text: the present invention will be further described , by way of example , with reference to the following figures : [ 0040 ] fig1 shows the facs fluorescent overlays for rantes - mediated ccr5 internalisation in 300 . 19 cells and the inhibition of internalisation by compound b at various concentrations : without compound removal ( a ), following compound removal by washing ( b ) and following removal and 1 . 5 hour rolling incubation ( c ). the fluorescence shift due to rantes - mediated ccr5 internalisation is depicted by trace d , and the vehicle control ( no compound or rantes , i . e ., total ccr5 ) is depicted by trace e . isotype control is depicted by trace f . [ 0041 ] fig2 shows the facs fluorescent overlays for rantes - mediated ccr5 internalisation in 300 . 19 cells and the inhibition of internalisation by compound c at various concentrations : without compound removal ( a ), following compound removal by washing ( b ) and following removal and 1 . 5 hour rolling incubation ( c ). the fluorescence shift due to rantes - mediated ccr5 internalisation is depicted by trace d , and the vehicle control ( no compound or rantes , i . e ., total ccr5 ) is depicted by trace e . isotype control is depicted by trace f . [ 0042 ] fig3 is a graph showing the relative inhibition (% relative to vehicle control ) of rantes - induced internalisation of ccr5 by compounds b , c and d , when incubation is followed by a wash step (+ wash ) or 1 . 5 hours incubation in the absence of compound (+ chase ). [ 0043 ] fig4 is a graph showing the mean day 1 % ccr5 occupancy on cd4 t cells for cohorts 1 , 4 and 6 subjects receiving placebo or a single 100 mg , 25 mg and 3 or 10 mg oral dose of compound a , respectively , administered as a solution . [ 0044 ] fig5 is a graph showing the mean day 12 % ccr5 occupancy on cd4 t cells for cohorts 1 , 4 and 6 subjects receiving placebo or 100 mg , 25 mg and 3 or 10 mg b . i . d . oral doses of compound a , respectively , on days 3 to 12 , administered as a solution . [ 0048 ] fig9 is a graph showing ccr5 receptor saturation over time in asymptomatic hiv seropositive male subjects administered with compound a 25 mg , 100 mg or placebo . the invention is exemplified with the use of four compounds , where : compounds were tested in an assay for ccr5 affinity following the procedures disclosed in combadiere et al , j leukoc . biol . 60 , 147 - 152 ( 1996 ). compounds a to d all had an ic 50 for ccr5 binding of less than 10 nm ( data not shown ). 1 . functional occupancy of ccr5 on 300 . 19 cells as measured by dynamic inhibition of rantes - mediated ccr5 internalisation by facs analysis physical occupancy of the ccr5 receptor by antagonists can be measured using radioligand dissociation studies . the functional occupancy of antagonists on the ccr5 receptor on whole cells can be assessed by measuring antagonist - dependent inhibition of cognate chemokine - mediated receptor internalisation . these measurements can be made using fluorescence activated cell sorting ( facs ) technology . the prolongation of either the inhibition of chemokine - mediated ccr5 internalisation , following the removal of antagonist by cell washing , is a measurement of the functional occupancy of ccr5 . cell culture : 300 . 19 cells ( mouse pre - b cell line , recombinantly expressing human ccr5 ) were cultured in 75 cm 2 cell culture flasks in growth medium at 37 ° c . for 2 - 3 days in the humidified 5 % ( v / v ) co 2 incubator . for cell passage , routine splits from 1 × 10 5 to 2 × 10 5 of the suspension cells were performed . cell preparation : cells from each 75 cm 2 flask used were centrifuged at 1500 rpm on a bench top centrifuge for 5 minutes . pelleted cells were resuspended in a minimal volume of rpmi ( 10 % fbs ) culture medium ( see materials ), counted on a cedex cell counter ( see materials ), and adjusted to a cell density of 5 × 10 6 / ml by dilution in the same medium . antagonist , antibody , and chemokine preparation : ccr5 antagonists were dissolved in 100 % dimethyl sulphoxide ( dmso ) to a concentration of 1 mm and diluted in rpmi ( 10 % fbs ) cell culture medium to enable testing in the facs assays at concentrations from 10 nm to 1000 nm in situ . anti - ccr5 antibody ( 2d7 — see materials ) was diluted 1 : 10 in 0 . 5 % bsa / pbs . antibody igg2a ( isotype control for the assay — see materials ) was used diluted 1 : 10 in 0 . 5 % bsa / pbs . the anti - 2d7 phyco - erythrin ( pe )- labeled goat anti - mouse secondary antibody ( see materials ) was used at a 1 : 20 dilution in 0 . 5 % bsa / pbs . the cognate agonist chemokine ligand for ccr5 , regulated on activation , normal t - expressed and secreted ( rantes ), was solubilised in pbs . lyophilised material ( 10 μg — see materials ) was resuspended in pbs to a concentration of 100 μm . dilutions were then made in rpmi ( 10 % fbs ) to give a final concentration of 1000 nm ( 100 nm final concentration in assay ). 300 . 19 cells ( 100 μl ) at 5 × 10 6 cells / ml were added to each assay tube . ccr5 antagonists ( 10 μl ) or vehicle ( rpmi ( 10 % fbs )) were added to appropriate assay tubes to enable profiling over concentrations varying from 10 - 1000 nm in situ . the tubes were incubated at 4 ° c . for 45 minutes to allow antagonist association to the cells . to investigate prolongation of ccr5 occupancy by antagonists , samples were either left with antagonist present or centrifuged ( 1500 rpm in a bench top centrifuge ) and washed twice in 1000 μl rpmi ( 10 % fbs ). half of the washed samples were resuspended in 1000 μl of the same media and placed on a rolling platform for one and a half hours before being processed through the facs assay . the other half of the washed samples , together with the unwashed samples ( i . e ., antagonist present ), were processed immediately . rantes ( 10 μl ) was added to the samples followed by incubation at 37 ° c . for 45 minutes to provoke ccr5 internalisation . the samples were centrifuged ( 1500 rpm in a bench top centrifuge ) and washed twice in 0 . 5 % bsa / pbs . washed samples were resuspended in 40 μl of the same buffer . then , 2d7 antibody or isotype control ( 10 μl ) was added to the samples followed by incubation for 45 minutes at 4 ° c . to enable antibody binding to ccr5 . the samples were then washed once in 0 . 5 % bsa / pbs followed by the addition of 75 μl phyco - erythrin ( pe )- goat anti - mouse secondary antibody ( 1 : 20 dilution in 0 . 