MXPA04011577A - Method for identification of a ligand whereby receptor residence time is measured. - Google Patents

Abstract

The present invention relates to the use of an assay that measures receptor residence time of a ligand on its receptor in vitro for the identification of a ligand for that receptor predicted to be efficacious in vivo in the treatment of a disease that responds to modulation of that receptor's natural function.

Description

METHOD FOR IDENTIFYING A BINDER, BY WHICH THE MEASURES THE RECEIVER'S RESIDENCE TIME DESCRIPTIVE MEMORY The present invention relates to the use of a test to determine receptor occupancy, as a means to identify ligands that are predicted to be clinically effective. In particular, the present invention relates to the identification of CCR5 receptor ligands, using a long residence time of the receptor as an indicator of antiviral activity, specifically anti-HIV activity. The production of an impressive medicine is becoming increasingly difficult, spending more and more money on research and development, while the number of new drugs that reach the patient rarely equals the investment. The depletion of new potential drugs typically results from safety problems, and the combination of survival rates from discovery to the clinic has become one of the most pressing problems facing the pharmaceutical industry. The costs up to the clinical phase can be around 20 million dollars (almost 13 million pounds sterling). For every 10 to 15 compounds, only one survives the clinical phase. Once a compound has shown sufficient in vitro activity and has already been tested on animals, the costs increase sharply.

Phase I tests in healthy volunteers cost an average of 20 million dollars (13 million pounds sterling) per compound studied, and about one of two compounds fails. Phase II trials in humans to study the correct dosage cost, on average, 40 · million dollars (26 million pounds sterling) per compound, and about one of two fail. Double-blind phase III trials around the world in large patient populations to determine efficiency against placebo or another frequently used product cost an average of 560 million dollars (360 million pounds sterling). The phase III tests at 560 million dollars is 70% of the total cost of a product and there is, on average, only a probability of survival of 58%. The final stage for a new drug is presenting approval or registration. Each patient of the thousands studied has a file; average of 50 pages, and costs are in the region of 103 million d & pounds sterling. Increasingly, strict and severe standards mean that only approximately 74% of new drugs are approved. Therefore, the depletion of those compounds that are less likely to survive, before they are taken to the clinical stage, is critical. Studies of the pharmacokinetics and metabolism of drugs, as well as the evaluation of drug safety, clinical priority and knowledge of the safety of a molecule or mechanism, provide valuable and essential data when evaluating a compound for its potential in the clinic. However, methods for predicting the potential clinical efficacy of an early developing compound are clearly desired. Although AIDS (acquired immunodeficiency syndrome) induced by HIV was first described almost two decades ago, it remains a disease of enormous proportions. It is estimated that there were more than 40 million people infected with HIV worldwide at the end of 2002 (UNAIDS / WHO (OS).) Epidemic AIDS Update: December 2002 http.7 / www.unaids.orq / worldaidsday /2002/press/epupdate.html). The human immunodeficiency virus type 1 (HIV-1) attacks the cells of the immune system, causing the body to lose the ability to fight infections and diseases. During the course of the infection, CD4 T cells (white blood cells that fight the infection) are disabled and destroyed as their number decreases. The infection with HIV-1 leads in the vast majority of cases, to progressive disease and finally, AIDS and death .. Unfortunately, even with the most powerful pharmacotherapies available today, the suppression of HIV-1 remains an incredibly frustrating challenge, due in part to the fact that HIV-1 is one of the viruses that mutate more quickly ever found. Recent advances have resulted in the use of a potent three-drug cocktail, known as highly active antiretroviral therapy (HAART), to reduce HIV-1 replication. HAART currently directs two different stages in the life cycle of the virus, and consists of two or three inhibitors of the reverse transcriptase of the virus, combined with at least one inhibitor of the viral protease. This combination antiretroviral therapy results in a dramatic reduction in viral load, decreasing the rate of CD4 cell decline and progression to AIDS in many patients. However, the side effect profiles for several of these drugs make long-term adherence difficult, and the emergence of drug resistance is a serious problem. In addition, performing sensitive tests shows that even the best treatments can not completely suppress viral replication. Since HIV-1 mutates at an incredibly fast rate, any viral replication allows drug-resistant mutants to appear. Once HIV-1 mutates, few viable treatment options remain for an infected individual. Currently, most patients will ultimately fail the drug combination therapies of the three existing classes, due to intolerance or resistance (or a combination of both), and therefore there continues to be a high medical need for better tolerated agents and administered conveniently to treat HIV-1 / AIDS. A new alternative is by inhibiting entry to host cells, one of its examples being blocking through the CCR5 co-receptor. To invade human cells, HIV-1 must bind to the differentiation group (CD) of the CD4 receptor and a chemokine co-receptor (either CCR5 and CXCR4) located on the surface of CD4 T cells. The only binding to the CD4 receptor is not enough to make the cells susceptible to infection by HIV-1. Isolates of tropic HIV for macrophages (tropics for M), regardless of subtype, predominantly use the beta-chemokine receptor CCR5, and are referred to as R5 virus. R5 viruses are transmitted preferentially and predominate in early asymptomatic disease. A change in the use of the chemokine receptor from CCR5 to CXCR4 may indicate the transmission of asymptomatic infection to AIDS. It is not known if the change of co-receptor favors the progression of the disease, or results from it. Inhibition by CCR5 is a particularly attractive drug target for two reasons. First, as mentioned above, viruses that establish a new infection are generally tropic for CCR5. In this way, successful inhibition of infection-using CCR5 could significantly decrease the likelihood of transmission. Second, individuals homozygous for a deletion of 32 base pairs of natural occurrence in the CCR5 gene (CCR5A32), and thus lacking this receptor, are apparently immunologically normal. These individuals are also resistant to infection by the R5 strains of HIV, and persons heterozygous for this defect of CCR5 exhibit a slower progression to AIDS and death. The frequency of different genetic polymorphisms varies among populations. CCR5A32 is more common in northern Europe (allelic frequency of 16%), less common in individuals in southern Europe (4%), and is extremely rare in African populations. Approximately 1% of Caucasians are homozygous for CCR5A32, and effectively represent human equivalents of experimental animals "with blocked expression". The chemokine receptors are seven molecules that span the membrane, present on the surface mainly of cells of the immune system. When these receptor molecules bind to their ligands, ie, chemokines, the end result is the recruitment of cells from the immune system to the site of disease or tissue damage. CCR5 (CC chemokine receptor 5) is the primary co-receptor for strains of tropic HIV-1 for macrophages (tropics for M), and appears to be also the primary co-receptor for primary isolates of HIV-1 tropics for microglia. The putative physiological ligands of the CCR5 receptor are the chemokines inflammatory protein of macrophages 1 (??? - 1 a), ??? - 1 ß and protein regulated after activation, normal tropic expressed and secreted (RANTES). Although a single domain consisting of the second extracellular loop appears to be predominantly responsible for chemokine binding, multiple extracellular domains of CCR5 intervene in the co-receptor activity of HIV-1. During the tropic phase for M of HIV infection, the macrophages favor the virus, which it invades by binding (through its gp120 protein) to the CD4 and CCR5 molecules on the surface of the macrophages. Finally, however, HIV-1 can become double tropic. Said strains produce gp120 molecules capable of recognizing the CXCR4 protein on T cells possessing CD4. During this phase, HIV-1 can infect macrophages and T cells. Even later, the volume of the viral population can change its preference for the CXCR4 receptor and become tropic for T. T-cell tropic viruses easily destroy cells T infected, favoring the collapse of the immune system and the onset of AIDS. Alternatively, some viruses, such as certain strains of HIV-2, could attack CXCR4, rapidly leading to AIDS. Since the absence of a biologically functional CCR5 receptor does not appear to be detrimental to the host, there is hope that agents that simulate the protective effect of the CCR5 deletion may develop. The possibilities include blocking the expressed receptors of wild type with agonists or antagonists suitably constructed for these molecules. The challenge remains that ensures that the analogs are potent, non-toxic, exhibit good pharmacological profiles and can be administered orally. Compounds which are potent antagonists of CCR5 have been identified, for example, in patent applications WO 00/38680, WO 00/39125 or EP 1013276, the disclosure of which is incorporated herein by reference. However, it has become apparent in the work of the present inventors that the high binding affinity of the CCR5 antagonists shown in preclinical studies does not always translate into equally high antiviral activity, especially in good clinical efficacy, of the compounds . Of course, the prediction of clinical efficacy using known preclinical methods remains a problem, resulting in unnecessary and expensive use of potentially ineffective compounds in clinical trials. The present invention is based on the observation that the compensation time, or functional occupancy, of a receptor by a ligand or modulator, provides a guide for the in vivo pharmacodynamics and efficacy of the ligand or modulator. In particular, the applicant has found that compounds with binding affinity very similar to a receptor, show marked differences in their residence time on the receptor, which can translate into improved antiviral potency. Thus, the purpose of the present invention is to provide a method whereby the potential for favorable pharmacodynamics and improved potency, and thus clinical efficacy of a compound, can be predicted more precisely from preclinical data. From one aspect, the present invention resides in the use of a test that measures the residence time of a receptor ligand on its receptor in vitro, for the identification of a ligand for that receptor that is predicted to be effective in vivo., in the treatment of a disease that responds to the modulation of the natural function of that receptor. Expressed in another way, the present invention relates to a method for identifying ligands with high potency and / or clinical efficacy for a disease that responds to the modulation of the natural function of a receptor, which comprises measuring its residence time on the receptor and select ligands based on the desired residence time. As used herein, a "ligand" for a receptor means any compound that binds to the receptor, and modulates in this way the natural function of the receptor. The term includes, but is not limited to, peptide, modified peptide, polypeptide, protein and small molecule ligands, such as synthetic chemical compounds, naturally occurring compounds or small organic molecules. Alternatively, the ligand can be an antibody or antibody fragment, or a nucleic acid or nucleic acid-derived material. As used herein, "residence time" means the average time that a ligand passes attached to its receptor, ie, the average time between the "activated" and "deactivated" states of the ligand with respect to its receptor. Stated another way, the term refers to time, during which the recipient is in a state that does not allow a natural ligand, such as a chemokine, or invasive molecule, such as HIV glycoprotein, to bind as a result of binding to the ligand. The residence time is also referred to as "receiver occupancy", and can be determined by any method that measures the rate of inactivation, dissociation rate or separation rate of a ligand. A typical method comprises the steps of incubating a labeled ligand with a suitable receptor, for example, cells expressing the receptor or membrane preparations derived from said cells, until equilibrium is reached. The excess unlabeled ligand is then added, and the amount of labeled ligand bound to the receptor is measured at desired intervals. The label can be any detectable label that does not influence the binding of the ligand to its receptor. Preferably, a radioactive label such as 3 H, 14 C, 32 P or 125 I is incorporated into the ligand, more preferably 3 H. Advantageously, the residence time of the receiver is at least one hour, at least three hours, at least six hours or, more advantageously, at least nine hours. In particular, the greater the functional occupancy of a ligand on a receptor, the greater the clinical effect of that ligand, thus reducing the number of doses that would be needed to administer to a patient. The ligands identified by the present invention can be agonists for the receptor but, preferably, they are antagonists. Preferably, the receptor is a G protein coupled receptor, more preferably the chemokine receptor CCR5. The residence time (or clinical efficacy) of the receptor of a ligand can be a mechanism for predicting antiviral activity, giving preference to anti-HIV activity. In particular, the present invention provides a method for selecting CCR5 antagonists, which show potent antiviral activity and clinical efficacy, by measuring the residence time of the antagonists on CCR5, and selecting ligands having a long residence time.

