Patent Application: US-25695295-A

Abstract:
calcineurin antagonists are disclosed for use in the treatment of hiv infection . preferred materials include cyclosporin a and fk 506 .

Description:
the present invention relates to the use of a compound for the manufacture of medicament for the treatment of human immunodeficiency virus ( hiv ) infection . infection by hiv , of which there are two distinct types hiv - 1 and hiv - 2 , leads to the development of acquired immune deficiency syndrome ( aids ) 1 , 2 . several compounds which interfere with the replication of the virus have been developed for the treatment of aids , however only one compound , azt ( azidothymidine ), has been widely used in the treatment of hiv infection . although azt is a highly effective drug in blocking hiv replication , it has two major drawbacks . firstly , it is frequently toxic inhibiting not only hiv replication but also the division of bone marrow cells causing anaemia , leukopenia , neutropenia and thrombocytopenia 3 , 4 . secondly , within six months of prolonged treatment , azt - resistant mutants of hiv appear 5 . thus , there is an urgent need to find drugs that will interfere with hiv replication without overt cytotoxicity . it is known that hiv replication depends on t - cell activation and proliferation . the aim of the present invention is to develop an agent which inhibits hiv replication . it was thought that if t - cell activation could be suppressed , hiv replication would be inhibited . this aim is achieved by affecting the signal transduction pathway for t - cell activation by administering calcineurin - antagonists and / or rotamase - antagonists and / or nf - at - antagonists . accordingly the present invention provides the use of calcineurin - antagonists for the manufacture of a medicament for the treatment of hiv infection . in another aspect , the present invention provides the use of rotamase - antagonists for the manufacture of a medicament for the treatment of hiv infection . in a further aspect , the present invention provides the use of nf - at - antagonists for the manufacture of a medicament for the treatment of hiv infection . suitable for use according to the present invention are compounds that act as calcineurin - antagonists , rotamase - antagonists or nf - at - antagonists , for example also included within the scope of the invention are medicaments for the treatment of hiv infection which comprise a compound of the invention as active ingredient in association with a pharmaceutically acceptable carrier or diluent . the medicament of the invention will normally be administered orally or by injection . compositions for parenteral administration will normally be solutions in aqueous saline , which is pyrogen free for human use . such compositions can be administered intravenously or intraperitoneally . compositions for oral administration will mostly be in solid or liquid form , mostly as tablets , capsules , lozenges , etc . liquid compositions can be solutions or dispersions in aqueous or non - aqueous media . ideal solutions are of neutral or alkaline ph and of low ionic strength , e . g 5 % dextrose . suitable daily doses of the antagonists when used in accordance with the invention range from about 10 mg to 1 g / m 2 body surface . fig2 a : effect of the drugs on the growth of non - infected cells . open triangle , control cells grown in the presence of growth medium only ; closed triangle , cells grown in the presence of 4 μg / ml csa ; open circle , cells grown in the presence of 10 μg / ml csa ; closed circle , cells grown in the presence of 4 μg / ml fk506 ; and , open square , cells grown in the presence of 10 μg / ml fk506 . fig2 b : effect of the drugs on the growth of chronically infected cells . open triangle , control cells grown in the presence of growth medium only ; closed triangle , cells grown in the presence of 4 μg / ml csa ; open circle , cells grown in the presence of 10 μg / ml csa ; closed circle , cells grown in the presence of 4 μg / ml fk506 ; and , open square , cells grown in the presence of 10 μg / ml fk506 . fig3 : effect of cyclosporin a and fk506 on cd4 expression . samples 1 - 5 were non - infected cells 1 ) in growth medium only , 2 ) with 1 μg / ml csa , 3 ) with 4 μg / ml csa , 4 ) with 1 μg / ml fk506 , and 5 ) with 4 μg / ml fk506 . samples 6 - 10 were hiv - 1 chronically infected cells 6 ) in growth medium only , 7 ) with 1 μg / ml csa , 8 ) with 4 μg / ml csa , 9 ) with 1 μg / ml fk506 , and 10 ) with 4 μg / ml fk506 . leu 3a / 3b okt4a monoclonal antibodies . bars indicate mean on three replicates with error bars . fig4 : effect of the drugs on the release of infectious hiv particles from chronically infected cells . open square , tissue culture infected dose ( tcid ) released from the infected cells in the presence of growth medium ; open circle , tcid released from the infected cells when grown in either 10 μg / ml csa or fk506 ; and , closed square , tcid released for a week after the drugs were removed . assay for the effect of cyclosporin a ( csa ) and fk506 on hiv cell fusion and replication method : a clone of the leukaemia t - cell line molt 4 , which expresses a very high level of cd4 , was selected . this was infected with the highly cytopathic ndk strain of hiv - 1 6 and a clone which is chronically infected with the virus but which readily replicates was selected . when these hiv - infected cells w ere co - cultivated with non - infected molt 4 cells at a ratio