5 % bsa / pbs ) with incubation at 4 ° c . for 45 minutes in the dark to enable binding . the samples were subsequently centrifuged ( 1500 rpm on a bench top centrifuge for 5 minutes ), washed twice in 100 μl 0 . 5 % bsa / pbs , and resuspended in 1000 μl of 1 % formaldehyde / pbs for fixing . the fixed cells were processed in a becton dickinson facscalibur , to measure the fluorescence levels on the cells in each assay to assess antagonist - dependent inhibition of rantes - mediated ccr5 internalisation and consequently functional ccr5 occupancy . the facscalibur assays were run using cell quest software according to the supplier &# 39 ; s instruction manual , using excitation / emission wavelengths of 488 nm / 530 nm respectively . culture medium : 1 × 500 ml rpmi - 1640 medium with nahco 3 without l - glutamine — gibco brl ( cat no : 31870 - 025 ); 5 ml 200 mm l - glutamine — gibco brl ( cat no : 25030 - 024 ); 5 ml penicillin / streptomycin ( 10 , 000u / ml pen , 10 , 000 μg / ml strep )— gibco brl ( cat no : 15140 - 122 ); 50 ml foetal bovine serum ( fbs )— sigma aldrich ( cat no : f7524 ); 5 ml 1 m hepes buffer — sigma h - 0887 ; 2 μl neat 2 - mercaptoethanol — sigma m - 3148 . 225 cm 2 cell culture flask tissue culture treated — costar ( cat no : 3001 ); cedex innovatis cell counter ; innovatis sample cups ( cat no : 600050000 ); incubator set @ 37 ° c ., 5 % co 2 humidified ; denley br401 refrigerated centrifuge . assay buffer : rpmi ( 10 % fbs ); rpmi — gibco brl ( cat no : 31870 - 025 ); fbs — sigma ( cat no : f7524 ). wash buffer : 0 . 5 % bsa / pbs ; bsa — albumin , bovine fraction v — sigma a4503 ; dulbecco &# 39 ; s phosphate buffered saline ( pbs ) without ca 2 + and mg 2 + — gibco brl ( cat no : 14190 - 094 ). consumables : dmso tissue culture grade — sigma ( cat no : d - 8418 ); assay tubes : micro centrifuge tubes — costar ( cat no : 3621 ); facs tubes : falcon tubes — becton dickinson ( cat no : 352054 ); labsystems finn pipettes thermo life sciences ( cat nos : 4500 / 090 / 050 ); 1 ml pipette tips — thermo life sciences ( cat no : 9401103 ); 250 μl pipette tips — thermo life sciences ( cat no : 9400263 ); lms cooled incubator ; becton dickinson facscalibur — cell quest software ; 1 % formaldehyde / pbs : formaldehyde — sigma cat no : f1635 , dulbecco &# 39 ; s phosphate buffered saline ( pbs ) without ca 2 + and mg 2 + — gibco brl ( cat no : 14190 - 094 ); iec micromax rf centrifuge ; rantes — r & amp ; d systems ( cat no : 278 - rn - 010 ); mouse anti - human ccr5 monoclonal antibodies : 2d7 — pharmingen ( cat no : 36461a ); isotype control antibodies : mouse igg2a — pharmingen ( cat no : 33031 a ); secondary pe labelled antibody : pe labelled goat anti - mouse antibody — sigma ( cat no : p9287 ). the cell quest software used for acquisition of fluorescence data was also used for data analysis . mean fluorescence values were determined for the cell population from each assay sample . the decrease in mean fluorescence due to rantes - mediated internalisation was measured relative to vehicle exposed 2d7 antibody control . this enabled the calculation of antagonist - dependent inhibition of rantes mediated ccr5 internalisation , and subsequently comparison of functional occupancy by various ccr5 antagonists . the functional occupancy as measured by compound b - dependent inhibition of rantes - mediated ccr5 internalisation by 300 . 19 cells is depicted by fig1 . rantes - mediated ccr5 internalisation was apparent as observed by the shift in fluorescence upon following incubation of 300 . 19 cells with chemokine . inhibition of this internalisation was observed at all concentrations tested , as shown by the overlay in fluorescence for incubations where compound b was present throughout the assay ( no wash ), compared to the fluorescence profile for ccr5 ( as recognised by antibody 2d7 ) in the vehicle control . retention of this inhibition ( i . e ., functional ccr5 occupancy ) was observed following removal of compound b by cell washing . dynamic retention of inhibition was observed to varying levels at all concentrations following a 1 . 5 hour rolling incubation post compound b removal . these results highlight the dynamic retention of compound b - dependent inhibition of rantes - mediated uptake of ccr5 on 300 . 19 cells , and therefore show that functional occupancy of ccr5 by this antagonist can be retained over time . quantification of this functional occupancy was undertaken by measuring the geometric mean fluorescence signal for each assay and calculating the percent inhibition of rantes - mediated internalisation compared to uninhibited vehicle control . this quantification was undertaken for compounds b , c and d , to determine their functional occupancy in this assay , and demonstrate the usefulness of the assay for the identification of compounds with slow functional offset . the results of this quantification are shown in fig3 . the percent inhibition under each test condition are plotted on the bar chart and listed in the attached table . where the results are average values from more than one experiment , error bars indicate the range . as described above , compound b dissociates slowly in these experiments , whereas compound c dissociates more rapidly . in addition , it is apparent that compound c does not inhibit receptor update completely even at concentrations well in excess of its receptor ic 50 . this is consistent with offset from ccr5 as measured by radioligand dissociation studies ( see example 3 ). compound b dissociates slowly from ccr5 upon the addition of excess unlabelled compound ( t 1 / 2 = 3 . 5 hours , fig7 ). conversely , compound c showed relatively rapid dissociation in parallel radioligand dissociation studies ( t 1 / 2 = 15 minutes , fig8 ). according to the data shown in fig3 compound d is a slow offset inhibitor of ccr5 . this facs assay is capable of differentiating compounds based upon their dynamic occupancy of ccr5 and can be used to identify both slow and fast ccr5 offset compounds . 