WO1997025350A1

WO1997025350A1 - Use of chemokines for the treatment and prevention of hiv infection

Info

Publication number: WO1997025350A1
Application number: PCT/GB1997/000061
Authority: WO
Inventors: Michael George Hunter; Lloyd George Czaplewski; Keith Martyn Dawson; Richard Mark Edwards
Original assignee: British Biotech Pharmaceuticals Limited
Priority date: 1996-01-11
Filing date: 1997-01-10
Publication date: 1997-07-17
Also published as: GB9600559D0; AU1389897A

Abstract

The use of a disaggregated mutant of the stem cell inhibitor molecule MIP-1alpha or LD78, for inhibiting HIV infection.

Description

USE OF CHEMOKINES FOR THE TREATMENT AND PREVENTION OF HIV INFECTION

This invention relates to the use of disaggregated mutants of the stem cell inhibitor molecule macrophage inflammatory protein-1 alpha (Ml P-1 alpha) or its human homologue LD78, alone or in conjunction with other known human immunodeficiency virus (HIV) inhibitors for the treatment or prevention of HIV infection.

The murine haematopoietic stem ceil inhibitor is termed macrophage inflammatory protein-1 alpha (MIP-1alpha(Graham et al., Nature. 344:442, 1990)). The human LD78 gene product is the functional homologue of murine MIP-1 alpha (Dunlop et al., Blood. 79:2221-2225, 1992). Alternative N-termini for mature human MIP-1 alpha exist. Theoretical peptide cleavage sites have variously predicted the mature peptide to be 66, 69 and 70 amino acids in length (Brown et al., J. Immunol. 142:679, 1989; Zipfel et al., J. Immunol. 142:1582, 1989; WO-A-9104274; Nakao et al., Mol. Cell. Biol. 10:3646, 1990). Various wild-type alleles of the first identified murine and human Ml P-1 alpha molecules also exist. Disaggregated mutants of any variant or allele of murine or human MIP-1 alpha find use in this invention. An allele will possess a high degree of amino acid sequence identity with the sequences identified above (see Graham et al. ibid and Dunlop et al., ibid), comprising no more than 10 amino acid differences and usually only a few substitution differences.

Suitable disaggregated mutants for use in this invention, and further disclosures relevant to their preparation and characterisation are described in WO-A-9313206. The preferred disaggregated mutant for use in this invention is now known in the art as BB-10010, and is the product of Example 7 of WO-A-9313206.

The invention provides the use of a disaggregated mutant of the stem cell inhibitor molecule MIP-1 alpha or LD78, in the preparation of a medicament for the treatment or prevention of HIV infection.

The invention also provides a method of treating an HIV infected patient or one suspected of recent HIV exposure or one at risk of infection, which comprises administering to the patient an effective amount of a disaggregated mutant of MIP- 1 alpha or LD78.

At birth, babies born to HIV-positive mothers are particularly susceptible to HIV infection, and therefore fall within the group "at risk of infection".

Many wild-type chemokine molecules (especially CC chemokines) at physiological ionic strength and at concentrations as low as 100μg/ml form large multimeric complexes which have a tendency to aggregate. These complexes show a broad heterogeneous mixture of molecular weights ranging from 100,000Da to »200,000Da and at higher concentrations in vitro, can form into cloudy precipitates. Wild-type MIP- 1 alpha and LD78 monomers have a molecular weight of about 8,000Da. Consequently, molecules with molecular weights >100,000Da will comprise multimers greater than dodecamers.

By the term "disaggregated mutant" as used herein is meant a molecule differing in amino acid sequence from the native protein (i.e. MIP-1 alpha, LD78, MIP-1beta, ACT-2 or RANTES), and which at physiological ionic strength is substantially incapable of forming multimers higher than a dodecamer as determined by sedimentation equilibration analytical ultracentrifugation (AUC).

The sedimentation equilibration analytical ultracentrifugation method is known in the art. In brief, it involves spinning the test sample until it reaches equilibrium, then determining the distribution of the sample across the sample tube by measuring the absorbance at an appropriate wavelength. From the resulting distribution the average molecular weight and the size homogeneity is estimated by successively fitting the distribution to models that assume the presence of only one size species, each model representing a different size species. The size model which correlates best with the data for the sample is taken to be the average molecular weight of the species in the test sample. The disaggregated mutant is considered substantially incapable of forming multimers higher than a dodecamer if a 0.5mg/ml sample of mutant protein in physiological solution possesses less than 20%, preferably less than 15%, more preferably 10% or less of any other multimer formation as determined by AUC.

The terms "physiological solution" or "physiological ionic strength" are well known to those skilled in the art. They are generally equivalent to about 137mM NaCl, 3mM KCl and about 10mM phosphate. Physiological pH is about 7.4.

As used herein, the terms "human immunodeficiency virus" or "HIV" includes any member of the retroviral family eg HTLV-1 , HTLV-2, HTLV-III (HIV-1) or HIV-2, but preferably refers to HIV-1.

