WO1996029067A1

WO1996029067A1 - Anti-viral and anti-cancer agents

Info

Publication number: WO1996029067A1
Application number: PCT/GB1996/000650
Authority: WO
Inventors: Washington Odur Ayuko
Original assignee: Radopath Limited
Priority date: 1995-03-17
Filing date: 1996-03-18
Publication date: 1996-09-26
Also published as: AU5115396A

Abstract

2-Chloro-5-nitrobenzoic acid and various other chloro and nitro substitute benzoic acid derivatives are disclosed for administration in tablet or injectable solution form for treatment of HIV infection. The active compounds have surprising efficacy in various treatment regimes and have particular application to effect regression of lesions in HIV-related Kaposi's sarcoma. The compounds act as costimulators of CD3-triggered T-lymphocyte proliferation to increase the immunological challenge to the retrovirus presented by the natural immune system.

Description

ANTI-VIRAL & ANTI-CANCER AGENTS

The present invention relates to arylating agents, in particular phenylating agents. which are suitable as therapeutic compounds in the treatment of disease caused by viral infections (eg retrovirus infections such as HIV). As shown by studies reported herein, the compounds of the invention show marked activity in assisting the human immune system to challenge HIV infection and specificially in AIDS and pre-AIDS therapy show the capacity to regress Kaposi's sarcoma, improve CD4 count in humans, increase the level of various blood parameters in murine patient models and to achieve weight gain and general patient condition by immunological action against HIV and/or its effects (eg opportunistic disease).

The invention provides use of a physiologically non-toxic reactive oxygen intermediate species (ROS) oxygen donor substance as defined below for the manufacture of a medicament for use in the proliferation of lymphocytes, eg the treatment of HIV infection, in a mammalian subject, the ROS being a substance having the following functional characteristics:-

(i) its molecules are relocatable from serum to T-lymphocyte cytoplasm: and

(ii) its molecules modify the intracellular signal transduced from the extracellular event comprised of interaction in CD4 cells between the T-cell CD3 receptor and MHC-transported peptide fragments of target cell surface target cell intracellular protein, the target cell being an antigen presenting cell which, in the case of an HIV patient, is cell infected with HIV, the modified signal, at least in the presence of a second intracellular signal transduced from the extracellular event comprised of interaction between the T-cell CD28 co-receptor with target cell B7 immunoglobulin. producing enhanced intracellular DNA synthesis and/or enhanced tyrosine kinase activation occassioning tyrosyl phosphorylation of protein substrates inhibitory of cytokine or cytokine receptor expression of eg interleukin-2 and/or interleukin-2 receptor by the IL-2 gene whereby the proliferative response of the lymphocyte is increased; and

(iii) The ROS is a compound comprising an aromatic (eg monocyclic) nucleus which is (a) substituted with a carboxylic moiety of formula -COOX in which X is a cation (eg an alkali metal cation) and (b) optionally substituted with one or more further substituents (eg halogen or nitro).

According to the invention, there is in particular provided use of a benzoic acid derivative (BAD) as defined below for the manufacture of a medicament for use in the treatment of HIV infection in mammalian subjects by costimulation of the proliferative response of T-lymphocytes to receptor triggering by a mechanism which comprises:-

(i) activating a T-lymphocyte by first and second intracellular signals delivered by a target antigen presenting cell in the form of a cell infected with HIV, the first signal being transduced by the T-cell CD3 receptor by interaction thereof with MHC- transported peptide fragments of intracellular protein and the second signal being transduced by the T-cell CD28 co-receptor by interaction thereof with target cell B7 immunoglobulin;

(ii) penetrating the T-lymphocyte cytoplasm by molecules of BAD;

(iii) generating by means of BAD molecules and in the T-lymphocyte cytoplasm a reactive oxygen species (ROI) by donation from the BAD molecules; and

(iv) promoting the first signal to effect tyrosine kinase activation and tyrosyl phosphorylation of protein substrates inhibitory of expression of interleukin-2 by the T-cell IL-2 gene; the BAD having the formula:

COORg IB

R.

R, o R<

R.

wherein R₍, R₂, R₃, R and R₅ are. each independently hydrogen. C₍ to C₄ alkyl, nitro or halo, and wherein R₄ is hydrogen, an ester-forming moiety or a salt-forming moiety; subject to the following two provisos:-

(a) at least one of R| , R₂, R₃, R₄ and R₅ is other than hydrogen, and

(b) the halo atom(s) when present is (are) chlorine or bromine.

The BAD may in particular be a compound of the following general formula:-

COOH

wherein X, and X₂ are, each independently, hydrogen or chloro or bromo and wherein X₃ and X₄ are, each independently, hydrogen or nitro, provided that at least two of X,, X₂, X₃ and X₄ are other than hydrogen, or a salt or ester thereof (eg a salt wherein -COOH is substituted by -COOR in which R is optionally substituted amino). Conveniently, X, is chloro or bromo. X₂ is chloro or bromo or hydrogen, X₃ is nitro and X₄ is nitro or hydrogen. The following are preferred compounds:-

2, 4-dichloro-3, 5-dinitrobenzoic acid 4-chloro-3, 5-dinitrobenzoic acid 2-chloro-3. 5-dinitrobenzoic acid 2-chloro-5-nitrobenzoic acid and its sodium salt 2, 5-dichlorobenzoic acid 2. 4-dinitrobenzoic acid 3, 5-dinitrobenzoic acid

2-bromo-5-nitrobenzoic acid and its sodium salt 2-nitrobenzoic acid and its sodium salt 2, 5-dichloro-4-nitrobenzoic acid

2. 4-dichloro-5-nitrobenzoic acid 2. 6-dichloro-4-nitrobenzoic acid

3. 5-dichloro-4-nitrobenzoic acid 4-chloro-3-nitrobenzoic acid 2-chloro-4-nitrobenzoic acid

3. 4-dichlorobenzoic acid

Since treatment in accordance with the invention is by what is believed to be an arylating mechanism, use is typically at relatively high concentrations and. consequently, doses. Generally, the recommended dosage is lmg/kg body weight to 30mg/kg body weight per day for 5 days weekly.

