptides from the resin and simultaneously deprotect them by anhydrous HF.
ung), 1975), Yuhina peptide, 60% (
Volume/Volume) Acetonitrile - 0,1% (Volume/Volume)
Extracted with trisfluoro#acid, rotary evaporated and lyophilized. The crude peptide was transferred to a low pressure column (2.5 x 30 cm, Amicon (3mlcon)).
Purified by reverse phase chromatography on C18 reverse phase resin, 445-701t, 250^ porosity) with a gradient end of 0-60% acetonitrile. The purified peptides were lyophilized and characterized by high pressure liquid chromatography and amino acid analysis. For extraction and purification of peptides suitable for therapeutic use, non-toxic solvents are required, as will be apparent to those skilled in the art.

In this example, the following peptide 99120 (105-125) HED (■SLWD
QSLKPCVKLTPLgp120 (105-11
7) tlEDIIslJDQsL and gp41
(572-591) IKQLQARILAVE
RYLKDQQ.

Explain the antiviral activity of.

Viral activity was induced in peripheral blood lymphocytes from patients with acute lymphoblastic leukemia (OEM cells) by particle-associated reverse transcriptase (R'r) activity and virus-associated syncytium formation (CPE). Evaluation was made using blood lymphocytes. The HIV isolate used in all experiments was HTLV-11.
It was lb.

OEM cells (4X 105/ml) at 29.2 μg/ml
RPMI6 containing ml glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin 2 mg/…l polybrene and 10% heat-inactivated fetal calf serum.
Preincubation was performed for 1 hour in 5% CO□ at 37° with synthetic peptides, /+l) in 40 medium. After preincubation, HTLF l
1ltl H9 cell-free supernatant using MT4M cells,
0.0005 PF (1/cell) was added for 2 h in multiple infections at 37°C according to the plaque method.

The virus inoculum and peptides were removed by washing once with Eagle's BME medium. CEM cells, total volume 2.0
Resuspend in fresh vehicle containing peptide (100 μg/ml) in Il+l.

Cells were harvested at 1 p.i. on days 4, 7.11 and 14 post-infection.
．． 0ml cell-free supernatant was supplied by replacing with fresh peptide-containing vehicle solution (100μg/ml). Cell-free supernatants (500 μm) were analyzed for reverse transcriptase activity. A positive control with no peptide and a non-specific peptide corresponding to gizzard myosin light chain kinase (RRKWQTGSAV) was used in this experiment along with a negative control without virus for cytotoxicity. Four sets of cell cultures were analyzed. The added synthetic peptide did not exhibit any cytotoxicity as cell viability and number were equal to cultures without the peptide.

The gp120 and gp41 test peptides showed significant virus inhibition as measured by inhibition of viral reverse transcriptase activity. Two orders of magnitude of virus suppression was observed. The control peptide showed no viral activity.

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Claims (1)

[Scope of Claims] 1. A compound used for the treatment or prevention of human immunodeficiency virus (HIV) infection, which comprises contacting with HIV particles and causing interaction between the gp41 and gp120 envelope proteins of the particles. A compound characterized in that it is able to prevent or destroy in the contact area of said protein. 2. Claim 1, wherein the HIV infection is an HIV I infection.
Compounds described in. 3. The compound according to claim 2, wherein the prevention or disruption of interactions between said envelope proteins is effected in the contact region defined by the following amino acid sequence: [there is a gene sequence]. 4. The compound according to claim 1, wherein said HIV infection is an HIV II infection. 5. The compound according to claim 2, wherein the prevention or disruption of the interaction between the envelope proteins is effected in the contact region defined by the following amino acid sequence: [there is a gene sequence]. 6. The compound of claim 3 or 5, which mimics at least a portion of the amino acid sequence of either gp41 or gp120 protein. 7. The compound according to claim 6, which contains the following amino acid sequence: [there is a gene sequence] or [there is a gene sequence] or a part thereof. 8. The compound according to claim 7, comprising the following amino acid sequence: [There is a gene sequence]. 9. having a basic functional group capable of interacting with acidic residues of gp120 protein in the contact region;
A compound according to claim 1. 10, having an acidic functional group capable of interacting with the basic residue of gp41 protein in the contact region;
A compound according to claim 1. 11, gp41 and/or g in the contact area
2. The compound according to claim 1, having a hydrophobic functional group capable of interacting with a hydrophobic region of p120 protein. 12. Acquired immunodeficiency syndrome (AIDS) or AID
2. A compound according to claim 1 for use in the treatment or prevention of S-related complex (ARC). 13. Used for the treatment or prevention of HIV-positive individuals,
A compound according to claim 1. 14. The following amino acid sequence: [There is a gene sequence] or [There is a gene sequence] or a peptide compound that has a part thereof. 15. A peptide compound with the following amino acid sequence: [There is a gene sequence]. 16. A pharmaceutical composition comprising a compound according to claim 1 and at least one pharmaceutically acceptable carrier or excipient. 17. A pharmaceutical composition comprising a peptide compound according to claim 14 or 15 and at least one pharmaceutically acceptable carrier or excipient therefor.