of 100 non - infected : 1 infected cell , very large giant cells were formed within 24 hours . the formation of giant cells in the mixed culture was used as a rapid assay to determine the anti - hiv effect of csa and fk506 . the effects of serum from healthy hiv - 1 infected individuals and aids patients on giant cell formation was also examined . in addition the level of cytotoxicity and anti - hiv - 1 activity of these substances was tested in an anti - hiv assay system which has been previously described 7 . this enabled us to establish the anti - viral activity and cytotoxicity in parallel and to compare it to the effects of azt on virus inhibition and cell division . results : 1 . giant cell assay : when various cells cultures were examined after 24 hours the cells which were co - cultivated with chronically infected cells contained a high proportion of very large and pleomorphic giant cell formations . in contrast , when the same cells were co - cultured in the presence of 10 μg / ml of csa or fk506 , only a few small giant cells could be observed . when chronically infected cells , cultured for one week in the presence of the drugs , were washed and then co - cultivated with non - infected cells in the absence of the drug , a few small giant cells could be seen during the first 24 hours . however after prolonged culture larger giant cells were formed . 2 . hiv inhibition : the effect of various concentrations of csa and fk as well as azt on hiv - 1 replication are outlined in fig1 . although azt is known to be toxic in vivo , it appears to be a far more effective drug in blocking replication of the virus . effect of cyclosporin a and fk506 on the growth - rate of non - infected and chronically infected t - cells method : molt 4 cells and the clone of the same cells , chronically infected with hiv - 1 , were seeded separately at an approximate concentration of 5 × 10 4 cells / ml . five separate cultures of each of the two cell populations were seeded . one was grown with growth medium alone , the others with 4 μg / ml of csa , 10 μg / ml of csa , 4 μg / ml of fk and 10 μg / ml of fk . at 24 hour intervals samples were taken for cell counting . feeding with or without the corresponding chemical was done after the fourth day . azt was also used in parallel . results : fig2 a shows that csa did not reduce the rate of t - cell division , either at a concentration of 10 or 4 μg / ml , when compared to cells which were grown in the presence of growth medium alone . likewise , for the first five days fk did not affect the growth of uninfected cells . thereafter there was only a marginal increase in the number of cells in the presence of 10 μg / ml fk while 4 μg / ml , did not appear to slow cell division . most interesting was the effect of the two drugs on the same t cells chronically infected with hiv - 1 . fig2 b shows that both drugs significantly inhibited the replication of the hiv - 1 infected cells . at a concentration of 10 μg / ml there was only a marginal increase in the number of cells after a week in culture . with a lower concentration of 4 μg / ml there was still a significant reduction in cell division . unlike csa and fk , azt was toxic to the chronically infected cells as it was to the uninfected cells . method : both the non - infected molt 4 cells as well as the hiv - 1 chronically infected cells were grown in the presence of csa and fk506 . each cell preparation was kept separately at a concentration of 1 and 4 μg / ml of each drug for 3 days . infected and uninfected cells were suspended at a concentration of 10 6 / ml in phosphate buffered saline ( pbs ) containing 0 . 1 % sodium azide . triplicate aliquots of 100 μl were incubated separately with 20 μl leu - 3a ( fitc ) ( ortho ( becton dickinson uk ltd ) or with 10 μl okt4a ( fitc ) diagnostic systems ). after 20 min at 22 ° c . the cells were washed twice with pbs and resuspended in 0 . 51 % paraformaldehyde / pbs ( sigma ). the preparations were fixed overnight at 4 ° c . and subsequently analysed by flow cytometry ( facscan , becton dickinson uk ltd ). the median channel fluorescence was recorded for each sample . results : fig3 shows that uninfected molt 4 cells expressed a high level of cd4 even when grown in the presence of 1 and 4 μg / ml of csa or fk . in contrast , fig3 also shows that the chronically hiv - 1 infected molt 4 cells expressed a very low level of cd4 which does not seem to be affected by either csa or fk . 2 . chronically infected cells grown in 10 μg / ml of csa for a week and for a week after removal of the compound ; 3 . chronically infected cells grown in 10 μg / ml of fk506 and for 1 week after removal of compound ; each culture was titrated at serial ten - fold dilutions . each dilution was used to infect molt 4 cells in duplicate . the end point was recorded after 10 days . results : fig4 shows that approximately 10 6 infectious hiv particles per ml can be recovered from the culture fluid of chronically infected cells . however , when the cell cultures were assayed for infectious particles after one week of growth in the presence of csa or fk506 there was about 100 - fold drop in the yield of infectious hiv in both culture fluids . when the drug - containing culture medium was replaced with growth medium alone , there was a recovery in the rate of cell division with an increase in the production of infectious hiv - 1 to the levels which preceded the drug treatment . method : cloned molt 4 cells , chronically infected with hiv - 1 