The compounds of the invention may be prepared by known process techniques for preparing benzene substituted compounds. Such techniques are described in various standard texts, for example, "Organic Syntheses" 1963 Collective Volume 4, pages 364 to 366, by Harry P Schultz and published by John Wiley and Sons Inc. Post- preparation sterilization may be required, at least following formulation of the compounds into pharmaceutical composition form. The compounds of the invention may be formulated for use as pharmaceutical compositions (eg for iv, ip, oral or sc administration) comprising at least one active compound and a diluent or carrier. Thus, the invention includes a pharmaceutical composition, which composition comprises a compound according to the invention and a pharmaceutically-acceptable diluent or carrier (eg aqueous).

Such a composition may be in bulk form or, more preferably, unit dosage form. Thus, for example, the composition may be formulated as a tablet, capsule, powder, solution or suspension. Soft gel capsules may be especially convenient. The composition may be a liposomal formulation or administered in a slow sustained release delivery system.

Compositions in accordance with the invention may be prepared using the active compounds defined herein in accordance with conventional pharmaceutical practice. The diluents, excipients or carriers which may be used are well known in the formulation art and the form chosen for any particular regimen will depend on the given context and the physician's choice.

Thus, for example, as illustrated below the compounds of the invention may be administered in solution in sterile deionised water. Also, if necessary, solution may be facilitated using dimethyl sulphoxide (DMSO) or alternatively an alcohol, a glycol or a vegetable oil. The compounds are most favourably administered in corn oil or as a solution in DMSO/sterile water. In the case of the use according to the invention of sodium salts of the active compounds, the active ingredient is preferably administered as an aqueous solution.

In using a compound of the invention, dosage guidance can be taken from animal studies such as that described below. In such studies doses of from about 50mg/kg typically up to about lOOOmg/kg (eg up to about 400mg/kg. conveniently about 200mg/kg or less). Thus it is to be expected that a typical dosage for humans will be from about 5mg/kg upwards (eg up to about 20mg/kg). The concentration and dose are to be sufficient to bring an arylating mechanism into play. For intravenous administration the active compound will be dissolved or dispersed in an aqueous or other injectable liquid bearing in mind the aforementioned requirements of dosage. Typically, concentration will be 250mg/ml or less. Patient tolerance was found to be of a higher order for concentrations below that threshold although concentrations of more than 250mg/ml may be used. Typically, a preferred concentration is less than lOOmg/ml. with concentrations below 50mg/ml (eg 40mg/ml) being preferred. For oral administration, enteric coated tablets will often be preferred. Maximum strength will be less than 500mg and preferably 400mg or less. Thus, for example, a range of strengths for oral administration (whether in tablet or other solid form) will be lOOmg, 200mg or 400mg. Patients who are subject to oral administration will generally fast for 8 hours prior to first dose with the fast continuing for 4 hours after first dose. Free access to fluid will generally be allowed during this fasting period. Intravenous treatment will generally take place by administration very slowly over a period, typically a period of approximately 20 minutes or more. Administration will typically be by catheter, for example a catheter left in situ after flashing with 2ml of saline. Of course, the catheter will in practice then be used for taking blood samples for pharmacokinetic measurements and removed prior to the patient's departure from the clinic.

As shown by the results reported below, 2-chloro-5-nitrobenzoic acid in the form of its sodium salt in particular shows considerable activity in vivo. This and the other compounds according to the invention operate as immunomodulators.

2-Chloro-5-nitrobenzoic acid sodium salt has been found potently to costimulate the anti-CD3 induced proliferation of human peripheral blood mononuclear cells (PBMC) in a dose dependent manner (p=0.001). Anti-CD3/compound costimulation induced the tyrosyl phosphorylation of a number of cellular protein substrates. The costimulatory effect was eliminated when PBMCs were simultaneously incubated with the antioxidant butylated hydroxytoluene. The inhibition of proliferation was associated with a corresponding decrease in tyrosine kinase activity. Studies with blocking monoclonal antibodies against B7-1 or B7-2 indicate that the immunopotentiatory effect of the compound requires the macromolecular interaction between CD28 (a costimulatory receptor on T-cells) and its counter receptor B7 expressed on antigen-presenting cells. It is thought that CNBA acts intracellularly through the generation of reactive oxygen intermediates which in turn activate signalling pathways that are involved in gene expression and cell cycle regulation.

Various oxidants including hydrogen peroxide (H₂O₂) have been shown to induce both cytoplasmic [1,2,3] and nuclear [4] signalling events that are involved in gene expression and surface receptor modification. As an oxygen free-radical donor, 2- chloro-5-nitrobenzoic acid sodium salt has selectivity in promoting lymphocyte proliferation when used in combination with an anti-CD3 activating antibody. Like H₂O₂, the compound has a marked costimulatory effect on protein tyrosine kinase activity, resulting in an antioxidant-sensitive increase in tyrosyl phosphorylation of various intracellular protein substrates.

Preparation of PBMC and lymphocyte costimulation assay.