2 . functional occupancy of ccr5 on peripheral blood mononuclear cells as measured by inhibition of mip - 1β - mediated ccr5 internalisation by facs analysis mip - 1β mediated ccr5 internalisation (% occupancy ) was assessed in cd4 t lymphocytes prepared from whole blood citrate cpt ( cell preparation tubes ) samples taken from healthy volunteers participating in a clinical study designed to investigate the administration of multiple oral doses of compound a . compound a is a ccr5 antagonist and prevents mip - 1β binding to and subsequently internalising the ccr5 receptor . the difference in ccr5 expression on the cell surface between stabiliser treated ( total ccr5 ) and untreated ( maximum internalisation ) peripheral blood mononuclear cells subjected to mip - 1β challenge will give an estimate of the proportion of free ccr5 present on the cell surface at any given plasma concentration of compound a . this data can then be used to estimate the degree of receptor occupancy obtained at different doses of compound a . single dose administrations ( 3 mg , 10 mg , 25 mg and 100 mg ) of compound a followed by the same doses , i . e ., 3 mg , 10 mg , 25 mg and 100 mg , respectively , as multiple oral doses , b . d . for 10 days or placebo were investigated in healthy male subjects aged 18 - 45 years . subjects were allocated to one of the six cohorts ( data from cohorts 1 , 4 and 6 only are illustrated here ). within each of these six cohorts , subjects were assigned to receive either compound a at the appropriate dosing regimen or to receive placebo . the cohorts are : prior to dosing , a blood sample was taken for tests including laboratory safety tests and genotyping evaluation . each subject received compound a or placebo on day 1 between 08 : 00 and 10 : 00 hours . no dose was given on day 2 . a solution containing compound a was taken by the subject while sitting or standing with water for a total volume of 250 ml . blood samples were taken pre - dose and at intervals over the next 48 hours post - dose ( day 3 ), as described below . on days 3 - 11 subjects were dosed with compound a or placebo between 08 : 00 and 10 : 00 hours in the morning and 20 : 00 and 22 : 00 in the evening according to the same procedures as on day 1 . each day a blood sample was taken immediately prior to the administration of the first dose of compound a for the determination of trough plasma concentrations of compound a . on day 12 , compound a or placebo was dosed according to the same procedures as day 1 ; subjects received only one dose on day 12 . blood was taken pre - dose and at intervals over the next 72 hours post - dose . subjects were discharged at 24 hours post last dose but returned for a follow - up physical examination 7 - 10 days following the final dose . blood samples from subjects in cohort 1 were taken ( into 4 ml citrate cpt tubes ) at the following times : 0 ( baseline ), 4 , 12 , 24 and 48 h post 100 mg sd . compound a dose on day 1 . further samples were taken on day 12 at time 0 ( baseline ) and at 24 , 48 , 59 , 72 , 97 and 120 h post 100 mg sd . compound a dose . for subjects in cohort 4 , additional samples were collected at 8 , 18 , 52 and 64 h post 25 mg sd . compound a on day 1 . on day 12 , additional samples were collected at 144 , 168 and 240 h post 25 mg sd . compound a dose . for subjects in cohort 6 , samples were obtained at time 0 ( baseline ) and at 4 , 8 , 12 , 18 , 24 and 48 h post 3 or 10 mg sd . compound a dose on day 1 ; 4 and 16 h post - dose day 3 ; 2 h post - dose on days 4 to 11 post 3 or 10 mg b . i . d . compound a dose and then on day 12 at time 0 ( baseline ) and at 8 , 12 , 24 , 48 , 96 and 120 and 144 h post 3 or 10 mg sd . compound a dose . prior to the first dose of compound a or placebo , a 6 ml edta blood sample was also collected . the residual ccr5 receptor expression on cd4 positive lymphocyte populations was determined by flow cytometric analysis of processed sodium citrate cpt anti - coagulated whole blood samples . sodium citrate cpt ( 4 ml draw ) blood tubes ( becton dickinson . cat no . 362760 ) sample processing tubes ( 12 × 75 mm polystyrene round bottomed ) and caps ( push fit ) ( sarstedt ltd , cat nos . 55 . 476 and 65 . 809 . 504 respectively ) 10 × concentrated pbs ; 10 % paraformaldehyde ; 10 % sodium azide solution ; lyophilized bsa ; mip - 1β working solution ( aliquots stored frozen at − 70 ° c . ; ccr5 stabilising solution ( contains 600 nm compound a in pbs ); stabilising control solution ( pbs ); ccr5 and igg isotope control antibody cocktails ( store at 2 - 8 ° c .) 1 ml 10 % sodium azide solution in di h 2 o 2 . dilute stock solution 1 : 500 with 1 × pbs to create a 20 μm solution ( 2 μl / ml pbs ); 3 . dilute 20 μm solution 1 : 20 with 1 × pbs to create a 1000 nm ( 1 μm ) solution ; 4 . dilute 100 nm solution 3 : 2 with 1 × pbs to create a 600 nm solution . 600 nm drug solution ( stabilizing solution ) should be added 1 : 6 in plasma to achieve a final concentration of 100 nm . as per protocol , stabilizing solution should be added at 50 μl to 250 μl of cell enriched plasma . control stabilising solution should be added in similar proportions to the relevant tube . the stabilising solution should be made up fresh for each cohort and vortexed prior to use to ensure that the drug is in solution . 1 . lymphocyte rich plasma was isolated by centrifugation at 1550 g for 25 mins ( rt ) in a swing out rotor . 2 . the buffy coat layer of cells was re - suspended in the plasma by gently inverting tube several times (× 5 ). 3 . 250 μl of cell enriched plasma was pipetted into 3 separately labelled 12 × 75 mm polystyrene round - bottomed tubes . [ tube 1 ( control ), tube 2 ( total ccr5 ), and tube 3 ( ccr5 mip1β )]. 4 . 50 μl of ccr5 stabilising solution was added to tube 2 , while 50 μl of control stabilizing solution was added to tubes 1 and 3 add , before briefly vortexing on a medium setting for 2 seconds and incubating at 37 ° c . for 30 minutes . 5 . all tubes were centrifuged at 400 × g for 5 minutes in a swing out rotor to isolate cells and the supernatant then decanted . 6 . to all tubes , 15 μl of mip - 1β working solution was added , then gently vortexed on a medium setting for 2 seconds to re - suspend pellet in fluid . the tubes were then incubated uncapped for 45 minutes at 37 ° c . in a 5 % co 2 , 98 % humidity atmosphere . 7 . 1 ml of 0 . 5 % paraformaldehyde in pbs was added to each tube , vortexed on a medium setting for 2 seconds and incubated for 10 minutes in the dark at room temperature . 8 . 2 ml of pbs with bsa was then added to all tubes , the tubes centrifuged at 400 × g for 5 minutes in a swing out rotor to pellet cells before decanting the supernatant . 9 . antibody reagents ( 50 μl ) were added in the following manner : to tube 1 — msigg r - phycoerythrin ( pe ) and cd4 - fluorescein ( fitc ) ( control ) was added to tube 2 & amp ; 3 — ccr5 - pe and cd4 - fitc ( total ccr5 and ccr5 mip1β ) was added . 