From another at, the present invention relates to a research method comprising measuring the residence time of the receptor of each of a plurality of ligands for that receptor, and selecting to further investigate at least one ligand whose time of residence is longer than that of at least some other ligand. From another at, the present invention resides in a research method comprising contacting a plurality of ligands for a given receptor with that receptor, measuring the binding affinity of the receptor and the residence time of the receptor of each ligand, assigning to each ligand a hierarchy value that is the product of the union affinity of its measurement and the residence time of its receiver, and selecting to further investigate one or more ligands that have a hierarchy value greater than a hierarchy value Limiting selected. The potency and / or clinical efficacy refers to the functional effect that a ligand exhibits while residing on its target receptor. This can be any effect that an agonist or an antagonist for a receptor can have, for example, the inhibition of the entry of a virus into T cells. In a preferred embodiment, the effect is an antiviral effect, preferably an anti-viral effect. HIV The desired residence time may be short for some conditions, or long for others. In a preferred embodiment of the invention, the desired residence time is at least one hour, preferably at least three hours, still more preferably at least six hours, most preferably at least nine hours. In another at, the invention relates to a ligand selected by the method of the invention. The term "receptor" will be understood broadly, and may be related to any molecule comprising a binding site for a ligand as defined above including, but not limited to, receptors, enzymes, ion channels, adhesion molecules or antibodies. Preferably, the receptor is a receptor present in or on a cell, preferably a mammalian cell, even more preferably a human cell. Preferably, the receptor is a cell surface receptor, even more preferably a G protein coupled receptor, even more preferably a chemokine receptor. The most preferred receptor for this invention is the chemokine receptor CCR5. The term "binding affinity" refers to the binding of a ligand that can be determined by traditional binding assays well-known in the art, such as a competition test. Typically, a membrane preparation of cells expressing the receptor of interest will be incubated with a known labeled ligand for this receptor, using one. concentration of labeled ligand giving approximately 50% of the total possible binding. In parallel test tubes, a competent non-labeled test ligand is included in varying amounts, to measure the ability of the test compound to compete for binding to the labeled ligand. Competency curves are then generated, plotting the concentration of the test ligand used along the x-axis, and the amount of tag attached along the y-axis. From these experiments, the KD value, the IC50 (ie, the concentration of competent ligand that displaces 50% of the labeled ligand) or the IC90 (ie, the concentration of competitive ligand that displaces 90%) can be calculated. of the labeled ligand). There are many ways to express the binding affinity of the ligands with which the skilled in; technique will be well versed in calculating, and which can be found in any basic pharmacology textbook. The term "chemokine receptor" refers to receptors for a large family of proteins that are chemotactic cytokines, that is, they have the ability to attract leukocytes as chemotactic factors of leukocytes. Chemokines share certain important structural characteristics, and bind to receptor families, most of which belong to the superfamily of G-protein coupled receptors. Chemokines and their receptors are central to the pathophysiology of inflammatory-and infectious diseases, and agents that are active to modulate, preferably antagonizing, the activity of chemokines and their receptors, are useful in the treatment of said inflammatory and infectious diseases. The term "CCR5" refers to the chemokine receptor which is the cellular receptor for the beta-chemokines RANTES, MIP-1cc and γ-1β. CCR5 has also been identified as an important receptor in HIV infection, binding to the gp120 glycoprotein of the HIV envelope. Basic teachings on CCR5 can be found in WO 97/32019, the disclosure of which is incorporated herein by reference. The term "HIV" refers to human immunodeficiency virus, which is presumed to be the agent that causes AIDS. There is an abundance of literature on this subject. One of the first references to HIV, originally called HTLV-III, is made in Ratner et al, Nature 313, 277-284, 1985. The present invention will be further described, by way of example, with reference to the following figures: Figures 1A-1C show the fluorescent coatings of FACS for RANTES-mediated incorporation of CCR5 into 300 cells. 9, and the inhibition of compound B incorporation at various concentrations: without compound removal (Fig. 1A), after removal of the compound by washing (Fig. 1 B), and after removal and oscillating incubation for 1.5 hours (Fig. 1 C). The change in fluorescence due to incorporation of CCR5 mediated by RANTES, is described by the trace D, and vehicle control (without compound or RANTES, ie total CCR5) is described by the trace E. Isotype control is described by the stroke F; Figures 2A-2C show the fluorescent covers of FACS for RANTES-mediated incorporation of CCR5 into 300.19 cells, and the inhibition of compound C incorporation at various concentrations: without compound removal (Figure 2A), after removal of the compound by washing (Fig. 2B), and after removal and oscillating incubation for 1.5 hours (Fig. 2C). The change in fluorescence due to incorporation of CCR5 mediated by RANTES, is described by the trace D, and vehicle control (without compound or RANTES, ie total CCR5) is described by the trace E. The isotype control 6 is described by the stroke F; Figure 3 is a graph showing the relative inhibition (percent relative to vehicle control) of incorporation of CCR5 induced by RANTES by compounds B, C and D, when the incubation is followed by a washing step (+ wash) or incubation for 1.5 hours in the absence of compound (+ removal); Figure 4 is a graph showing the average occupancy rate of CCR5 on day 1 on CD4 T cells for subjects of groups 1, 4 and 6 receiving placebo or a single oral dose of 100 mg, 25 mg and 3 or 10 mg of compound A, respectively, administered as a solution; Figure 5 is a graph showing the average occupancy rate of CCR5 on day 12 in CD4 T cells for subjects of groups 1, 4 and 6 receiving placebo or oral doses twice daily of 100 mg, 25 mg and 3 or 10 mg of compound A, respectively, on days 3 to 12, administered as a solution; Figure 6 is a dissociation curve for compound A; Figure 7 is a dissociation curve for compound B; Figure 8 is a dissociation curve for compound C; and Figure 9 is a graph showing the saturation of the CCR5 receptor over time in asymptomatic HIV seropositive male subjects administered with compound A, 25 mg, 100 mg or placebo.