PBMCs were isolated using Ficoll-Paque gradient from Buffy coat preparations obtained from blood donors (North East London Transfusion service) and cultured in pyrogen-free RPMI-complete medium as described [5]. Flat-bottomed microtitre plates were precoated with goat anti-mouse IgG Fc(Fab')2 fragments (Sigma, Poole, England) (10 μ/ml; 200 μ/well in sodium bicarbonate buffer, (pH 9.6) overnight at 4°C. Each treated well was washed twice with phosphate buffered saline (PBS). An azide free anti-CD3 mAb (5 μg/ml) (UCHT1), Autogen Bioclear, Potterne. England) was immobilised in the wells for 4h at 4°C, and the plates were washed twice with PBS. 2x10⁵ PBMC (in 200 μl) were then added to each well. Compound C 17 as identified hereinafter (see Table 3.1) was dissolved in water as its sodium salt and added to cultures at a final concentration of 1 -600 μg/ml. All samples were in triplicates. Controls included cultures containing PBMC alone and PBMC stimulated with anti-CD3 mAb only. Plates were incubated in a humidified 5% CO₂ atmosphere. Proliferative responses were measured on day 3 by pulsing the cultures with - .5μCi/well of [methyl ³H] thymidine 18h before harvesting of cells for the measurement of radiolabel incorporated into newly synthesised DNA. The effect of a lipid-soluble antioxidant butylated hydroxytoluene (BHT) on anti- CD3/C17-costimulated lymphocyte proliferation was examined by the simultaneous addition of various concentrations of BHT (1,5,10 and 20 μg/ml) into PBMC cultures.

B7-1 (CD80) and B7-2 (CD86) molecules on antigen-presenting cells (APC) are ligands for CD28. a major costimulatory receptor on T lymphocytes [6]. The effects of their blockade on anti-CD3/C17 costimulation were investigated by preincubation of lymphocytes with azide-free anti-B7-l or anti-B7-2 mAb (20 μg/ml) (Autogen Bioclear. Potterne. England) at 4°C for 30 minutes prior to transfer to anti-CD3 antibody coated wells.

Cell stimulation and preparation of cell extracts for Western blotting

PBMC (8-9 x 10 in 1 ml) were stimulated for 10 min in 24-well tissue culture plates (Life Technologies. Paisley, England) precoated with an anti-CD3 mAb (UCHT-1), 5 μg/ml) as described in section 2.1. Prior to anti-CD3 stimulation, cells were incubated in serum-free RPMI medium containing 300 or 600 μg/ml CNBA for 20 min at 37°C. The effect of antioxidant was examined by preincubating PBMC, before addition of CNBA in serum free medium containing BHT (5 μg/ml) for 30 min at 37°C. Following a 10 minute incubation in the anti-CD3 antibody-coated wells, reactions were terminated by the addition of 1 ml stop solution (cold PBS containing 5mM EDTA, lOmM NaF, lOmM Na pyrophosphate and 0.4mM Na vanadate). Cells were pelleted and then lysed in hypotonic buffer (20mM HEPES-NaOH [pH 7.4], 5mM KCI, 5mM MgCl₂. 5mM dithiothreitol, 0.1 mM EDTA. 50 mM NaF, 10 μg/ml aprotinin and 0.1% Nonidet P-40) at 4°C for 30 min. After removing insoluble material by centrifugation at 20,000g for 10 min, supematants each containing 15 μg of protein (measured according to Bradford [7]) were separated on a 10% Tris-glycine gel under reducing conditions and electrophoretically transferred to PVDF membrane, then probed with an anti-phosphotyrosine mAb 4G10 (1 μg/ml (UBI, Lake Placid. NY, USA) followed by a horseradish peroxidase-conjugated goat anti-mouse Ig(l :4000) (Sigma). The immunoblots were developed using the ECL detection system (Amersham International. Little Chalfont, UK).

Statistical analysis

Statistical analysis was performed using the SPSS/PC+ Statistics 4.0 package (SPSS, Inc.. Chicago, Illinois, USA) as specified in the results below.

Results

C17 was shown in the above to augment anti-CD3-stimulated lymphocyte proliferation in a dose-dependent manner (Figure 1 ). The mean increases in ³H- thymidine uptake were 25% (p=0.025) and 56% (p=0.001) respectively for anti- CD3/C17 (300 μg/ml) and anti-CD3/C17 (600 μg/ml) compared to anti-CD3 stimulation alone. (p=0.025 and 0.001 for the corresponding increase by Student's t- test). C17 alone did not significantly stimulate ³H-thymidine uptake in resting PBMC. Table 1 below shows the results for a number of assays using PBMC from different blood donors (n=9). A representative of three studies with the antioxidant BHT at 20 μg/ml is shown in Figure 2: BHT abolished the costimulatory effect of C17 (300 μg/ml) on anti-CD3-induced lymphocyte proliferation (p=0.004 by the

Mann- Whitney test). Since BHT was solubilised in ethyl alcohol (final concentration 1%). the effect of 1 :100(v/v) alcohol an anti-CD3-induced proliferation was investigated and found not to be significant.

Table 1

The average and standard deviations values of anti-CD3/C17 induced thymidine uptake in PBMC are shown for a number of different blood donors. C17 was used at concentrations of 300 and 600 μg/ml. Results were pooled and statistical analysis was carried out. AssayNo. Anti-CD3/17 Standard Anti-CD3 Standard

(300ug/ml) deviation aloneAverage deviation

Averagecpm cpm

1 101787 981 82626 2929

2 92981 11873 57720 1744

3 132343 1551 90549 13247

4 99686 4688 86829 3677

5 101895 4436 85518 7417

6 134635 2712 117128 10584

7 112476 2098 88441 7013 (600ug/ml)

8 126112 5308 88441 7013

9 158199 9795 102089 6081

10 126804 16529 57720 1744

11 144037 18714 117128 10584

Immunoblot studies show that the addition of C17 augmented anti-CD3-inducted tyrosyl phosphorylation of a number of protein substrates (molecular weights between 56-127 kDa) in a dose-dependent manner. This costimulatory effect was negated in cells that had been pre-incubated with BHT (5 μg/ml). Figure 3 shows a representative of three experiments using PBMC from different donors. An increase in the phosphotyrosine content of a 57kDa tyrosine kinase substrate (pp57) was consistently found following anti-CD3/C17 costimulation (Figure 3a). Figure 3b shows the antioxidant-inhibitable increase in tyrosyl phosphorylation of pp57 by densitometric analysis, using the Image Analysis Systems (Bio-Rad Laboratories. Hercules, CA, USA) in conjunction with the Molecular Analyst/PC software. BHT was dissolved in DMSO (final concentration 1 :2000). RPM1 medium containing 0.05% (v/v) DMSO had no demonstrable effect on tyrosine kinase activity of PBMC compared to medium alone (data not shown). Also, in the absence of anti-CD3 stimulation, C17 alone did not induce any significant increase in tyrosine kinase activity (data not shown).