The invention also provides the use of a disaggregated mutant of the stem cell inhibitor molecule MIP-1 alpha or LD78 in conjunction with one or more other human immunodeficiency virus (HIV) inhibitors in the preparation of a medicament for the treatment or prevention of HIV infection.

Suitable HIV inhibitors for use in conjunction with the disaggregated MIP-1 alpha or LD78 mutants in the preparation of a medicament for the treatment or prevention of HIV infection according to the invention, are selected from the group consisting of: RANTES, ACT-2, MIP-1beta and non-proteinaceous inhibitory molecules.

Suitable non-proteinaceous inhibitory molecules include but are not limited to: HIV protease inhibitors such as ritonavir and indinavir or reverse transcriptase inhibitors such as AZT, or 3TC.

Cytokine molecules such as MIP-1beta, ACT-2 and RANTES which in in vitro systems, are known to inhibit replication of HIV, the causative agent of the acquired immuno-deficiency syndrome (AIDS) (Canque and Gluckman. Blood. 84(10) Suppl 1:p480a, Abstract No. 1907, 1994; Cocchi et al., Science 270:1811-1815, 1995; Paxton et al., Nat Med 2: 412-417, 1995), may thus be co-administered with a disaggregated MIP-1 alpha or LD78 mutant, to an HIV infected patient, or can be used in conjunction with the disaggregated MIP-1 alpha or LD78 mutant in the preparation of a medicament for the treatment or prevention of HIV infection.

Co-administration may be by separate, sequential or simultaneous means.

Variants of known HIV inhibitors which are also capable of inhibiting HIV are also useful for co-administration with the disaggregated MIP-1 alpha or LD78 mutants according to this invention. Suitable variants include N-terminal or C-terminal truncated mutants and disaggregated mutants.

WO-A-9313206, herein incorporated by reference, is concerned with disaggregated mutants of stem cell inhibitory proteins such as LD78. Because of the extensive amino acid homology and near identical tertiary structures of the chemokines, by analogy to the mutation sites leading to disaggregated LD78 mutants, WO-A- 9313206, teaches how to construct disaggregated mutants of other chemotactic cytokine molecules. Thus, by following the teaching in WO-A-9313206, suitable disaggregated mutants of other chemotactic cytokine molecules such as: MIP-1beta, ACT-2 and RANTES can be made.

According to the invention there is also provided a pharmaceutical composition comprising a disaggregated mutant of a MIP-1 alpha or LD78 stem cell inhibitor molecule, and one or more HIV inhibitor molecules selected from the group consisting of MIP-1beta, ACT-2 and RANTES, together with a pharmaceutically acceptable carrier.

The invention also includes a method of treating an HIV infected patient or one suspected of recent HIV exposure or one at risk of infection, which comprises the simultaneous, separate or sequential administration of effective amounts of a disaggregated MIP-1alpha or LD78 stem cell inhibitor molecule, and one or more HIV inhibitor molecules selected from the group consisting of: MIP-1beta, ACT-2 and RANTES.

An effective amount is one that, in infected individuals, is capable of at least stabilising and preferably reducing the HIV burden of the patient, and in those at at risk of infection is one that prevents infection.

Dosage of the agent(s) in accordance with any aspect of the invention will be such as is accepted to be effective following the clinical trials approved for that purpose by the relevant health regulatory authorities, and will be under the control of the physician or clinician considering various factors such as the condition, sex, weight of the patient and the degree or severity of any infection.

Administration of the agent(s) can be by injection, preferably via intra-venous, intra- peritoneal, intra-muscular or sub-cutaneous routes. Other routes such as topical, transdermal, oral, intranasal or by inhalation may also be possible.

The following figures and examples support the invention. They demonstrate the ability of disaggregated stem cell inhibitor mutant BB-10010 to inhibit HIV replication and demonstrate its increased potency relative to wild-type MIP-1 alpha (LD78).

Brief description of figures:

Figure 1.

Comparison of neutralisation activity of wild-type LD78 and BB-10010 for HIV infection

(molecular clone HXB2-US3) of PM1 cells.

Figure 2.

(molecular clone HXB2-US4) of PM1 cells. Example 1.

BB-10010 inhibition of H V replication.

BB-10010 was prepared according to Example 7 of WO-A-9313206.

The C8166 CD4+ T-cell line is used for acute assays of virus replication. C8166 cells are incubated with 100μl of supernatant from chronically-HIV-IIIB/LAI-infected H9 cells, for 2-4 hours at 37°C, then washed twice. The cells are then plated out at 5 x 10⁴ cells/well in flat-bottomed 96-well plates (Nunc) in R/10 supplemented with 10 U/mi rlL-2 (Cetus Corp.). HIV-infected C8166 cells are either cultured alone or in the presence of medium containing varying concentrations of BB-10010. Culture supernatants are harvested at 4-6 day intervals and assayed for virus-associated reverse transcriptase (RT).

BB-10010 at a concentration of 1000ng/ml inhibited HIV replication by 50% in this assay.

Using the RF strain of HIV (HIV-1_RF) in the same assay system, virus replication was inhibited by 50% at 1000ng/ml and 100% at 2000ng/ml of BB-10010.