10 . all tubes were then vortexed on a medium setting for 2 seconds and incubated for 20 mins in the dark at room temperature . 11 . added 2 ml of pbs with bsa to all tubes . centrifuged at 400 × g for 5 minutes . decanted supernatant , then added 0 . 5 ml of 1 % paraformaldehyde in pbs and vortexed on a medium setting for 2 seconds to re - suspend pellet . 12 . finally , tubes were capped appropriately and stored at 2 - 8 ° c . until analysis . the mean percentage occupancy of ccr5 receptors on the surface of cd4 t cells prepared from whole blood ( cpt ) samples and corresponding standard deviations ( sd ) at each nominal time point for healthy male subjects receiving a single oral dose of compound a or placebo on day 1 followed by b . i . d . doses on days 3 to 12 are given in table 1 . ccr5 occupancy was related to dose . volunteers receiving 100 mg b . i . d . demonstrated ccr5 receptor occupancy in excess of 90 % throughout the dosing period , while in those subjects receiving 3 mg , mean receptor saturation was & lt ; 80 %. physical occupancy of the ccr5 receptor by antagonists may be measured using radioligand dissociation studies . radiolabelled antagonist is incubated to equilibrium with ccr5 expressed on hek - 293 cells or membrane preparations thereof . dissociation , and hence physical occupancy or residence time , is subsequently measured by adding excess unlabelled antagonist to prevent re - association of dissociated radiolabelled antagonist . thus , dissociation can be measured by counting retained radiolabel on whole cells or membranes over time following addition of unlabelled antagonist . cell culture : hek - 293 cells stably expressing ccr5 ( produced by standard techniques ) were grown in 225 cm 2 cell culture flasks to a confluency between 50 - 70 % in culture medium ( see materials ) at 37 ° c . for 2 - 3 days in the humidified 5 % ( v / v ) co 2 incubator . the cells were grown at low density because of their tendency to clump , with less expression of ccr5 at full confluency . each 225 cm 2 flask used for the binding assays was washed once by removal of culture medium and replacement of 20 ml phosphate buffered saline ( pbs ) at room temperature . the supernatant was removed and the cells were dislodged by rapping the side of the culture flask in the presence of 10 ml binding buffer at room temperature ( see materials ). the cells were placed into a 50 ml centrifuge tube . a further 10 - 20 ml binding buffer ( room temperature ) was added into the culture flask then transferred to the centrifuge tube to harvest residual cells . the cells were centrifuged at 350 g for 10 minutes at 20 ° c . the cells were resuspended in 3 ml binding buffer , counted in a modified neubauer haemocytometer and resuspended to a density of 2 × 10 6 cells / ml . membrane preparation : each 225 cm 2 flask used for the binding assay was washed once by discarding the culture medium and replaced by 20 ml pbs ( room temperature ) and resuspended in 5 - 10 ml pbs ( room temperature ). the cells were transferred into a 50 ml centrifuge tube . a further 10 - 20 ml binding buffer ( room temperature ) was added into the culture flask then transferred to the centrifuge tube to harvest residual cells . the cells were centrifuged at 350 g for 10 minutes at 4 ° c . the cells were resuspended in 15 ml lysis buffer ( see materials ) at room temperature , and homogenised with a polytron handheld homogeniser ( 5 - 10 seconds on ice , 3 - 4 times on the high setting ). the homogenate was transferred to oakridge tubes and centrifuged in the beckman ultracentrifuge ( in t865 rotor ) at 25000 rpm ( 40 , 000 g ) for 30 minutes at 4 ° c . the supernatant was discarded and the pellet resuspended in a minimal volume of lysis buffer ( room temperature ). the protein concentration was estimated using the bradford microassay for proteins and adjusted to 0 . 25 mg / ml in binding buffer . this enabled the addition of 12 . 5 g of membrane protein to be used in each radioligand binding assay compound preparation : 1 - 2 mg compound a , b or c was dissolved in 100 % dimethyl sulphoxide ( dmso ) to a concentration of 10 mm and diluted in binding buffer to enable compound addition to the binding assays over a concentration range of 0 . 41 nm to 50 μm in situ . 20 μl of [ 3 h ] compound a ( 62 . 5 μm , specific activity 16 ci / mmol and radioactive concentration of 1 mci / ml ) was diluted in binding buffer to enable radioligand addition to the binding assay over a concentration range of 0 . 41 - 100 nm in situ . similar dilutions were performed with [ 3 h ]- labelled compounds b and c . dissociation studies : binding buffer ( 25 μl ) was added to wells of a 96 well assay plate , followed by 50 μl cells or cell membrane preparation . [ 3 h ] compound a , b or c dilutions ( 25 μl ) were added to designated wells to enable radioligand association . non - labelled compound ( 100 ×[ 3 h ] compound concentration ) was added designated wells ( 25 μl ) in place of the binding buffer to determine non - specific radiolabel binding in the assays added prior to the radiolabel . the reaction mixtures were set up in duplicate to enable non - radiolabelled compound to be added to one of the pair , to measure dissociation , and binding buffer to the other to act as an association control . this control was set up to account for dissociation occurring as a result of ccr5 or membrane / cell denaturation . the plates were incubated at room temperature for 1 hour post addition of [ 3 h ] compound . this allowed radioligand binding to ccr5 to reach equilibrium . after this incubation , 25 μl non - labelled compound dilutions were added to the dissociation wells . the well contents were harvested after incubations at room temperature up to 48 hours post - association . for harvest of cells and membranes , the unifilter plates were blocked by applying 25 ml blocking agent ( room temperature ) ( see materials ) for 30 minutes . the unifilter plates were washed once with 50 ml wash buffer ( room temperature ), the reactions were harvested by aspiration and the plate washed 3 times ( 1 ml / well ) with wash buffer ( room temperature ) on the packard harvester . the unifilter plate was allowed to dry overnight . microscint 0 ( 50 μl — room temperature ) was added to each well and a topseal cover placed over each plate . the plates were read ( 1 minute per well ) on the nxt packard topcount scintillation counter . growth medium : 1 × 500 ml dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) with sodium pyruvate , without l - glutamine , with pyridoxine — gibco brl ( cat no : 21969 - 035 ) containing 5 ml 200 mm l - glutamine — gibco brl ( cat no : 25030 - 024 ); 5 ml penicillin / streptomycin ( 100u / ml pen / 10 mg / ml strep )— sigma ( cat no : p - 7539 ); 50 ml foetal calf serum ( fcs )— paa laboratories , austria ( cat no : al 5 - 042 ); and 6 . 