Prior blockade of B7-1 (Figure 4a) of B7-2 (Figure 4b) with specific antibodies also appeared to eliminate the costimulatory effect of C17 on anti-CD3 -induced H- thymidine uptake, indicating that the latter requires the macromolecular interaction between CD28 on T-lymphocytes (and/or CTLA-4 on activated T-cells [6] and its ligand B7 expressed on APC. (p=0.004 by the Mann- Whitney test for both anti- CD80-and anti-CD86-mediated inhibition of anti-CD3/C 17 [300 μg/ml]-induced lymphocyte proliferation).

As will be appreciated from the foregoing, 2-chloro-5-nitrobenzoic acid sodium salt has a significant effect on anti-CD3-induced lymphocyte proliferation in a dose dependent enhancement on the cytoplasmic tyrosine kinase activity. C 17-induced enhancement of DNA synthesis and tyrosyl phosphorylation of cellular protein substrates was shown to be inhibited by a lipid-soluble antioxidant BHT. It is apparent that C 17 acts by generating reactive oxygen intermediates which have potent stimulatory effects on a number of intracellular signal transduction targets. This concept is supported by findings in relation to a number of chloro-. nitro- and methyl- substituted benzoic acid derivatives and inhibition of the costimulatory function ofC17 by BHT.

A number of studies have demonstrated that butylated hydroxyanisole (BHA), a lipid- soluble antioxidant similar to BHT, inhibits the interleukin-2 receptor (IL-2R) expression and proliferation of T-lymphocytes stimulated by mitogens or in mixed lymphocyte reactions (MLT) [8,9]. Recently, the expression of the IL-2Ra gene encoding the a chain of the IL-2R (CD25) was shown to be regulated by the binding of the transcription factor NF-kB (and its composition) to the kB element of the IL-2R gene promoter region [10]. NF-kB belongs to the Rel family of proteins which share a highly conserved 300-amino acid domain (Rel homology domain) [1 1]. In most cells these proteins are cytoplasmic and exist in an inactive complex with inhibitory proteins of the I-kB family. T-cell stimulation with phorbol ester, a non-specific mitogen, promotes NFkB activation possibly by inducing the phosphorylation and dissociation of I-kB and hence allowing the nuclear translocation of NFkB [4,12]. The activation of NFkB in T-cells [4] and other Rel proteins including Rel A in astrocytes [13] appears to be dependent on the generation of oxygen free radicals.

Figure 5 presents a model of how C17 is thought to exert its immunopotentiary effects on T lymphocytes by acting on a number of signal transduction targets, through the generation of reactive oxygen intermediates (ROI). Enhancement of tyrosine kinase activity by C17 possibly the generation of ROI may improve the coupling of surface receptors such as TcR CD3 complex to downstream signalling events including the breakdown of inositol lipid to generate secondary biochemical messengers [14]. Tyrosine phosphorylation may also lead to the inactivation of protein serine-threonine phosphatase PP2A [15] which has been postulated to have a negative regulatory effect on NF-kB activation [16].

Anti-B7 blocking studies indicate that the potentiatory effect of C17 on anti-CD3 induced proliferation of PBMC requires the presence of costimulatory signals provided by CD28. CD28 ligation by B7-1 has also been shown to increase the tyrosyl phosphorylation of cellular substrates in activated T-cells [reviewed in 6]. Rather than providing the initial biochemical signal, C17 predominantly acts intracellularly by modifying downstream events. Its mode of action differs from tucaresol [(4(2-formyl-3-hydroxy-phenoxymethyl)benzoic acid)] which costimulates T-cells extracellularly by forming a Schiff base on CD4⁺T-cell-surface amines ( 17).

2-Chloro-5-nitrobenzoic acid sodium salt has potent T-cell costimulatory effects and at the same time a good safety profile in animal studies (950mg/kg/day and 900mg/kg/day are the MTDs for mice and rats, respectively)

Figure 1 shows the effect of anti-CD3/C 17 on PBMC proliferation. Each point on the graph is the mean of three triplicates. The bars represent the standard deviation. Similar results have been obtained on eight separate occasions. Values for both anti- CD3 stimulated and unstimulated cells in the absence of C 17 are shown as broken lines.

Figure 2 shows the effect of adding butylated hydroxytoluene (1-20 μg/ml) to PBMC cultures and shown to inhibit C17-costimulated increase in anti-CD3-induced lymphocyte proliferation. The effect was most pronounced at 20 μg/ml. Figure 3a shows as a Western blot the dose-dependent anti-CD3/C17-costimulated increase of tyrosyl phosphorylation of cellular protein substrates (56-127 kDa) in donor PBMC. Inhibition of C17-induced tyrosine kinase activity by a lipid-soluble antioxidant BHT (5 μg/ml). Concentrations of Cl 7 were measured in μg/ml. Lanes 4-6 show the effect of anti-CD3/C 17 stimulation. Lanes 4-6 show the effect of anti- CD3/C17 in the presence of BHT. Maximum phosphorylation of protein substrates is shown in lane 4 with C 17 at a concentration of 600 μg/ml.