The invention is exemplified by the use of four compounds, wherein: compound A is example 4 in WO 01/90106; compound B is example 34 in WO 01/38680; Compound C is not described; and compound D is example 33 in PCT / IB / 03/01220.

EXAMPLE 1 Binding of the compounds to CCR5 Compounds were tested in an affinity test of CCR5 following the procedures described in Combadiere et al, J Leukoc. Biol. 60, 147-152 (1996). Compounds A to D had an IC50 for binding to CCR5 less than 10 nM (data not shown).

EXAMPLE 2 Antagonist-dependent inhibition of chemokine-mediated CCR5 incorporation 1 . Functional occupancy of CCR5 in 300.19 cells measured by dynamic inhibition of RANTES-mediated CCR5 incorporation by FACS analysis The physical occupancy of the CCR5 receptor by antagonists can be measured using radioligand dissociation studies. The functional occupation of antagonists on the CCR5 receptor in whole cells can be evaluated by measuring the antagonist-dependent inhibition of chemokine-mediated cognate receptor incorporation. These measurements can be made using fluorescence activated cell distribution technology (FACS). The prolongation of the inhibition of CCR5 incorporation mediated by chemokine, after the removal of H antagonist by washing cells, is a measure of the functional occupancy of CCR5.

Methods All tests / reagents were carried out / used at room temperature, unless otherwise indicated. Cell culture: 300.19 cells (line of pre-B cells of mouse, expressed in recombinant human CCR5 form) were cultured in 75 cm2 cell culture flasks in growth medium at 37 ° C for 2-3 days in the incubator of C02 at 5% (v / v) humidified. For the passage of cells, routine separations were performed from 1x105 to 2x105 of the cells in suspension. Cell preparation: Cells were centrifuged from each used 75 cm2 flask, at 1500 rpm in a bench-top centrifuge for 5 minutes. The pelleted cells were resuspended in a minimum volume of RPMI culture medium (10% FBS) (see materials), counted in a Cedex cell counter (see materials), and adjusted to a cell density of 5x106 / ml by dilution in the same medium. Antagonist, antibody and chemokine preparation: CCR5 antagonists were dissolved in dimethyl sulfoxide (DMSO) at 100% at a concentration of 1 mM, and diluted in RPMI cell culture medium (10% FBS) to allow the testing in FACS tests at concentrations of 10 nM to 1000 nM in situ. Anti-CCR5 antibody (2D7 - see materials) was diluted 1: 10 in 0.5% BSA / PBS. IgG2a antibody (isotype control for the test - see materials) diluted 1: 10 in 0.5% BSA / PBS was used. The goat anti-mouse secondary antibody labeled with anti-2D7 phycoerythrin (PE) (see materials), was used at a 1: 20 dilution in 0.5% BSA / PBS. The chemokine agonist ligand cognate for CCR5, protein regulated upon activation, normal expressed and secreted tropic (RANTES), was solubilized in PBS. Lyophilized material (10 μg - see materials) was resuspended in PBS at a concentration of 100 μ ?. Dilutions were then made in RPMI (10% FBS) to give a final concentration of 1000 nM (final concentration of 100 nM in the test). Cells 300.19 (100 μ?) Were added at 5 × 10 6 cells / ml to each test tube. CCR5 antagonists (10 μ?) Or vehicle (RPMI (10% FBS)) were added to suitable test tubes to allow profiling on concentrations ranging from 10-1000 nM in situ. The tubes were incubated at 4 ° C for 45 minutes to allow the antagonist to be associated with the cells. To investigate the prolongation of the occupation of CCR5 by antagonists, samples were left with antagonist present or centrifuged (1500 rpm in a bench centrifuge), and washed twice in 000 μ? of RPMI medium (10% FBS). Half of the washed samples were resuspended in 1000 μ? of the same means, and was placed on a rotating platform for 1.5 hours, before being processed through the FACS test. The other half of the washed samples, together with the unwashed samples (ie, antagonist present), were processed immediately. RANTES (10 μ?) Was added to the samples, followed by incubation at 37 ° C for 45 minutes, to induce incorporation of CCR5. The samples were centrifuged (1500 rpm in a bench-top centrifuge), and washed twice in 0.5% BSA / PBS. The washed samples were resuspended in 40 μ? of the same pH regulator. Then 2D7 antibody or isotype control (10 μm) was added to the samples, followed by incubation for 45 minutes at 4 ° C to allow binding of the antibody to CCR5. The samples were then washed once in 0.5% BSA / PBS, followed by the addition of 75 μ? of goat anti-mouse secondary antibody labeled with phycoerythrin (PE) (1: 20 dilution in 0.5% BSA / PBS), with incubation at 4 ° C for 45 minutes in the dark, to allow binding. The samples were subsequently centrifuged (1500 rpm in a bench-top centrifuge for 5 minutes), washed twice in 100 μ? of BSA at 0.5% / PBS, and resuspended in 1000 μ? of formaldehyde at 1% / PBS for fixation. The fixed cells were processed in a Becton Dickinson FACScalibur to measure the levels of fluorescence in the cells in each test to evaluate the inhibition dependent of the antagonist, the incorporation of CCR5 mediated by RANTES, and consequently the occupation of functional CCR5. The FACScalibur tests were carried out using the Cell Quest program according to the supplier's instruction manual, using excitation / emission wavelengths of 488 nm / 530 nm, respectively.

Materials Culture medium: 1 x 500 ml of RPMI-1640 medium with NaHCO3 (without L-glutamine - Gibco BRL (catalog number: 31870-025); 5 ml of L-glutamine at 200 mM - Gibco BRL (No of catalog: 25030-024), 5 ml of penicillin / streptomycin (10,000 U / ml of penicillin, 10,000 μg / ml of streptomycin) - Gibco BRL (Catalog No.: 15140-122); 50 ml of bovine serum Fetal (FBS) - Sigma Aldrich (Catalog No.: F7524); 5 ml of pH regulator HEPES at 1 M - Sigma H-0887; 2 μ? of pure 2-mercaptoethanol - Sigma M-3148. 225 cm2 cells with treated tissue culture - Costar (Catalog No.: 3001), Cedex Innovatis cell counter, Innovatis sample cups (Catalog No.: 600050000), incubator set at 37 ° C, CO2 at 5 %, humidified, refrigerated centrifuge Denley BR401.