Example 2.

BB-10010 is a more potent inhibitor of HIV-1 replication than wild-tvpe human MIP- 1 alpha (LD78,.

The ability of BB-10010 and wild-type rhMIP-1 alpha (R&D Systems, Abingdon, U.K.) to inhibit the replication of HIV-1_RF in MT-4 cells was assessed using a tetrazolium- based colorimetric assay (Pauwels et al., J. Virological Methods. 20(4):309-321 , 1988; Brennan et al., Antiviral Research. 26:173-187, 1995).

At 1000ng/ml, BB-10010 inhibited HIV-1_RF replication by 50% whereas 1000ng/ml of wild-type MIP-1 alpha had no inhibitory effect. Example 3.

Dose-dependent inhibition of HIV-1 infection in PM1 cells bv wild-type recombinant LD78 and the disaggregated LD78 mutant BB-10010.

The ability of wild-type and disaggregated mutant (BB-10010) LD78 chemokine to inhibit PM1 cell infection by two different HIV-1 viral isolate molecular clones (HXB2- US3 and HXB2-US4, both sequences present in the Los Alamos database) were tested. These isolates are based on the clone HXB2, with the gp120 ORF exchanged for that of the primary virus derived U3 or U4. Both chimeric viruses bind the CCR-5 receptor.

WO-A-9313206 discloses the vector construction, expression and purification of the recombinant wild-type LD78 and BB-10010 used in this example.

The method followed was essentially as described in Fig. 3 of Cocchi et al., (Science. 270:1811-1815, 1995).

2 x 10⁴ cells per 100μl of PM1 cells were incubated with increasing concentrations (in triplicate wells) of the test chemokine for 1hr at 37°C. 100 TCID₅₀ (50% tissue-culture inhibitory dose) of virus was added to each well and incubated at 37°C. 24 hours post infection, the cells were washed twice to remove unbound viral antigen and chemokine and fresh medium lacking chemokine was added (10%FCS/RPMI). Seven days post infection, the extracellular fluid was tested for p24 antigen by antigen capture (p24 EIA method according to McKeating et al., J. Virol. 67:4932-4944, 1993).

The results are presented in the table below as % p24 antigen production, and in Figures 1 and 2.

HXB2-US3 and HXB-2US4 gave p24 values in the absence of chemokine of 1025 and 1550 pg/50μl, respectively. Chemokine % p24 production by HXB2- % p24 production by HXB2- concentration US3 US4

(ng/ml) wild-type LD78 BB-10010 wild-type LD78 BB-10010

500 0 0 0 0

250 0 0 0 0

125 8 1 4 1

62 16 4 13 2

31 25 10 29 7

16 55 24 58 21

8 82 64 83 58

4 92 78 95 72

1 100 100 100 100

Wild-type LD78 had an ID₉₀ (90% inhibition dose) of 105ng/ml against HXB2-US3. BB-10010 had an ID₉₀ of 30ng/ml against HXB2-US3. Wild-type LD78 had an IDgo of 80ng/ml against HXB2-US4. BB-10010 had an ID₉₀ of 28ng/ml against HXB2-US4.

These results demonstrate that the disaggregated LD78 mutant BB-10010, is 2 - 4 fold more potent at inhibiting HIV-1 infection (at 90% level) of PM1 cells than wild-type LD78.

Wild-type MIP-1 alpha, RANTES and MIP-1beta have already been proposed as potential agents for inhibiting HIV, it was not previously known that disaggregated analogues (mutants) of wild-type LD78 could also inhibit HIV. Moreover, the inventors have surprisingly found that the disaggregated LD78 analogue BB-10010 is more potent at inhibiting HIV-1 infection of cells in vitro than wild-type LD78. Wild-type MIP-1 alpha, RANTES and MIP-1beta are believed to inhibit HIV-1 infection by blocking viral entry through the CCR-5 receptor. BB-10010 has been shown to be capable of binding the CCR-5 receptor and therefore, other disaggregated LD78 analogues capable of binding the CCR-5 receptor should also be useful in this invention.

Claims

Claims:

1. The use of a disaggregated mutant of MIP-1 alpha or LD78, in the preparation of a medicament for the treatment or prevention of HIV infection.

2. A method of treating an HIV infected patient or one suspected of recent HIV exposure or one at risk of infection, which comprises administering to the patient an effective amount of a disaggregated mutant of MIP-1 alpha or LD78.

3. The use of a disaggregated mutant of MIP-1 alpha or LD78 in conjunction with one or more other human immunodeficiency virus (HIV) inhibitors in the preparation of a medicament for the treatment or prevention of HIV infection.

4. A pharmaceutical composition comprising a disaggregated mutant of MIP-1 alpha or LD78, and one or more HIV inhibitor molecules selected from the group consisting of: MIP-1 beta, ACT-2 and RANTES, together with a pharmaceutically acceptable carrier.

5. The use as claimed in claim 1 or claim 3 or the method as claimed in claim 2 or a composition as claimed in claim 4, wherein the disaggregated mutant is BB-10010.