Figure 3b shows densitometric analysis of the phosphotyrosine content of a 57kDa tyrosine kinase substrate (pp57) following anti-CD3/C 17 costimulation with or without the addition of BHT.

Figures 4a and 4b both show that anti-B7-l(CD80) and anti-B7-2(CD86) (20 mg/ml) both significantly reduced the effect of anti-CD3/C17 costimulation back to baseline control levels.

Figure 5 is a schematic diagram of a T-lymphocyte activated through its surface receptors in the presence of C17-2-chloro-5-nitrobenzoic acid, indicating the hypothetical target sites for C17 through the generation of reactive oxygen intermediates (ROI). Induction of tyrosyl phosphorylation may inactivate PP2A. a serine/threonine phosphatase. which has been postulated to have a negative regulatory effect on NF-kB activation. Dissociation of the Rel protein from Ik-B in the presence of ROI allows it to translocate to the nucleus and bind to the IL-2 receptor promoter.

Figure 6 is referred to in the primary assay below.

Figures 7 to 14 show the results of murine haematology assays.

Figures 15 and 16 show anti-CD3/C17-induced thymidine uptake in PBMC for a number of different donors at C 17 concentration 300μg/ml. The following primary assay was used to investigate the anti-viral activity of compounds in accordance with the invention: -

Primary Assay (i) Acute Infection Assay. High titre virus stocks of the human immunodeficiency virus HIV- 1 up were grown in H9 cells with RM1 1640 (Flow laboratories) supplemented with 10% fetal calf serum, penicillin (lOOIU/ml). Cell debris was removed by low speed centrifugation, and the supernatant stored at -70°C until required. In a typical assay C8166 T-lymphoblastoid CD4+ cells were incubated with 10xTCID50 HIV-I _RF at 37°C for 90 minutes and then washed three times with phosphate buffered saline (PBS). Cell aliquots (2 x 10⁵) were resuspended in 1.5ml growth medium in 6ml tubes, and active compounds in log dilutions [200μM to 0.2μM] were added immediately. 20mM stock solutions of each compound were made up in 70% alcohol. The compounds were stored as a powder and made up freshly in distilled water before each experiment or were stored as a 90 mM stock solution in 70% alcohol. The final concentration of alcohol in the tissue culture medium was 1%. The cells were then incubated at 37°C in 5% CO₂. At 72 hours post-infection 200μl of supernatant was taken from each culture and assayed for HIV (Kingchington et al, 1 89, Robert et al 1990) using an antigen capture ELISA which recognizes all the core proteins equally (Coulter Electronics. Luton. UK). The following controls were used: supernatants taken from uninfected and infected cells, infected cells treated with AZT (Roche Products UK Ltd) and ddC (Roche) and RO31-8959 (Roche) an inhibitor of HIV proteinase. The IC₅₀ activities of 8959, AZT and ddC in infected cells were 1, 10, 20 nM and 200nM respectively (accompanying Figure 6). The ELISA plates were read with a spectrophotometer. Compounds were tested in duplicate at each concentration, and the data shown is the average of at least two assays. This assay assesses the activity of compounds by measuring their inhibition of HIV core antigen levels.

(ii) Chronically Infected Cell Assay. Chronically infected cells (H9rf) were washed three times to remove extracellular virus and incubated with the active compounds (200-0.2 μM) for four days. HIV-1 antigen in the supernatant was then measured using an ELISA.

To test for compound toxicity, uninfected H9 cells were incubated with the compounds for four days. Supematants were discarded and the cells resuspended in 200μl pg growth medium containing C protein hydrolysate. After 6 hours the cells were harvested and the C incorporation measured.

(iii) Toxicity Assay. To test for compound toxicity, aliquots of 2 x 10⁵ of uninfected cells were cultured with the compounds in the same dilutions for 72 hours. The cells were then washed with PBSA and resuspended in 200μl of growth medium containing C protein hydrolysate. After 12, hours the cells were harvested and the C incorporation measured. Uninfected, untreated cells were used as controls.

Toxicity is expressed as inhibition of uptake of 1 , C protein hydrolysate

The results of these assays are summarized in Table 2 below. The IC₅₀ is the drug concentration that causes a 50% reduction in HIV core antigen levels as detected by the Coulter P24 antigen assay and is determined by doubling dilutions of supernatant taken from tubes containing untreated acutely infected cells. The CD₅₀ is the concentration of drug that causes a 50% inhibition of cells as measured by ^l4C protein hydrolysate uptake. The therapeutic index (TI) is determined by dividing the CD₅₀ by the IC₅₀.

TABLE 2

ode Compound I£₅₀ CJ2₅₀ TJ

C4 4-chloro-3, 5-dinitrobenzoic acid 30μm 70μm 2.33

Following the same methodology, more extensive assays were performed as reported in Tables 3 below. TABLE 3.1

STRUCTURE ACTIVITY RELATIONSHIP AGAINST HIV VIRUS

CODE COMPOUNDS

C l 2. 4-dichloro-3. 5-dinitrobenzoic acid

C3 2, 4-dichloro-3. 5-dinitrobenzoic acid methyl ester

C4 4-chloro-3, 5-dinitrobenzoic acid

C6 4-chloro-3, 5-dinitrobenzoic acid methyl ester

C7 2-chloro-3. 5-dinitrobenzoic acid

C8 2-chloro-3. 5-dinitrobenzoic acid methyl ester

C9 4-chloro-3-nitrobenzoic acid

CIO 2-chloro-4-nitrobenzoic acid

Cl l 3, 4-dichlorobenzoic acid

C12 2, 5-dichlorobenzoic acid

C17 2-chloro-5-nitrobenzoic acid

IΔfiLE. 2

IC50 CC50 SI

(Antiviral) (Toxicity) (Selectivity Index)