5 ml 50 mg / ml geneticin — gibco brl ( cat no : 10131 - 019 ) 225 cm 2 cell culture flask tissue culture treated — costar ( cat no : 3001 ) dulbecco &# 39 ; s phosphate buffered saline ( pbs ) without ca 2 + and mg 2 + — gibco brl ( cat no : 14190 - 094 ). binding buffer : 50 mm hepes in distilled water — sigma ( cat no : h - 0763 ). 1 mm cacl 2 — sigma ( cat no : c - 3881 ). 5 mm mgcl 2 — sigma ( cat no : m - 1028 ). 0 . 5 % bovine serum albumin ( bsa )— sigma ( cat no : a - 4503 ). buffer adjusted to ph 7 . 4 ( 2m . hcl ) and 0 . 2 μm filtered . lysis buffer : 20 mm hepes in distilled water — sigma ( cat no : h - 0763 ). 1 mm cacl 2 — sigma ( cat no : c - 3881 ). 1 tablet complete ™ protease inhibitors per 50 ml lysis buffer — boehringer mannheim ( cat no : 1697498 ). buffer adjusted to ph 7 . 4 ( 2m hcl ) and 0 . 2 μm filtered . wash buffer : 10 mm hepes in distilled water — sigma ( cat no : h - 0763 ). 0 . 5m nacl — sigma ( 5m solution cat no : s - 5150 ). 0 . 5 % bovine serum albumin ( bsa )— sigma ( cat no : a - 4503 ). buffer adjusted to ph 7 . 4 ( 2m hcl ) and 0 . 2 μm filtered . stock solutions made up at 1 m ( hepes , cacl 2 and mgcl 2 ) and stored at room temperature , the above buffers are prepared fresh each time . blocking agent : 0 . 3 % polyethyleneimine ( pei ) in distilled water — sigma ( cat no : p - 3143 ). consumables : dmso tissue culture grade — sigma ( cat no : d - 2650 ). polypropylene deep well blocks ( 1 ml / well )— porvair ( cat no : 219008 ). multichannel pipettes — labsystems finnpipette ( cat no : 4510 - 000 / 020 / 030 / 040 / 050 ). 1 ml pipette tips — sigma ( cat no : p7174 ). 250 μl pipette tips — sigma ( cat no : p7049 ). reagent reservoirs for multichannel pipettes — costar ( cat no : 4870 ). packard filtrate universal harvester ( 96 well head ). packard unifilter gf / b 96 well filter plates with bottom seal — packard ( cat no : 6005177 ). microscint 0 — packard ( cat no : 6013611 ). topseal - a microplate press - on adhesive sealing film — packard ( cat no : 6005185 ). nxt packard topcount scintillation counter . as shown in fig6 % of compound a was still bound after 9 hours of incubation with excess unlabelled compound a . after 3 hours , 50 % of compound b was still bound ( fig7 ), whereas 50 % of compound c was still bound after 15 minutes ( fig8 ). anti - hiv activity in peripheral blood lymphocytes ( pbls ) for compounds was tested by infecting these cells with hiv ( isolate ba - l ) and measuring compound - dependent reduction in supernatant reverse transcriptase ( rt ) activity following incubation for 5 days . the hiv - 1 ba - l and iiib viruses used in the antiviral assays were obtained from the aids reagent project , nibsc , potters bar , herts , uk ( repository references arp118 and arp101 ). the virus was stored in 1 ml vials at − 80 ° c . for expansion of hiv - ba - l virus stock ( 1 ml ) was removed from − 80 ° c . storage , and rapidly thawed in a 37 ° c . incubator for 10 minutes . virus stock ( 0 . 5 ml ) was placed in a labelled well of a 24 well plate with 1 ml of fresh pbls ( pre - stimulated 3 days with phytohaemagglutinin ( pha ) at a final concentration of 1 . 5 μg / ml ) at a cell density of 1 . 0 × 10 7 / ml in complete rpmi 1640 growth medium ( supplemented with 10 % fcs , 2 mm l - glutamine , and 1u / ml penicillin , 0 . 1 mg / ml streptomycin ). the 24 - well plate was incubated at 37 ° c . in a humidified 5 %( v / v ) co 2 incubator for 1 hour to allow infection . after 1 hour , the cells and supernatant from this plate were centrifuged at 225 g for 10 minutes at 25 ° c ., and the supernatant discarded . the cells were suspended in 10 ml of rpmi 1640 growth medium at room temperature and transferred to a 25 cm 2 tissue culture flask . il - 2 was added to a final concentration of 10 ng / ml . the infected pbl culture was incubated in an upright position at 37 ° c . in humidified 5 %( v / v ) co 2 incubator for 3 days . at day 3 , 5 mls of fresh rpmi 1640 growth medium were added together with il - 2 at a final concentration of 10 ng / ml . after 7 days the infected cells were transferred to an 80 cm 2 tissue culture flask . a further 7 ml of il - 2 - stimulated pbls ( 1 . 0 × 10 7 cells / ml ) in rpmi 1640 growth medium was added , and incubated in a humidified 5 %( v / v ) co 2 incubator until day 10 . fresh rpmi 1640 growth medium ( 25 ml ), containing 10 ng / ml il - 2 , was added at day 10 followed by gentle mixing . the culture was separated into two top 80 cm 2 tissue culture flasks , and incubated at 37 ° c . in a humidified 5 %( v / v ) co 2 incubator until day 15 . viral expansion was measured by reverse transcriptase ( rt assay ), and counts of ≧ 4000 cpm per 20 μl supernatant from the rt assay were deemed as active . cells were expanded further by dividing the infected culture in to fresh 80 cm 2 tissue culture flasks , followed by the addition of 13 ml of fresh il - 2 - stimulated pbls ( 1 . 0 × 10 7 cells / ml )/ ml . the flasks were incubated at 37 ° c . in a humidified atmosphere of 5 % co 2 until day 18 . at day 18 , 20 ml of rpmi 1640 growth medium ( containing il - 2 at long / ml ) was added . the cultures were incubated at 37 ° c . in a humidified atmosphere of 5 % co 2 until day 23 . virus samples were deemed active if rt counts in the supernatant ( 20 μl ) were & gt ; 5000 cpm at day 23 . these supernatants were clarified by centrifugation at 850 g for 10 minutes at 250 ° c ., and the resultant supernatant mixed , and aliquoted into 4 ml and 1 ml cryotubes for storage at − 80 ° c . the tissue culture infectious dose 50 ( tcid 50 — assumed number of viral particles required to infect 50 % of cells ) was calculated for the hiv ba - l stock ( harvested dec . 17 , 1999 ), by the reed and muench equation (( 1938 ) am . j . hyg . 27 , 493 - 497 ), ( using rt as a positive marker for hiv infection ). titration of virus stock hiv - 1 ba - l ( harvest dec . 17 , 1999 ) produced a tcid 50 5 . 