Test reagents: Test pH regulator: RPMI (10% FBS); RP I - Gibco BRL (Catalog No.: 31870-025); FBS - Sigma (Catalog No.: F7524). Wash pH regulator: 0.5% BSA / PBS; BSA - albumin, bovine fraction V - Sigma A4503; saline solution regulated in its pH with phosphate (PBS) of Dulbecco without Ca2 + or Mg2 + - Gibco BRL (Catalog No.: 14190-094). Consumables: DMSO, tissue culture grade - Sigma (Catalog No.: D-8418); test tubes: microcentrifuge tubes - Costar (Catalog No.: 3621); FACS tubes: Falcon tubes - Becton Dickinson (Catalog no .: 35 2054); Labsystems Finn Termo Life Sciences pipettes (Nos. catalog: 4500/090/050); pipette tips of 1 ml - Termo Life Sciences (Catalog no .: 9401 103); pipette tips of 250 μ? - Termo Life Sciences (No. catalog: 9400263); LMS cooled incubator; Becton Dickinson FACScalibur - Cell Quest program; 1% formaldehyde / PBS; formaldehyde -Sigma, Catalog No.: F1635, saline regulated at its pH with Dulbecco's phosphate (PBS) without Ca2 + or Mg2 + - Gibco BRL (Catalog No. 14190-094); IEC icromax RF centrifuge; RANTES - R & D Systems (catalog no .: 278-RN-010); mouse anti-human CCR5 monoclonal antibodies: 2D7-Pharmingen (Catalog No. 36461A); Isotype control antibodies: mouse IgG2a - Pharmingen (Catalog No. 33031 A); antibody labeled with secondary PE: goat anti-mouse antibody labeled with PE-Sigma (Catalog No. P9287).

Data analysis The Cell Quest program used for the acquisition of fluorescence data was also used for data analysis. Mean fluorescence values were determined for the cell population of each test sample. The decrease in mean fluorescence due to incorporation mediated by RANTES with respect to the control of 2D7 antibodies exposed to vehicle was measured. This allowed the calculation of antagonist-dependent inhibition, RANTES-mediated incorporation of CCR5, and the subsequent comparison of functional occupancy by several CCR5 antagonists.

Results The functional occupancy measured by compound B-dependent inhibition of RANTES-mediated incorporation of CCR5 by cells 300.19 is described in Figures 1A-1C. The incorporation of CCR5 mediated by RANTES was apparent, observed by the change in fluorescence after incubation of 300.19 cells with chemokine. Inhibition of this incorporation was observed at all concentrations tested, as shown by the fluorescence cover for incubations where compound B was present throughout the test (without washing), compared to the fluorescence profile for CCR5 (recognized by antibody 2D7) in vehicle control. Retention of this inhibition (ie, occupancy of functional CCR5) was observed after removal of compound B by cell washing. Retention of dynamic inhibition was observed at variable levels at all concentrations after an oscillating incubation of 1.5 hours after the removal of compound B. These results highlight the dynamic retention of compound B-dependent inhibition., of RANTES mediated uptake of CCR5 in cells 300.19, and therefore show that the functional occupancy of CCR5 by this antagonist can be retained over time. The quantification of this functional occupancy was made, measuring the geometric average fluorescence signal for each test, and calculating the percent inhibition of RANTES-mediated incorporation, compared to the non-inhibited vehicle control. This quantification was carried out for compounds B, C and D, to determine their functional occupancy in this test, and to demonstrate the usefulness of the test for the identification of compounds with slow functional displacement. The results of this quantification are shown in Figure 3. The percent inhibition under each test condition is plotted in the bar chart, and is included in the accompanying table. Where the results are average values of more than one experiment, the error bars indicate the scale. As described above, compound B dissociates slowly in these experiments, while compound B dissociates more rapidly. Furthermore, it is evident that compound C does not completely inhibit receptor uptake, even at concentrations greater than the IC 50 of its receptor. This is consistent with the displacement of CCR5 measured by radioligand dissociation studies (see example 3). Compound B slowly dissociates from CCR5 upon addition of the excess unlabelled compound (ti / 2 = 3.5 hours, Figure 7). Conversely, elk 'compound C showed relatively rapid dissociation in parallel radioligand dissociation studies (t1 2 = 15 minutes, figure 8). According to the data shown in figure 3, compound D is a slow displacement inhibitor of CCR5.

Conclusion This FACS test is able to differentiate compounds based on their dynamic occupancy of CCR5, and can be used to identify slow and fast CCR5 displacement compounds. 2. Functional occupancy of CCR5 in peripheral blood mononuclear cells, measured by inhibition of CCR5 incorporation mediated by ??? - 1? By FACS analysis The incorporation (occupancy percent) of CCR5 mediated by ??? - 1 was evaluated ß on CD4 T lymphocytes prepared from CPT samples (cell preparation tubes) with whole blood citrate taken from healthy volunteers who participated in a clinical study designed to investigate the administration of multiple oral doses of compound A. Compound A is a CCR5 antagonist and prevents the binding of ??? - 1β to the CCR5 receptor, and subsequent incorporation thereto. The difference in the expression of CCR5 on the cell surface between peripheral blood mononuclear cells treated with stabilizer (total CCR5) and untreated (maximal incorporation) subjected to challenge with ??? - 1 ß, will give a calculation of the proportion of CCR5 free present on the cell surface, at a certain concentration of compound A in plasma. These data can then be used to calculate the degree of receptor occupancy obtained at different dosages of compound A.

Methods Individual dose administrations (3 mg, 10 mg, 25 mg and 100 mg) of compound A were followed by the same doses, ie 3 mg, 10 mg, 25 mg and 100 mg, respectively, as multiple oral doses , twice a day for 10 days or placebo, in healthy male subjects from 18 to 45 years of age. Subjects were assigned to one of the six groups (data for groups 1, 4 and 6 are illustrated only here). Within each of these six groups, subjects were assigned to receive compound A at the appropriate dosing regimen, or to receive placebo. The groups are: group 1: 100 mg twice daily of compound A or placebo; group 4: 25 mg twice daily of compound A or placebo; group 6: 3 mg twice daily or 10 mg twice daily of compound A or placebo. Prior to dosing, a blood sample was obtained for tests that included 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 administered 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 before the dose and at intervals for the next 48 hours after the dose (day 3), as described below. On days 3 to 11, the subjects were dosed with compound A or placebo between 08:00 and 10:00 am in the morning, and at 8:00 pm and 10:00 pm in the early hours of the night, according to the same procedures followed on day 1. Each day, a blood sample was taken immediately before the administration of the first dose of compound A, for the determination of the concentrations of compound A in plasma. On day 12, compound A or placebo was dosed according to the same procedures followed on day 1; subjects received only one dose on day 12. Blood was obtained before the dose and at intervals for the next 72 hours after the dose. The subjects were discharged 24 hours after the last dose, but returned for a supplementary physical examination 7 to 10 days after the final dose. Blood samples were taken from the subjects of group 1 (in CPT tubes with 4 ml of citrate) at the following times: 0 (reference line), 4, 12, 24 and 28 hours after 100 mg on the same day of the dose of compound A on day 1. Other samples were taken on day 12 at time 0 (baseline) and 24, 48, 59, 72, 97 and 120 hours after 100- 'mg on the same day as the dose of compound A. For the subjects of group 4, other samples were obtained at 8, 18, 52 and 64 hours after 25 mg on the same day of compound A on day 1. On day 12, other samples were collected at 144, 168 and 240 hours after 25 mg on the same day of the dose of compound A. For the subjects in group 6, samples were obtained at time 0 (baseline) and at 4, 8, 12, 18, 24 and 48 hours after 3 or 10 mg on the same day of the dose of compound A on day 1; 4 and 16 hours after the dose on day 3; 2 hours after the dose on days 4 to 1 1 after 3 or 10 mg twice daily of the dose of compound A, and then on day 2 at time 0 (baseline) and to 8, 12, 24, 48, 96 and 120 and 144 hours after 3 or 10 mg on the same day of the dose of compound A. Before the first dose of compound A or placebo, a blood sample was also collected with 6 ml of EDTA. Residual CCR5 receptor expression was determined in CD4 positive lymphocyte populations, by flow cytometric analysis of processed whole blood samples anticoagulated in CPT with sodium citrate.