Against HIV-I IIIB

Cl 5 70 14

36 70 2

33 70 2

35 60

Average 27 70 3

Cl 10 30 3

2.5 20 8 Against HIV-IRF

Cl 7 60 8.5 56

16 56 3.5

Average 11.5 57 5

Against Chronicall' v infected cells

Cl 16 30 2

16 95 6

Average 16 63

Against HI V-l -ITTF

C2 2 70 35

C3 0.3 7 23

C4 40 100 2.5

30 70 2.3

Average 35 85 2.4

C5 5 50 10

C6 5 60 12

C7 23 150 6

5 >200 >10

Average 22 >175 8

C7 >1 35 >35

10 30 3

C8 10 60 5

C9 >200 >200

CIO >200 >200

Cl l >200 >200

C12 >200 >200

Against HTV-T TUB

C17 >1 >1000 >1000

5 >1000 >200 Patient Tests

A pilot study was conducted for safety, pharmacokinetics and preliminary activity of C17 in patients with HIV-1 infection and HIV-related Kaposi's sarcoma. In the pilot study, 10 patients were enrolled but only 6 were eligible for evaluation. These patients were positive for serum antibody to HIV-1 as determined by both enzyme- linked immunosorbent assay (ELISA) and Western Blot. These patients had WHO Clinical Stage 2 to 3 for HIV infection and disease and fulfilled the inclusion and exclusion criteria as per the Clinical Trial Protocol. Their CD4 ranged from 0.072 to 0.812 x 10³ cells/mm³ (normal range 1.0 x 1.3 x 10³ cells/mm³). Patient 6 had AIDS- related Kaposi^'s sarcoma confirmed on histology.

C17 was prepared for intravenous administration under GMP (good manufacturing practice) in strength of 40 mg/ml following the method set forth below for producing a 250mg/ml solution, additional water being added at Step 7:-

Method of Preparation

To 275ml water for injection add 266.4ml 4N sodium hydroxide solution and mix.

Add with stirring 182.5g of active compound.

Add additional 4N sodium hydroxide if needed to obtain solution.

Filter solution through a Sterivex GV 0.22μm filter.

Dilute to approximately 680ml with water for injection.

Adjust to pH 7.2-7.6 with hydrochloric acid.

Make-up to 730ml water for injection. Adjust pH if necessary.

Transfer solution to aseptic room and filter through Sterivex GV 0.22μm filter into injection vials. Seal with rubber stoppers and aluminium closures.

Effect terminal sterilization by autoclave. The patients received lOmg/kg body weight/day of C17 by deep intramuscular injection daily for 5 days weekly. The CD4 counts were estimated before and at about 2 weeks after the beginning of the therapy.

The CD4 counts in all 6 patients showed an increase which was associated with clinical improvements in patient general condition including weight gain and a marked decrease in opportunistic infections and diaorrhea. Patient observations are shown in Tables 4 below.

In the case of Patient 6, the sarcoma lesions disappeared.

Murine Haematology

Preliminary data on the administration of C 17 in mice and rats showed increases in red blood cell count, haemoglobin concentration, macrophage count, white blood cell count (WBC), lymphocyte count and haematocrit (HTC) on day 7 when maximum tolerated dose (MTD) was given by 5 consecutive daily intravenous injections. These values returned to normal on day 14. The MTD values were 950mg and 900mg/kg body weight/day respectively in mouse and rat. The data are reported in Figures 7 to 14.

TABLE 4,1 PATIENT 1 IMMUNOLOGY:-

DAY 20

Lymphocytes x 10³ mm -3 2.1 2.0

CD4 % 19 38

CD4 ABS/mm³ 0.399 0.760

CD8 ABS/mm³ 0.567 0.840

CD4/CD8 0.70 0.90

TABLE 4.2 PATIENT 2 IMMUNOLOGY:-

DAY 14

Lymphocytes x 10^Jmm^" 2.0 1.9

CD4 % 18 27

CD4 ABS/mm³ 0.360 0.513

CD8 ABS/mm³ 0.940 0.874

CD4/CD8 0.38 0.59 TABLE 4.3 PATIENT 3 IMMUNOLOGY:-

DAY 1 20

Lymphocytes x 10³mm^'3 0.9 1.1

CD4 % 8 17

CD4 ABS/mm³ 0.072 0.187

CD8 ABS/mm³ 0.270 0.682

CD4/CD8 0.27 0.27

TABLE 4,4 PATIENT 4 IMMUNOLOGY:-

DAY 14

Lymphocytes x 10³mm^'3 1.8 2.0

CD4 % 6 22

CD4 ABS/mm³ 0.108 0.440

CD8 ABS/mm³ 1.350 1.260

CD4/CD8 0.08 0.35 TABLE 4.5 PATIENT 5 IMMUNOLOGY:-

DAY 21

Lymphocytes x 10³ mm^"3 2.8 3.1

CD4 % 29 33

CD4 ABS/mm³ 0.812 1.023

CD8 ABS/mm³ 1.456 1.550

CD4/CD8 0.56 0.66

TABLE 4.6 PATIENT 6 IMMUNOLOGY:-

DAY 34

Lymphocytes x 10³mm^"3 2.1 1.9

CD4 % 18 23

CD4 ABS/mm³ 0.378 0.437

CD8 ABS/mm³ 1.218 0.931

CD4/CD8 0.31 0.47 REFERENCES

1. Miller. C.C.. Hale. P. and Pentland. A.P. (1994) J. Biol. Chem. 269. 3529- 3533. 2 Sundaresan, M., Yu, Z.X.. Ferrans, V.J., Irani, K. and Finkel. T. (1995)

Science 270. 296-299.