76 × 10 4 / ml using this method . all titrations were performed using freeze - thawed virus stocks , in fresh - pooled pha - stimulated pbls . to prepare pbls , cells were pelleted by centrifugation for 10 minutes at 500 g and 25 ° c ., checked for viability by removing 100 μl of cells , followed by the addition of 100 μl of cell dissociation solution ( see materials ) and 100 μl of 0 . 4 %( v / v ) trypan blue . viable ( uncoloured ) cells were counted in a kova counting chamber ( see materials ). only cell suspensions showing & gt ; 95 % viability progressed in assays . viable cells were resuspended to a density of 2 . 0 × 10 6 cells / ml in rpmi growth medium ( 10 ng / ml il - 2 added for pbl cultures ). expanded frozen hiv ba - l stock ( 2 × 1 ml ) were rapidly thawed in a 37 ° c . incubator for 10 minutes . one sample was progressed for titration assessment and the other progressed for antiviral testing in parallel . for titration , 25 μl of thawed expanded virus stock , was progressed through 8 x 0 . 5 log serial dilutions in rpmi growth medium . each viral dilution ( 20 μl ) was transferred to a 96 - well assay plate together with 180 μl of pbls . the assay plate was incubated at 37 ° c . in humidified atmosphere of 5 % ( v / v ) co 2 for 5 days , after which 100 μl of supernatant was harvested and tested for rt activity , to enable tcid 50 determination . the titred virus was deemed to be positive with respect to virus infectivity when rt counts were greater (& gt ; 2 × standard deviations ) than the uninfected cell counts . viral titre was calculated using the method reported by reed and muench (( 1938 ) am . j . hyg . 27 , 493497 ). this enables the standardisation of viral input for the antiviral assays and subsequent antiviral potency testing of the compounds . the tcid 50 for hiv - ba - l in the antiviral assays in this study was 5 . 76 × 10 4 / ml . the north london blood transfusion centre supplied single donor buffy coats containing pbls , from hiv and hbv sera - negative donors . serological status was determined by north london blood transfusion services . pbls were prepared from 4 buffy coat samples that were individually separated and expanded in culture . each of the 4 buffy coats ( 50 ml ) were transferred into an 80 cm 2 tissue culture flask with an equal volume of sterile phosphate - buffered saline ( pbs ). aliquots ( 25 ml ) of cell suspension were gently layered onto 25 ml ficoll - paque in separate 50 ml centrifuge tubes . following centrifugation for 30 min at 1000 g at 25 ° c ., the pbl layer was removed by pipetting from between the erythrocyte and plasma layers . the separated pbls were transferred to fresh centrifuge tubes and washed twice with pbs ( 4 ° c .) and centrifugation for 10 minutes at 850 g at 4 ° c . contaminating erythrocytes were removed by adding 9 ml sterile water to the pbl pellet together with × 10 hanks balanced salt solution ( see materials ). pbs ( 4 ° c .) was added to give a final volume of 45 ml . the pbls were centrifuged for 10 min at 500 g at 4 ° c . the pellet was suspended in 30 ml rpmi 1640 growth medium ( room temperature ). cell viability was checked as described above , and only cell suspensions showing & gt ; 95 % viability were processed for antiviral assay . the cell suspension was adjusted to a density of 1 × 10 6 cells per ml by the addition of rpmi 1640 growth medium supplemented 1 . 5 μg / ml pha . the cells ( 50 ml ) were transferred to 80 cm 2 tissue culture and incubated for 3 days at 37 ° c . in a humidified 5 % co 2 ( v / v ) incubator , in preparation for antiviral assay . the pbls were prepared and tested for viability by trypan blue staining as described above . pbl preparations with & gt ; 95 % viability were transferred to 80 cm 2 culture flasks in a total volume of 50 ml and cell density of 1 × 10 6 / ml . the flasks were incubated for 1 hour at 37 ° c . in a humidified 5 % co 2 ( v / v ) incubator to facilitate monocyte adhesion to the flask surface . the non - adherent pbls were carefully decanted into 50 ml centrifuge tubes and pelleted by centrifugation at 500 g for 10 min at 25 ° c . the pbl pellet was adjusted to a density of 1 × 10 6 cells per ml by the addition of fresh rpmi 1640 growth medium containing 1 . 5 μg / ml pha . the cells ( 50 ml ) were transferred to 80 cm 2 tissue culture and incubated for 3 days at 37 ° c . in a humidified 5 % co 2 ( v / v ) incubator , in preparation for antiviral assay . the pha - stimulated cell cultures were dispersed by gentle shaking and transferred to a sterile 50 ml centrifuge tube . the cells were pelleted by centrifugation for 10 minutes at 500 g at 25 ° c . and resuspended in 50 ml rpmi growth medium . cell viability was checked as before . only cell suspensions showing & gt ; 95 % viability were processed for antiviral assay . for cell infection , expanded ba - l stock of known titre was added to the pbl cell pellet , at a ratio of 250 μl stock per 1 . 0 × 10 6 cells , tcid 50 5 . 76 × 10 4 / ml followed by the addition of 125 μl rpmi growth medium ( containing 10 ng / ml il - 2 ) per 1 . 0 × 10 6 cells . the cells were then incubated for 1 hat 37 ° c . in a humidified 5 %( v / v ) co 2 incubator , followed by centrifugation at 500 g for 10 minutes at 25 ° c . the cell pellet was resuspended in rpmi growth medium ( containing 10 ng / ml il - 2 ) at a density of 2 . 0 × 10 5 / ml . 1 . 8 ml aliquots of infected cell suspension were transferred to a 24 - well assay plate , containing 200 μl of compound . the final concentration range of compound in the assay plate was 0 - 100 nm ( 0 . 1 % dmso ). the assay plate was placed in a humidified 5 % co 2 ( v / v ) incubator at 37 ° c . for 5 days . non - infected pbl controls were processed in parallel , together with a positive control containing the anti - hiv inhibitor rantes tested over a 0 - 100 nm and 0 - 33 nm concentration range . following the 5 day incubation , 200 μl of supernatant from each well was transferred to a 96 well plate , for quantification of virus yield by reverse transcriptase activity , and subsequent determination of the compounds &# 39 ; antiviral potency . quantification of viral yield by direct measurement rt activity in hiv infected cell cultures was performed using a scintillation proximity assay kit from amersham ( see materials ). rt activity was assayed by monitoring [ 3 h ] ttp incorporation into biotin - labelled dna primer linked to the surface of the spa beads . reagent volumes for rt assay depended on the number of assays to be run . to enable 100 assays , 1 ml of stock [ 3 h ] ttp ( 3700 kbq ) was diluted in 2 ml assay kit buffer , and transferred to an 80 cm 2 