Materials CPT blood tubes with sodium citrate (4 ml extraction) (Becton Dickinson, Catalog No. 362760); Sample processing tubes (12 x 75 mm, polystyrene, round bottom) and lids (soft fit) (Sarstedt Ltd, Nos. Catalog 55,476 and 65,809,504, respectively). Reagents: 10X concentrated PBS; 10% paraformaldehyde; 10% sodium azide solution; BSA lyophilized; working solution of MIP-1 ß (stored aliquots frozen at -70 ° C); CCR5 stabilization solution (containing compound A at 600 nM in PBS); stabilization control solution (PBS); CCR5 and control antibody cocktail of IgG isotypes (stored at 2-8 ° C).

Reagent preparation: 1X PBS (stable for 1 month at 2-8 ° C) 100 ml_. of 10X PBS 899 mL of deionized water 1 mL of 10% sodium azide solution in deionized water pH 7.4 1X PBS with 1% BSA (stable for 2 weeks at 2-8 ° C) 1 L of 1X PBS 10 g of lyophilized BSA pH 7.4 paraformaldehyde at 1% (stable for 2 weeks at 2-8 ° C) 180 mL of 1X PBS 20 mL of paraformaldehyde at 10% paraformaldehyde at 0.5% (stable for 2 weeks at 2-8 ° C) 190 mL of 1X PBS 10 mL of 10% paraformaldehyde.

Preparation of the CCR5 stabilization solution: The control stabilization solution is PBS. 1. Provide solution of compound A at 10 mM in DMSO; 2. Dilute 1: 500 supply solution with 1X PBS to create a solution at 20 μ? (2μ? /? T ?? of PBS); 3. Dilute the solution to 20 μ? 1: 20 with 1X PBS to create a solution at 1000 nM (1 μ?); 4. Dilute the solution to 1000 nM 3: 2 with 1X PBS to create a solution at 600 nM. Drug solution should be added to 600 nM (stabilization solution) 1: 6 in plasma, until reaching a final concentration of 100 nM. As for the protocol, stabilization solution should be added to 50 μL · to 250 μ? _ Of plasma enriched with cells. Control stabilization solution should be added in proportions similar to the relevant tube. The stabilization solution should be prepared fresh for each group, and vortexed before use to ensure that the drug is in solution.

Sample processing: 1. Lymphocyte-rich plasma was isolated by centrifugation at 1550 g for 25 minutes (RT) on a rotating rotor. 2. The cell layer of the yellow coat was resuspended in the plasma by gently inverting the tube several times (x 5). 3. 250 μL · of cell-enriched plasma were pipetted into three separate labeled 12 x 75 mm polystyrene round bottom tubes. [Tube 1 (control), tube 2 (total CCR5) and tube 3 (CCR5 ??? ß). 4. 50 μ? of stabilization solution of CCR5 to tube 2, while adding 50 μ? of control stabilization solution to tubes 1 and 3, before subjecting them briefly to swirl in a medium positioning for 2 seconds and incubation at 37 ° C for 30 minutes. 5. All tubes were centrifuged at 400 xg for 5 minutes in a rotating rotor to isolate the cells, and the supernatant was then decanted. 6. To all tubes, 15 μl of working solution of? -1-ß was added, and then gently swirled in a medium setting for 2 seconds, and the pellet was resuspended in fluid. The tubes were then incubated uncovered for 45 minutes at 37 ° C in an atmosphere of C02 at 5% and 98% humidity. 7. To each tube was added 1 ml of 0.5% paraformaldehyde in PBS, swirled in a medium position for 2 seconds, and incubated for 10 minutes in the dark at room temperature. 8. Then, 2 mL of PBS with BSA were added to all the tubes, and the tubes were centrifuged at 400 xg for 5 minutes in a rotating rotor to transform the cells to pellets before decanting the supernatant. 9. Antibody reagents (50 μ?) Were added in the following manner: MsIgG R-phycoerythrin (PE) and CD4-fluorescein (FITC) (control) were added to tube 1. To tubes 2 and 3, CCR5-PE and CD4-FITC (total CCR5 and CCR5 MIP1 p) were added. 10. All tubes were then vortexed in a medium setting for 2 seconds, and incubated for 20 minutes in the dark at room temperature. 11. 2 ml_ of PBS with BSA was added to all the tubes. They were centrifuged at 400 xg for 5 minutes. The supernatant was decanted, and then 0.5 ml of paraformaldehyde at 1% in PBS was added, and it was subjected to swirling action in a medium positioning for 2 seconds to resuspend the pellet. 12. Finally, the tubes were adequately capped and stored at 2-8 ° C until analysis.

Results The average percent occupancy of CCR5 receptors on the surface of CD4 T cells prepared from whole blood samples (CPT), and the corresponding standard deviations (SD) at each nominal time point for healthy male subjects who received a Individual oral dose of compound A or placebo on day 1 followed by dosing twice daily on days 3 to 12, are given in table 1.

TABLE 1 TABLE 1 (Continued) Time Group 1 Group 4 Group 6 Group 6 Group 12 (h) Day 12 Day 12 Day 12 Day 12 (placebo) (100 mg) (25 mg) (3 mg) (10 mg) DesMedia SD Media SD Media SD Media SD SD after dose 0 97.2 6.1 93.0 5.9 70.7 8.4 82.4 18.4 27.6 9.6 8 NS NS DK DK 76.0 4.5 91.4 4.0 33.0 4.2 12 NS DK DK NS 73.3 3.8 86.6 4.0 35.5 3.5 24 90.9 25.5 90.5 7.5 67.8 8.5 93.8 21.1 37.3 5.2 32 NS NS NS NS (53.0) - NS NS NS H 36 NS NS NS NS (54.0) - NS NS NS H 48 94.0 14.7 84.6 7.9 63.0 16.6 74.6 8.9 26.4 5.8 59 87.6 7.4 NS NS NS DK DK NS 35.7 5.7 72 86.3 6.6 77.5 9.9 (45.0) - NS DK 31 .0 15. 8 96 83.4 9.4 68.9 6.5 41.8 8.8 46.4 5.7 28.7 6.4 120 77.1 11.9 55.9 8.6 31.6 6.1 34.6 6.8 29.5 4.3 144 NS NS 54.7 5.5 41.2 9.0 34.5 8.3 25.6 3.8 168 NS NS 47.8 6.5 (45.0) - NS NS 25.7 2.3 Codes: NS: no samples were taken at this point. -: Insufficient data for the calculation of standard deviation.

Conclusion The occupation of CCR5 was related to the dose. Volunteers who received 100 mg twice daily demonstrated CCR5 receptor occupancy greater than 90% throughout the dosing period, whereas in those subjects who received 3 mg, the mean saturation of the receptor was less than 80%.

EXAMPLE 3 Determination of the residence time of the compounds on CCR5 The physical occupation of the CCR5 receptor by antagonists can be measured using radioligand dissociation studies. Radiolabelled antagonist is incubated until equilibrium with CCR5 expressed on HEK-293 cells or membrane preparations thereof. Dissociation, and hence physical occupation or residence time, is subsequently measured by adding excess unlabeled antagonist to prevent re-association of the dissociated radiolabelled antagonist. In this way, the dissociation can be measured by counting the radioactive label retained in whole cells or membranes over time, after the addition of the unlabeled antagonist.