3. Nakamura. K.. Hori, T.. Sato. N.. Sugie, K.. Kawakami. T. and Yodoi. J.. (1993) Oncogene 8. 3133-3139.

4. Schreck. R.. Rieber. P. and Baeuerle. P.A. (1991) EMBO J. 10. 2247-2258. 5. Kinchington. D. and Ng, T. ( 1996) in: Animal Cell Culture (Walker. J.M. and

Pollard. J.W. Eds.) Humana Press Inc.. New Jersey. USA. (in press).

6. June. C.H.. Bluestone, J.A.. Nadler. L.M. and Thompson. C.B. ( 1994) Immunology Today 15, 321-331.

7. Bradford. M.M. ( 1976) Analytical Biochemistry 72. 248-254. 8. Chaudhri. G., Clar, LA,. Hunt, N.H.. Cowden. W.B. and Ceredig. R. ( 1986) J.

Immunol. 137, 2646-2652.

9. Dornand, J. and Gerber. M. ( 1989) Immunology 68. 384-391.

10. Algarte. M., Lecine, P., Costello. R.. Plet. A.. Olive. D. and Imbert. J. (1995) EMBO J. 14. 5060-5072. 1 1. Ghosh, G., Van Duyne. G.. Ghosh. S. and Sigler. P.B. ( 1995) Nature 373. 303- 310.

12. Ghosh. S. and Baltimore. D. ( 1990) Nature 344. 678-682.

13. Diehl. J.A.. Tong W.. Sun. G. and Hannink, M. (1995) J. Biol. Chem. 270. 2703-2707. 14. Khan. M.A., Jeremy, J.Y., Hallinan. T.. Tateson. J.E.. Hoffbrand. AN. and Wickremasinghe, R.G. (1993) Biochim. Biophys. Acta. 1178. 215-220.

15. Chen. J., Martin, B.L. and Brautigan. D.L. (1992) Science 257, 1261-1264.

16. Suzuki, Y.J.. Mizuno, M. and Packer. L. (1994) J. Immunol. 153. 5008-5015.

17. Rhodes, J., Chen, H., Hall. S.R.. Beesley, J.E., Jenkins. D.C.. Collins. P. and Zheng. B. (1995) Nature 377, 71-75.

Claims

1. Use of a physiologically non-toxic reactive oxygen intermediate species (ROS) oxygen donor substance as defined below for the manufacture of a medicament for use in the treatment of HIV infection in a mammalian subject, the ROS being a substance having the following functional characteristics:-

(i) its molecules are relocatable from serum to T-lymphocyte cytoplasm

(ii) its molecules promote the intracellular signal transduced from the extracellular event comprised of interaction between the T-cell CD3 receptor and MHC-transported peptide fragments of target cell surface target cell intracellular protein, the target cell being an antigen presenting cell in the form of a cell infected with HIV and the signal promotion, in the presence of a second intracellular signal transduced from the extracellular event comprised of interaction between the T-cell CD28 co-receptor with target cell B7 immunoglobulin. producing enhanced tyrosine kinase activation occassioning tyrosyl phosphorylation of protein substrates inhibitory of expression of interleukin-2 and or interleukin-2 receptor by the IL-2 gene.

2. Use of a benzoic acid derivative (BAD) as defined below for the manufacture of a medicament for use in the treatment of HIV infection in mammalian subjects by costimulation of the proliferative response of T-lymphocytes to receptor triggering by a mechanism which comprises:-

(i) activating a T-lymphocyte by first and second intracellular signals delivered by a target antigen presenting cell in the form of a cell infected with HIV, the first signal being transduced by the T-cell CD3 co-receptor by interaction thereof with MHC-transported peptide fragments of intracellular protein and the second signal being transduced by the T-cell CD28 co-receptor by interaction thereof with target cell B7 immunoglobulin;

(ii) penetrating the T-lymphocyte cytoplasm by molecules of BAD; (iii) generating by means of BAD molecules and in the T-lymphocyte cytoplasm a reactive oxygen species (ROI) by donation from the BAD molecules; and

(iv) promoting the first signal to effect tyrosine kinase activation and tyrosyl phosphorylation of protein substrates inhibitory of expression of interleukin-2 by the T-cell IL-2 gene;

the BAD having the formula:-

wherein R,, R₂, R₃, R₄ and R₅ are. each independently hydrogen. C, to C₄ alkyl. nitro or halo, and wherein R₄ is hydrogen, an ester-forming moiety or a salt-forming moiety: subject to the following two provisos:-

(a) at least one of R,, R₂, R₃, R₄ and R₅ is other than hydrogen, and

(b) the halo atom(s) when present is (are) chlorine or bromine.

3. Use as claimed in Claim 1 wherein the BAD has the general formula:-

COOH

wherein X_! and X₂ are. each independently, hydrogen or chloro and. wherein X₃ and X₄ are. each independently, hydrogen or nitro, provided that at least two of X,, X₂, X₃ and X₄ are other than hydrogen, or a salt or ester thereof.

4. Use as claimed in Claim 1 or Claim 3 wherein the BAD is a compound of the formular presented in Claim 2 or that presented in Claim 3 wherein X| is chloro. X₂ is chloro or hydrogen, X₃ is nitro and X₄ is nitro or hydrogen.

5. A compound as claimed in Claim 4 wherein the BAD is a compound of the formula presented in Claim 2 or that presented in Claim 3 wherein X| and X₂ are other than 4-chloro.