culture flask together with 5 ml sterile water and 1 ml spa bead from assay kit . this solution was mixed thoroughly and 80 μl aliquots were added to each well in the rt reaction plate , followed by 20 μl of the hiv - infected pbl supernatants . the reaction plates were sealed and placed in a 37 ° c . incubator for 1 hour . to stop the rt reaction , 100 μl of assay kit stop buffer ( 2 % ( w / v ) sds , ph 8 ) was added . the plates were centrifuged at 250 g for 10 minutes at 4 ° c . scintillation was measured for each well over a 60 second period using a 1450 microbeta liquid scintillation counter , following a 20 minute incubation period in situ to enable bead settlement . background counts for uninfected control samples were subtracted from infected sample counts . a standard curve for rt activity was generated by running purified recombinant rt samples in parallel to the hiv - infected cell samples , to ensure rt levels from the supernatants were in the linear range for the assay . for the rt standard curve , 20 μl of stock recombinant reverse transcriptase ( 10u / μl — see materials ) was defrosted and diluted with 9980 μl complete rpmi 1640 medium 4 ° c . the enzyme was stored at − 20 ° c . until used to produce serial dilutions in complete rpmi 1640 medium at 4 ° c . ( 20 , 10 , 5 , 2 . 5 , 1 . 25 , 0 . 62 , 0 . 31 and 0 . 15 mu / μl ) for a standard curve . each enzyme dilution ( 20 μl ) was sampled in duplicate in the rt assay . potential non - specific cytotoxicity of compounds in pbls was investigated as a possible cause for apparent antiviral activity . the assay was performed in 96 - well format , using a calorimetric cell proliferation kit from promega ( see materials ). in this assay , the tetrazolium compound 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 5 -( 3 - carboxymethoxyphenyl )- 2 -( 4 - sulfophenyl )- 2h - tetrazolium ( mts ) in combination with the electron - coupling reagent phenazine methosulphate ( pms ), is reduced by dehydrogenase activity in viable cells to generate formazan . the absorbance of the formazan at 492 nm is directly proportional to the density of living cells in the culture . this assay was performed on uninfected pbls separately cultured in parallel to those used in the antiviral assays . the pbls were prepared for the cytotoxicity assay as described for the antiviral assays , prior to hiv infection . the pbls were suspended in fresh culture medium to a final density of 2 × 10 5 cells / ml . the cell suspensions ( 180 μl ) were transferred to a 96 - well plate , in addition to 20 μl of compound to yield a final concentration range of 0 - 10 μm in situ ( 0 . 1 %( v / v dmso ). the assay plates were incubated at 37 ° c . in a humidified 5 % co 2 ( v / v ) incubator for 5 days . following this incubation 40 μl of mts / pms mixture from the cytotoxicity assay kit was added to each well , and incubated in the dark for 3 hours at 37 ° c . humidified 5 % ( v / v ) co 2 incubator . the plates were then shaken by hand , and absorbance at 492 nm measured spectrophotometrically to assess the viability of the cells and non - specific cytotoxicity of the compounds . antiviral activity was determined by plotting a graph of percent reduction in rt activity or p24 levels ( relative to no compound control ), against compound concentration ( log scale ). the ic 50 and ic 90 values were determined using the fit curve option of microsoft excel for duplicate datapoints at each concentration . the ic 50 and ic 90 values for independently prepared concentration ranges tested of compound were determined and the geometric mean calculated using standard software packages . these experiments were independently repeated with different preparations of pbl cells , and the overall geometric mean ic 50 and ic 90 values , together with the 95 % confidence intervals were calculated . the same analysis was performed for the antiviral standard rantes . individual ic 50 and ic 90 values which fell outside the concentration range of compound , or rantes were not included in further calculation for geometric mean ic 50 or ic 90 . hiv - 1 strain ba - l : a cell free supernatant was obtained from aids reagent project ( nibsc , potters bar , herts , uk .). rantes : obtained from r & amp ; d systems ( cat no . 278 - rn - 010 or 278 - rn - 050 ) cell culture medium : rpmi medium was obtained from sigma ( cat no : r0883 ), l - glutamine from life technologies ( cat no : 25030 - 024 ), fcs from sigma for pbl cells ( cat no : f2524 batch 77h3399 ), penicillin & amp ; streptomycin from life technologies ( cat no 15140 - 123 ). additives for pbl culture medium : pha suspended in sterile water to 1 mg / ml , and used at 1 . 5 μg / ml final concentration , murex - abbott laboratories ( cat no : ha16 ). il - 2 , human recombinant dissolved in 4 mm hcl plus 0 . 1 % ( v / v ) fcs to 2 μg / ml ( approx . 6000 u / ml ) and used at 10 ng / ml final concentration .— r & amp ; d systems ( cat no : 202 - il - 050 ) pbl isolation reagents : buffy coats supplied by north london blood transfusion centre ( colindale centre , ficoll - paque solution from pharmacia biotech ( cat no : 17 - 0840 - 02 ), pbs from sigma ( cat no : d8537 ), sterile distilled water from sigma ( cat no : w - 3500 ) reverse transcriptase assay kit : quan - t - rt assay system from amersham life science ( cat no : trk 1022 ). recombinant hiv reverse transcriptase , supplied as 200u ( 10u / μl ) from amersham , ( cat no : t3610y ). cytotoxicity assay kit : celltiter 96 ® aq ueous non - radioactive assay — promega ( cat no : g5430 ) consumables : 24 well sterile flat bottom falcon plates — becton dickinson ( cat no : 3047 ). 96 well sterile flat bottom falcon plates — becton dickinson ( cat no : 3072 ). 