Methods Cell culture: HEK-293 cells stably expressing CCR5 (produced by standard techniques) were grown in 225 cm2 cell culture flasks to 50-70% confluence in culture medium (see materials) at 37 ° C for 2-3 days in the humidified 5% (v / v) CO2 incubator. The cells developed at low density due to their tendency to accumulate, with less expression of CCR5 at full confluence. Each 225 cm2 flask used for the binding tests was washed once by removing the culture medium and replacing 20 ml of saline buffered at pH with phosphate (PBS) at room temperature. The supernatant was removed, and the cells were dislodged by striking the side of the culture flask in the presence of 10 ml of binding pH regulator at room temperature (see materials). The cells were placed in a 50 ml centrifuge tube. A further 10-20 ml of binding pH buffer (room temperature) was added to the culture flask, and transferred? Then to the centrifuge tube to harvest the residual cells. The cells were centrifuged at 350 g for 10 minutes at 20 ° C. The cells were resuspended in 3 ml of binding buffer, counted in a modified Neubauer hemocytometer, and resuspended to a density of 2 × 10 6 cells / ml. Membrane preparation: Each 225 cm2 flask used for the binding test was washed once the culture medium was discarded, and this was replaced by 20 ml of PBS (room temperature) and resuspended in 5-10 ml of PBS (room temperature) ). The cells were transferred to a 50 ml centrifuge tube. Another 10-20 ml of binding pH buffer (room temperature) was added to the culture flask, and then transferred to the centrifuge tube to harvest the residual cells. The cells were centrifuged at 350 g for 10 minutes at 4 ° C. The cells were resuspended in 15 ml of pH lysis buffer (see materials) at room temperature, and homogenized with a Polytron manual homogenizer (5-10 seconds on ice, 3 to 4 times at high positioning). 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 was resuspended in a minimum volume of lysis pH regulator (room temperature). The protein concentration was calculated using the Bradford microassay for proteins, and adjusted to 0.25 mg / ml in binding buffer. This allowed the addition of 12.5 μg of membrane protein for use in each radioligand binding assay. Preparation of the compound: 1 to 2 mg of compound A, B or C were dissolved in dimethyl sulfoxide (DMSO) at 100% to a concentration of 10 mM, and diluted in binding buffer to allow addition of the compound to the binding tests on a concentration scale of 0.41 nM to 50 μ? In situ. They were diluted 20 μ? of [3H] compound A (62.5 μ ?, specific activity of 16 Ci / mmol and radioactivity concentration of 1 mCi / ml in binding pH regulator to allow the addition of the radioligand to the binding test on a concentration scale of 0.41-100 nM in situ Similar dilutions were made with compounds B and C marked with [3H] Dissociation studies: Binding pH regulator (25 μ?) Was added to the cavities of a 96-well test plate , followed by 50 μ? of cells or cell membrane preparation Dilutions of [3H] compound A, B or C (25 μ?) were added to cavities designed to allow radioligand association. 100x [3H] compound) to designed cavities (25 μ?) In place of the binding pH regulator, to determine the binding of the non-specific radioactive label in the tests added before the radioactive label. to allow q The radiolabelled compound was added to one of the pair, to measure the dissociation, and the pH regulator to bind to the other to act as an association control. This control was established to account for the dissociation that occurs as a result of CCR5 or denaturing of the membrane / cell. The plates were incubated at room temperature for 1 hour after the addition of [3 H] compound. This allowed the radioligand to bind to CCR5 until equilibrium was achieved. After this incubation, 25 μ? of dilutions of the unlabeled compound to the dissociation cavities. The contents of the cavities were harvested after incubations at room temperature up to 48 hours after the association. For harvesting cells and membranes, the Unifilter plates were blocked by applying 25 μ? of blocking agent (room temperature) (see materials) for 30 minutes. The Unifilter plates were washed once with 50 ml of wash buffer (room temperature), the reactions were harvested by aspiration, and the plate was washed 3 times (1 ml / well) with wash buffer (room temperature ) on the Packard harvester. The Unifilter plate was left to dry overnight. Microscint 0 (50 μ? - room temperature) was added to each cavity, and a TopSeal cover was placed on each plate. The plates were read (1 minute per well) in the NXT Packard Top Count scintillation counter.

Materials Growth medium: 1 x 500 ml of Dulbecco's modified Eagle's medium (DMEM) with sodium pyruvate, without L-glutamine, with pyridoxine-Gibco BRL (Catalog No.: 21969-035) containing 5 ml of L- Glutamine at 200 mM - Gibco BRL (Catalog No. 25030-024); 5 ml of penicillin / streptomycin (100 U / ml penicillin / 10 mg / ml streptomycin) - Sigma (Catalog No. P-7539); 50 ml of fetal calf serum (FCS) -PAA Laboratories, Austria (Catalog No. A15-042); and 6.5 ml of 50 mg / ml Geneticin - Gibco BRL (Catalog No. 10131-019). Cell culture flask of 225 cm2, with treated tissue culture - Costar (Catalog No.: 3001). Saline solution regulated in its pH with phosphate (PBS) of Dulbecco without Ca2 + or Mg2 + - Gibco BRL (Catalog No.: 14190-094).

Cell counting chamber, Improved Neubauer - Weber Scientific International (Catalog No.: AC1000). Connection pH regulator: HEPES at 50 mM in distilled water -Sigma (catalog no .: H-0763). CaCl2 at 1 mM - Sigma (Catalog No.: C-3881). MgCl 2 at 5 mM - Sigma (Catalog No.: M-1028). Bovine serum albumin (BSA) at 0.5% - Sigma (Catalog No.: A-4503). PH regulator adjusted to pH 7.4 (HCI to 2M) and filtered to 0.2 μ ?? Lysis pH regulator: HEPES at 20 mM in distilled water -Sigma (Catalog no .: H-0763). CaCl2 at 1 mM - Sigma (Catalog No.: C-3881). One tablet of COMPLETE ™ protease inhibitors per 50 ml of lysis pH regulator - Boehringer Mannheim (Catalog No. 1 697 498). PH regulator adjusted to pH 7.4 (HCI to 2M) and filtered to 0.2 μ? T ?. Wash pH regulator: HEPES at 10 mM in distilled water - Sigma (Catalog No. H-0763). NaCl at 0.5 M - Sigma (5M solution, catalog no .: S-5150). Bovine serum albumin (BSA) at 0.5% - Sigma (Catalog No.: A-4503). PH regulator adjusted to pH 7.4 (HCI to 2M) and filtered at 0.2 μp ?. Supply solutions prepared at 1 M (HEPES, CaCI2 and MgCI2) and stored at room temperature; the above pH regulators are prepared fresh each time. Blocking agent: polylenimine (PEI) at 0.3% in distilled water - Sigma (Catalog No.: P-3143). Consumables: DMSO, tissue culture grade - Sigma (Catalog No.: D-2650). Polypropylene deep cavity blocks (1 ml / cavity) - Porvair (catalog no .: 219008). Multi-channel pipettes - Labsystems Finnpipette (Catalog No.: 4510-000 / 020/030/040/050). Pipette tips of 1 ml - Sigma (catalog no .: P7174). Pipette tips of 250 μ? - Sigma (Catalog No.: P7049). Reagent reservoirs for multi-channel pipettes - Costar (catalog no .: 4870). Universal Packard filter harvester (96 cavity head). Packard UniFilter GF / B 96-well filter plates with lower seal - Packard (catalog no .: 6005177). Microscint 0 - Packard (Catalog No.: 6013611). Sealing film of printed microplate adhesive TopSeal-A -Packard (Catalog No.: 6005185). NXT Packard TopCount scintillation counter. Results As shown in Figure 6, 50% of compound A was still bound after 9 hours of incubation with excess unlabelled compound A. 50% of compound B was still bound after 3 hours (figure 7), while 50% of compound C was still bound after 15? minutes (figure 8).

EXAMPLE 4 Antiviral activity The anti-HIV activity was tested in peripheral blood lymphocytes (PBLs) for compounds, infecting these cells with HIV (Ba-L isolate), and measuring the compound-dependent reduction in the transcriptase activity (RT) of the supernatant after of incubation for 5 days.