6. A compound as claimed in anyone of Claims 1 to 5 wherein the BAD is a compound of the formula presented in Claim 2 or that presented in Claim 3 wherein X| and X together represent 2-chloro-. 4-chloro-. 2.5-dichloro-. 2. 4-dichloro-. 2, 6- dichloro-, 3, 5-dichloro- or 3, 4-dichloro- and wherein X₃ and X together represent two hydrogen atoms, 3-nitro-. 4-nitro-, 5-nitro-. 2, 4-dinitro- or 3. 5-dinitro-, provided that:-

(i) when X₃ and X₄ together represent 3, 5-dinitro-, X, and X₂ together represent

2-chloro-, (ii) when X₃ and X₄ together represent 5-nitro-. X, and X₂ together represent 2- chloro- or 2, 4-dichloro-, (iii) when X₃ and X₄ together represent two hydrogen atoms. X, and X₂ together represent 3. 4-dichloro-,

(iv) when X₃ and X₄ together represent 4-nitro-, Xi and X₂ together represent 2, 5- dichloro-. 3, 5-dichloro-2-chloro- or 2. 6-dichloro-, (v) when X₃ and X₄ together represent S-nitro-. X| and X₂ together represent 4- chloro-.

7. Use as claimed in Claim 5 wherein the BAD is a compound of the formula presented in Claim 2 or that presented in Claim 3 wherein X X₂, X₃ and X₄ are as there defined subject to provisos (I), (ii), (iv) and (v) and subject to the further proviso that when X₃ and X₄ together represent 5-nitro-, X, and X₂ together represent 2. 4- dichloro-.

8. Use as claimed in Claim 1 wherein the BAD is a compound as set forth by name below or an ester or salt thereof: -

8.1 2. 4-dichloro-3. 5-dinitrobenzoic acid

8.2 4-chloro-3, 5-dinitrobenzoic acid 8.3 2-chloro-3, 5-dinitrobenzoic acid

8.4 2. 5-dichlorobenzoic acid

8.5 2. 4-dinitrobenzoic acid

8.6 3, 5-dinitrobenzoic acid

8.7 2. 5-dichloro-4-nitrobenzoic acid 8.8 2. 4-dichloro-5-nitrobenzoic acid

8.9 2. 6-dichloro-4-nitrobenzoic acid

8.10 3. 5-dichloro-4-nitrobenzoic acid

8.1 1 4-chloro-3-nitrobenzoic acid

8.12 2-chloro-4-nitrobenzoic acid 8.13 3. 4-dichlorobenzoic acid

8.14 2-chloro-5 -nitro benzoic acid

9. Use as claimed in Claim 1 wherein the BAD is a compound as set forth by name below or an ester thereof: -

9.1 2-chloro-5-nitrobenzoic acid

9.2 2,5-dichloro-4-nitrobenzoic acid

9.3 2.5-dichloro-5-nitrobenzoic acid

9.4 2.6-dichloro-4-nitrobenzoic acid 9.5 3,5-dichloro-4-nitrobenzoic acid

10. A pharmaceutical composition for use in the treatment of viral infection, the composition comprising a compound as defined in any preceding claim together with a pharmaceutically-acceptable carrier or diluent.

1 1. A composition as claimed in Claim 10 wherein the carrier or diluent is aqueous.

12. A composition as claimed in Claim 10 wherein the carrier is a solid excipient.

13. A composition as claimed in Claim 10 which is in tablet, capsule, powder, solution or suspension form.

14. A composition as claimed in any one of Claims 10 to 13 which is in unit dosage form.

15. A composition as claimed in any one of Claims 10 to 14 and comprising 2- chloro-5-nitrobenzoic acid, or a salt or ester thereof, encapsulated by a soft gel capsule or contained by a sustained release drug delivery system.

16. A composition as claimed in Claim 9 and in the form of enteric coated tablets comprising a compound as defined in any one of Claims 1 to 9 in strength 100 to 400mg, the compound being formulated as a mixture comprising the compound together with an inert pharmaceutically-acceptable solid tabletting carrier.

17. A composition as caimed in Claim 10 and in the form of an injectable solution or dispersion of a compound as claimed in any one of Claims 1 to 9 in an aqueous or other injectable liquid carrier in a concentration of less than 250mg/ml.

18. A method of preparing a pharmaceutical composition as claimed in any one of Claims 10 to 17 which method comprises combining a compound as defined in any one of Claims 1 to 8 with aqueous sodium hydroxide solution to form a solution. adjusting the pH of the solution to an alkaline pH of not more than 8 by addition to the solution of an acidic material, optionally diluting the pH-adjusted solution with physiologically injectable water and sterilizing the solution.

19. A method of treatment by prophylaxis or therapy of neoplasm or viral injection in mammalian subjects, which method comprises administering to a mammalian subject an effective dose or dosage regime of a compound as defined in any one of Claims 1 to 9.

20. Use as claimed in any preceding claim wherein the medicament is specifically intended for use in the treatment of AIDS-related Kaposi's sarcoma.

21. A method of treatment of HIV infection in a mammalian subject, which method comprises administering to the subject the ROS being a substance having the following functional characteristics.

(i) its molecules are relocatable from serum to T-lymphocyte cytoplasm

(ii) its molecules promote the intracellular signal transduced from the extracellular event comprised of interaction between the T-cell CD3 receptor and MHC-transported peptide fragments of target cell surface target cell intracellular protein, the target cell being an antigen presenting cell in the form of a cell infected with HIV and the signal promotion, in the presence of a second intracellular signal transduced from the extracellular event comprised of interaction between the T-cell CD28 co-receptor with target cell B7 immunoglobulin, producing enhanced tyrosine kinase activation occassioning tyrosyl phosphorylation of protein substrates inhibitory of expression of interleukin-2 by the IL-2 gene.

22. A method as claimed in Claim 21 wherein the ROS donor substance is a benzoic acid derivative.

23. A method as claimed in Claim 22 wherein the benzoic acid derivative is a benzoic acid derivative as defined in any one of Claims 1 to 19.