96 square well plate polypropylene 2 ml capacity per well sterile ( beckman coulter cat no 609681 ). cell counting chamber - kova biostat diagnostics ( cat no : 887144 ). in the antiviral assay , compound d has an ic 90 of 1 nm , compound a and compound b have an ic 90 in the antiviral assay of around 2 nm , whereas compound c has a potency in the antiviral assay of & gt ; 100 nm . although compounds a to d have very similar binding affinity to ccr5 , the antiviral effect is markedly improved for ligands that have a slow dissociation rate , or a long residence time on ccr5 . therefore , the residence time of a ligand on ccr5 may be used as a predictor for the potency of the ligands for functional activity , in this case antiviral activity , and predicts clinical efficacy for the ligand as an antiviral agent . effect of short - term monotherapy with compound a on viral load in hiv - infected patients for standard antiretroviral agents that target the virus directly , there is a direct relationship between antiviral effects ( as measured by a decrease in plasma viral load ) and plasma drug concentrations . likely clinically efficacious dose ranges can therefore be selected based on in vitro antiviral data i . e ., ic 50 and ic 90 . however , an argument can be made that for this approach , saturation of the ccr5 receptor could be a better predictor of efficacy and may not be directly related to plasma drug concentrations . data from healthy volunteer studies demonstrated that even at a dose of 25 mg where the drug concentrations rapidly fell to well below the antiviral ic 90 , essentially complete saturation of the receptor was achieved for & gt ; 24 hours post - dose . the rationale for this study was therefore , inter alia , to determine whether saturation of the ccr5 receptor correlates with antiviral effects in vivo . twenty - four asymptomatic hiv seropositive male subjects with cd4 t cell count & gt ; 250 cells / mm 3 and plasma viral load & gt ; 5000 copies / ml received compound a 25 mg o . d ., 100 mg b . i . d . or placebo for 10 days . main exclusion criteria were liver enzyme abnormalities , cd4 t cell count & lt ; 250 cells / mm 3 , viral load & lt ; 5000 copies / ml and the presence of cxcr4 or dual tropic hiv . receptor saturation was evaluated using the ccr5 / mip - 1β , internalisation assay described above at the following time points : viral load was determined using an rt - pcr ( roche amplicor v1 . 5 ) assay with a lower limit of detection of 400 copies / ml as standard . for samples with a reading of & lt ; 400 copies / ml , the ultrasensitive method with a lower limit of detection of 50 copies / ml was used . samples were taken at the following timepoints : day 1 : 0 ( pre - morning dose ) and 4 , 12 and 18 hours post - dose mean ccr5 receptor saturation in patients receiving 100 mg b . i . d . was in excess of 90 % throughout the dosing period , but in subjects receiving 25 mg od mean receptor saturation fell to & lt ; 80 % by day 10 pre - dose ( steady state ). fig9 demonstrates receptor saturation over time ( in hours post - first dose ) per dose group . seven of eight patients receiving 100 mg b . i . d . of compound a showed a good viral load response with a viral load decline of & gt ; 1 log 10 . the mean drop in viral load from baseline to day 11 in this group was 1 . 200 log 10 . one subject in the 100 mg b . i . d . dose group , in whom co - existence of viruses using ccr5 and / or cxcr4 as their entry coreceptor was demonstrated on day 1 , had no response to compound a . if this individual is excluded from the analysis the drop in viral load in the 100 mg group is 1 . 419 log 10 . in the 25 mg o . d . dose group a mean viral load reduction of 0 . 4251 log 10 was demonstrated ( table 2 ). following termination of dosing ( day 11 onwards ) viral load returned to baseline over time and by day 40 most patients have returned to baseline . mean ccr5 receptor saturation in patients receiving 100 mg b . i . d . was in excess of 90 % throughout the dosing period , but in subjects receiving 25 mg o . d ., mean receptor saturation fell to & lt ; 80 % by day 10 pre - dose . subjects receiving 100 mg b . i . d . had a mean decrease in viral load of 1 . 200 log 10 from baseline to day 11 , and there was evidence of a response in subjects receiving 25 mg o . d . clinical data shows that compound a exhibited potent antiviral effects when given as short - term monotherapy and thus supports the in vitro data and its prediction of clinical efficacy . from the examples given above , it can be seen that ccr5 residence , or occupancy , time in vitro may be correlated with receptor occupancy in vivo . more importantly , ligands identified by in vitro receptor occupancy show significant decreases in viral load both in vitro and in vivo . thus , measurement of occupancy of a ligand on its receptor in vitro may be used to identify a ligands predicted to be efficacious in vivo . while this theory has been tested and proved in an hiv scenario , it will be appreciated that the prediction of the clinical efficacy of putative ligands from in vitro receptor occupancy may be extended to other ccr5 - mediated medical indications , including respiratory disorders , including adult respiratory distress syndrome ( ards ), bronchitis , chronic bronchitis , chronic obstructive pulmonary disease , cystic fibrosis , asthma , emphysema , rhinitis and chronic sinusitis . other conditions for which a correlation with ccr5 or ccr5 chemokines has been established include inflammatory bowel disease , including crohn &# 39 ; s disease and ulcerative colitis , multiple sclerosis , rheumatoid arthritis , graft rejection , in particular but not limited to solid organ transplants , such as heart , lung , liver , kidney and pancreas transplants ( e . g . kidney and lung allografts ), endometriosis , type i diabetes , renal diseases , such as glomerular disease , fibrosis , such as liver , pulmonary and renal fibosis , chronic pancreatitis , inflammatory lung conditions , encephalitis , such as hiv encephalitis , chronic heart failure , psoriasis , stroke , obesity , cns diseases , such as aids related dementias and alzheimer &# 39 ; s disease , anaemia , atherosclerotic plaque , atopic dermatitis , chronic pancreatitis , cancer , such as non - hodgkin &# 39 ; s lymphoma , kaposi &# 39 ; s sarcoma , melanoma and breast cancer , and pain , such as nociceptive pain and neuropathic pain ( e . g . peripheral neuropathic pain ). infectious diseases where modulation of the ccr5 receptor is implicated include acute and chronic hepatitis b virus ( hbv ) and hcv infection , bubonic , septicemic , and pneumonic plague , pox virus infection , such as smallpox , toxoplasmosis infection , mycobacterium infection , trypanosomal infection such as chagas &# 39 ; disease , pneumonia , and cytosporidiosis . while the invention has been illustrated by reference to specific and preferred embodiments , those skilled in the art will recognize that variations and modifications may be made through routine experimentation and practice of the invention . thus , the invention is intended not to be limited by the foregoing description , but to be defined by the appended claims and their equivalents .