Mds The HIV-1 Ba-L and IIIB viruses used in the antiviral tests were obtained from the AIDS Reagent Project, NIBSC, Potters, Bar, Herts, United Kingdom (references of the depositary ARP1 18 and ARP101). The virus was stored in 1 ml containers at -80 ° C. For expansion of HIV-Ba-L, the virus supply material (1 ml) was removed from storage at -80 ° C, and thawed rapidly in an incubator at 37 ° C for 10 minutes. The virus supply material (0.5 ml) was placed in a marked cavity of a 24 cavity plate with 1 ml of fresh PBLs (pretimulated 3 days with phytohemagglutinin (PHA) at a final concentration of 1.5 μg / ml) at a density of 1.0 x 107 cells / ml in complete growth medium of RPMI 1640 (supplemented with 10% FCS, 2 mM L-glutamine and 1 U / ml penicillin, 0.1 mg / ml streptomycin). The 24-well plate was incubated at 37 ° C in a 5% (v / v) C02 incubator humidified for 1 hour to allow infection. After 1 hour, the cells and the supernatant of this plate were centrifuged at 225 g for 10 minutes at 25 ° C, and the supernatant was discarded. The cells were suspended in 10 ml of growth medium of RPMI 1640 at room temperature, and transferred to a 25 cm2 tissue culture flask. IL-2 was added to a final concentration of 10 ng / ml. The culture of infected PBLs was incubated in a vertical position at 37 ° C in a 5% (v / v) C02 incubator humidified for 3 days. On day 3, 5 ml of fresh growth medium of RPMI 1640 was added together with IL-2 at a final concentration of 10 ng / ml. After 7 days, the infected cells were transferred to a 80 cm2 tissue culture flask. Another 7 ml of PBLs stimulated with IL-2 (1.0 x 10 7 cells / ml) were added in growth medium of RPMI 1640, and incubated in a humidified 5% (v / v) CO 2 incubator until day 10. Fresh growth medium of RPMI 1640 (25 ml) containing 10 ng / ml of IL-2 was added on day 10, followed by gentle mixing. The culture was separated in two 80 cm2 higher tissue culture flasks, and incubated at 37 ° C in a humidified 5% (v / v) C02 incubator until day 15. The viral expansion was measured by means of a test reverse transcriptase (RT test), and the counts >were considered as active; 400 cpm for 20 μ? of supernatant from the RT test. Cells were further expanded by dividing the infected culture in 80 cm2 fresh tissue culture flasks, followed by the addition of 13 ml of fresh PBLs stimulated with IL-2 (1.0 x 107 cells / ml) / ml. The flasks were incubated at 37 ° C in a humidified atmosphere of C02 at 5% until day 18. On day 18, 20 ml of growth medium of RPMI 1640 (containing IL-2 at 10 ng / ml) was added. The cultures were incubated at 37 ° C in a humidified atmosphere of C02 at 5% until day 23. Virus samples were considered as active if the RT counts in the supernatant (20 μ?) Were > 5000 cpm on day 23. These supernatants were clarified by centrifugation at 850 g for 10 minutes at 25 ° C, and the resulting supernatant was mixed and aliquoted into cryotubes of 4 ml and 1 ml for storage at -80 ° C. .

Virus titration The infectious dose 50 of the tissue culture (TCIDgo - assumed number of viral particles required to infect 50% of the cells) for the HIV-1 Ba-L supply material (harvested on 17/12) was calculated. / 99), using the equation of Reed and Muench ((1938) Am. J. Hyg. 27, 493-497) (using RT as a positive marker for HIV infection). The titration of the HIV-1 Ba-L supply material (harvested on 12/17/99) produced a TCID50 of 5.76 x 104 / ml using this method. All titrations were carried out using frozen / thawed virus supply materials in PBAs stimulated with PHA grouped in fresh. To prepare PBLs, the cells were pelleted by centrifugation for 10 minutes at 500 g and 25 ° C, and checked for viability by removing 100 μ? of the cells, followed by the addition of 100 μ? of cell dissociation solution (see materials) and 100 μ? from tripane blue to 0.4% (v / v). The count of viable cells (not stained) was made in a Kova count chamber (see materials). Only suspensions of cells that showed more than 95% viability progressed in the tests. Viable cells were resuspended at a density of 2.0 x 106 cells / ml in RPMI growth medium (10 ng / ml IL-2 added for PBL cultures). The expanded frozen Ba-L HIV supply material (2 x 1 mL) was thawed rapidly in an incubator at 37 ° C for 10 minutes. One sample was developed for titration evaluation, and the other was developed for parallel antiviral testing. For the titration, 25 μ? of the supply material of the defrosted expanded virus through gradual dilutions of 8 x 0.5 log in the middle of growth of the RPMI. Each viral dilution (20 μ?) Was transferred to a 96-well test plate along with 180 μ? of PBLs. The test plate was incubated at 37 ° C in a humidified atmosphere of C02 at 5% (v / v) for 5 days, after which 100 μ? of supernatant, and were tested for RT activity, to allow the determination of TCID50. It was considered as positive to. virus titrated with respect to virus infectivity, when the RT counts were higher (> 2x standard deviations) than the counts of uninfected cells. The viral titer was calculated using the method reported by Reed and Muench ((1938) Am. J. Hyg. 27, 493-497). This allows the standardization of the use of viruses for antiviral tests, and the performance of antiviral potency tests subsequent to the compounds. The TCID50 for HIV-Ba-L in the antiviral tests in this study was 5.76 x 104 / ml.

Preparation of cultures of PBLs with monocytes The North London Blood Transfusion Center provided yellow covers of individual donors containing PBLs from negative donors for sera with HIV and HBV. The serological status was determined by the North London Blood Transfusion Services. PBLs were prepared from 4 yellow cover samples that were separated and expanded individually in culture. Each of the 4 yellow covers (50 ml) was transferred to an 80 cm2 tissue culture flask with an equal volume of pH regulated saline with sterile phosphate (PBS). Little by little, aliquots (25 ml) of the cell suspension were stratified on 25 ml of Ficoll-Paque in separate 50 ml centrifuge tubes. After centrifugation for 30 minutes at 1000 g at 25 ° C, the PBLs layer was removed by pipetting between the erythrocyte and plasma layers. The separated PBLs were transferred to fresh centrifuge tubes, and washed twice with PBS (4 ° C) and centrifuged for 10 minutes at 850 g at 4 ° C. Removed contaminating erythrocytes, adding 9 ml of sterile water to the PBL pellets together with 10x 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 minutes at 500 g at 4 ° C. The pellet was suspended in 30 ml of growth medium of RPMI 1640 (room temperature). The viability of the cells was verified as described above, and only suspensions of cells showing more than 95% viability for antiviral test were processed. The cell suspension was adjusted to a density of 1 x 106 cells per ml by the addition of RPMI 1640 growth medium supplemented with 1.5 μg / ml of PHA. The cells (50 ml) were transferred to 80 cm2 tissue culture flasks and incubated for 3 days at 37 ° C in a humidified 5% (v / v) CO2 incubator in preparation for antiviral testing.

Separation and growth of PBLs without monocytes PBLs were prepared and tested for viability with tripan blue staining as described above. Preparations of PBLs with more than 95% viability were transferred to 80 cm2 culture flasks in a total volume of 50 ml and cell density of 1 x 106 / ml. The flasks were incubated for 1 hour at 37 ° C in a humidified 5% (v / v) CO 2 incubator to facilitate the adhesion of the monocytes to the surface of the flask. The non-adherent PBLs were carefully decanted into 50 ml centrifuge tubes, and pelleted by centrifugation at 500 g for 10 minutes at 25 ° C. The pellet of PBLs was adjusted to a density of 1 x 106 cells per ml by the addition of fresh growth medium of RPMI 1640 containing 1.5 μg ml of PHA. The cells (50 ml) were transferred to a 80 cm2 tissue culture flask and incubated for 3 days at 37 ° C in a humidified 5% (v / v) CO 2 incubator in preparation for antiviral testing.