ndk , and the same cells grown for 5 days in the presence of csa were fixed in 3 % glutaraldehyde . ultrathin sections of cytoplasm were examined for the presence of budding virus particles in csa treated and untreated cells . results : systematic electron microscopic examination of the chronically hiv - 1 infected cells revealed that most ultrathin sections contained readily recognisable and morphologically distinctive virus particles . in contrast most of the sections from the cultures which were grown in the presence of csa were devoid of detectable virus particles : the frequency of viral detection was lower than 1 in 50 cell sections and only a few particles could be seen . the experiments demonstrate an inhibition of and a delay in the onset of cytopathic effects , in particular giant cell formation , on co - cultivation of uninfected cells with chronically infected cells which had been grown in the presence of either drug for five days and then washed ( fig1 ). in addition , using the anti - hiv assay system previously developed 7 , we were able to detect marginal antiviral activity at non - toxic concentrations . the inhibition of hiv - 1 replication by either csa or fk506 was , however , far lower than that of azt . furthermore at 1 μg csa and / or fk506 there was only a partial decrease in hiv - 1 replication . the effect of both drugs on the expression of cd4 was studied . as can be seen in fig3 concentrations of 1 and 4 μg / mg csa caused no reduction in the expression of cd4 on the surface of the non - infected cells . the experiments also demonstrate that during acute infection , when uninfected cells were co - cultivated with infected cells in the presence of the drugs , both csa and fk506 had anti - hiv - 1 and anti - hiv - 2 activity within 24 hours , as shown in the delay of the cytopathic effect ( fig1 ). the drugs were not found to inhibit or slow down the replication of the uninfected leukaemia t - cells ( fig2 a ). by contrast , both drugs inhibited the replication of the cells chronically infected with hiv - 1 ( fig2 b ). with azt the inhibitory effect on growth did not discriminate between infected or non - infected t - cells . it is not known why cyclosporin a and fk506 should inhibit the replication of hiv - 1 infected cells at concentrations which do not inhibit uninfected cells . nevertheless it is not unreasonable to suppose that the same phenomenon may also occur in vivo in hiv - infected individuals . the mechanism of inhibition of hiv production by these drugs is thought to be threefold : 1 ) through the inhibition of synthesis of active nf - at , an important transcription factor in activated t - cells 8 . no active nf - at is found in resting t - cells but it is produced in response to antigen recognition and signal transduction by the t - cell receptor complex . since the productive replication of hiv depends on activated t - cells , the inhibition of nf - at production could explain the significant reduction of hiv synthesis . our chronically infected cells , which grew in the presence of the drugs , produced only about 1 / 100th the amount of the infectious particles released by non - treated cells into the culture fluid . likewise , electron microscopic examination of ultrathin sections of cells revealed nearly 100 - fold reductions in the frequency of distinct hiv particles in cell cultures treated with the drugs . these virological and ultrastructural studies suggest that both drugs also interfere with transcription of the hiv provirus . staining of the drug - treated cells for hiv - 1 antigen was also weaker when compared to non - treated cells . 2 ) through the inhibition of the cytoplasmic enzyme peptidyl - prolyl cis - transisomerase ( rotamase ) which facilitates protein folding . it is thought that the formation of a rotamase - drug complex is responsible for the inhibition of hiv replication . 3 ) through the inhibition of calcineurin , a protein which is present in the cytoplasm of t - cells . how and why the drugs inhibit the replication of the chronically infected cells at concentrations which do not affect the replication of non - infected cells is not clear . in conclusion the effects of the drugs cyclosporin a and fk506 have been studied on chronically hiv - 1 infected cells , as well as on non - infected and newly infected cells . both drugs were found to inhibit the growth of the chronically infected cells at concentrations which did not inhibit the growth of the non - infected cells . in addition there was approximately at 100 - fold reduction in the yield of infectious hiv - 1 when the infected cells were grown in the presence of these drugs , a finding consistent with the decrease in hiv expression . when cells chronically infected with hiv - 1 or with hiv - 2 were co - cultivated with uninfected cells in the presence of csa or fk506 there was a delay in the formation of giant cells ( syncytia ) and cytopathic effects ( cpe ). this inhibitory effect was not due to decrease in the expression of membrane cd4 . the results , demonstrating the inhibition of hiv - 1 and 2 production by csa and fk506 , suggest that both drugs could be used to treat hiv - infected individuals . for patients who have already developed hiv associated disease and aids , these drugs could be used in combination with passive immunization , an established therapy which has been used in treatment of various conditions over the years 9 , 10 . 1 ) barre - 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