Antiviral assay in PBLs (with or without monocytes) The PHA-stimulated cell cultures were dispersed by gentle agitation, 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 of RPMI growth medium. The viability of the cells was verified as previously done. Only suspensions of cells that showed more than 95% viability were processed for antiviral test. For infection of the cells, expanded Ba-L HIV supply material of known titer was added to the cell pellet of PBLs, at a ratio of 250 μ? of supply material for 1.0 x 106 cells, TCID50 of 5.76 x 10 / ml, followed by the addition of 125 μ? of RPMI growth medium (containing 10 ng / ml of IL-2) per 1.0 x 10 6 cells. The cells were then incubated for 1 hour at 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 growth medium of the RPMI (containing 10 ng / ml of IL-2) at a density of 2.0 x 10 5 / ml. Aliquots of 1.8 ml of the suspension of infected cells were transferred to a 24-well test plate containing 200 μ? of compound. The final concentration scale of the compound on the test plate was 0-100 nM (0.1% DMSO). The test plate was placed in a 5% (v / v) CO2 incubator humidified at 37 ° C for 5 days. Controls of uninfected PBLs were processed in parallel, along with a positive control containing the anti-HIV RANTES inhibitor tested on a concentration scale of 0-100 n and 0-33 nM. After incubation for 5 days, 200 μ? of supernatant from each cavity to a 96-well plate for quantification of the virus produced by reverse transcriptase activity and subsequent determination of the antiviral potency of the compounds.

Inverse transcriptase test The viral yield was quantified by direct measurement of RT activity in cultures of HIV-infected cells, using a scintillation proximity test kit from Amersham (see materials). The RT activity was tested by monitoring the incorporation of [3 H] TTP into biotin-labeled DNA primer bound to the surface of the SPA spheres. The volume of the reagents for the RT test depended on the number of tests to be performed. To allow 100 tests, 1 ml of [3H] TTP (3700KBq) supply material was diluted in 2 ml of pH regulator from the test set, and transferred to an 80 cm2 culture flask along with 5 ml of water sterile and 1 ml of SPA spheres of the test equipment. This solution was completely mixed and aliquots of 80 μ? to each cavity in the RT reaction plate, followed by 20 μ? of supernatants of PBLs infected with HIV. The reaction plates were sealed and placed in an incubator at 37 ° C for 1 hour. To stop the RT reaction, 100 μ? of pH regulator for stopping the test equipment (SDS at 2% (w / v), pH 8). The plates were centrifuged at 250 g for 10 minutes at 4 ° C. Scintillation was measured for each cavity for a period of 60 seconds using a 1450 Microbeta liquid scintillation counter, after an incubation period of 20 minutes in situ to allow the spheres to settle. Bottom counts for the uninfected control samples were subtracted from the counts of infected samples. A standard curve for RT activity was generated using purified recombinant RT samples and in parallel to the samples of HIV infected cells, to ensure that the RT levels of the supernatants were on the linear scale for the test. For the standard RT curve, 20 μ? of recombinant reverse transcriptase supply material (IOU / μ? - see materials), and were diluted with 9980 μ? of complete medium of RPMI 1640 at 4 ° C. The enzyme was stored at -20 ° C until use, to produce gradual dilutions in complete medium of RPMI 1640 at 4 ° C (20, 10, 5, 2.5, 1.25, 0.62, 0.31 and 0.15 mU / μ) for a standard curve. A sample was taken in duplicate of each enzyme dilution (20 μ?) In the RT test.

Cytotoxicity test Potential non-specific cytotoxicity of compounds in PBLs was investigated as a possible cause for apparent antiviral activity. The test was carried out in a 96-cavity format, using a Promega cell proliferation colorimetric equipment (see materials). In this test, the tetrazolium compound 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS) in combination with the reagent of electron coupling phenazine methosulfate (PMS), is reduced by dehydrogenase activity in viable cells to generate formazan. The absorbance of formazan at 492 nm is directly proportional to the density of live cells in the culture. This test was carried out in uninfected PBLs grown separately in parallel to those used in the antiviral tests. PBLs were prepared for the cytotoxicity test as described for antiviral tests, before HIV infection. The PBLs were suspended in fresh culture medium at a final density of 2 x 10 5 cells / ml. Cell suspensions (180 μ?) Were transferred to a 96-well plate, in addition to 20 μ? of compound, to give a final concentration scale of 0-10 μ? N situ (DMSO at 0.1% (v / v)). The test plates were incubated at 37 ° C in a 5% (v / v) CO2 incubator humidified for 5 days. After this incubation, 40 μ? Were added to each cavity. of MTS / PMS mixture from the cytotoxicity test set, and incubated in the dark for 3 hours at 37 ° C in a humidified 5% (v / v) C02 incubator. The plates were then manually shaken, and the absorbance was measured spectrophotometrically at 492 nm to evaluate the viability of the cells and the nonspecific cytotoxicity of the compounds.

Data analysis The antiviral activity was determined by plotting a percentage reduction in the activity of RT or levels of p24 (compared to the control without compound), against concentration of the compound (logarithmic scale). The IC50 and IC90 values were determined using the Microsoft Excel curve adjustment option to duplicate data points at each concentration. IC50 and IC90 values were determined for concentration scales of the compound tested independently, and the geometric mean was calculated using standard program packages. These experiments were repeated independently with different preparations of PBLs, and the IC50 and ICgo values of the general geometric mean were calculated together with the 95% confidence intervals. The same analysis was performed for the RANTES standard antiviral. The individual IC50 and IC90 values that were outside the compound concentration scale, or RANTES, were not included in the additional calculation for the IC50 or, IC90 of the geometric mean.

Materials Strain of HIV-1 Ba-L: A cell-free supernatant was obtained from the AIDS Reagent Project (NIBSC, Potters Bar, Herts, United Kingdom). RANTES: Obtained from R & D systems (Catalog No. 278-RN-010 Ó 278-RN-050). Cell dissociation solution: (Sigma, 1x conc C5789 lot 59H0890). Cell culture medium: Sigma RPMI medium (catalog number: R0883), L-glutamine from Life technologies (catalog no .: 25030-024), Sigma FCS for PBLs (catalog no .: F2524 lot 77H3399), penicillin and streptomycin from Life Technologies (Catalog No. 15140-123). Additives for the culture medium of PBLs: PHA suspended in sterile water at 1 mg / ml and used at a final concentration of 1.5 μg / ml, Murex-Abbott Laboratories (Catalog No. HA 6). Human recombinant IL-2 dissolved in 4 mM HCl plus 0.1% (v / v) FCS at 2 g / ml (approx 6000 U / ml), and used at a final concentration of 10 ng / ml - R &D Systems (Catalog No. 202-IL-050). PBLs isolation reagents: Yellow covers provided by the North London Blood Transfusion Center (Colindale Center, Ficoll-Paque solution from Pharmacia Biotech (Catalog No.: 17-0840-02), Sigma PBS (Catalog No.: D8573), Sterile Distilled Water from Sigma (Catalog No. W-3500). Reverse transcriptase test kit: Quan-T-RT test system from Amersham Life Science (Catalog No. TRK 1022). Recombinant HIV reverse transcriptase, provided as 200 U (10 U / μ?) Of Amersham (Catalog No.: T3610Y). Cytotoxicity test kit: CelITiter 96® AQueous non-radioactive test - Promega (Catalog No.: G5430).

Consumables: 24-cavity sterile flat bottom Falcon plates - Becton Dickinson (Catalog No. 3047). 96-well Sterile Flat Bottom Falcon Plates - Becton Dickinson (Catalog No .: 3072). Square plate with 96 cavities of polypropylene with a capacity of 2 ml per cavity, sterile (Beckman Coulter, Catalog No. 609681). Cell counting chamber - Kova Biostat Diagnostics (Catalog no .: 887144).

Results In the antiviral test, Compound D has an IC90 of 1 nM, Compound A and Compound B have an ICgo in the antiviral test of about 2 nM, while Compound C has a potency in the antiviral test greater than 100 nM.

Conclusion Although compounds A to D have binding affinity very similar 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 can be used as a prediction mechanism for the potency of the ligands for functional activity, in this case antiviral activity, and predicts the clinical efficacy for the ligand as an antiviral agent.

EXAMPLE 5 Effect of short-term monotherapy with compound A on viral carcases in HIV-infected patients For standard anti-retroviral agents directing the virus, there is a direct relationship between antiviral effects (measured by a decrease in viral load in plasma) and plasma drug concentrations. Also, therefore, clinically effective dose scales can be selected based on in vitro antiviral data, i.e., IC50 and IC90. However, the argument can be made that for this procedure, saturation of the CCR5 receptor could be a better predictor of efficacy, and can not be directly related to plasma drug concentrations. Data from studies conducted in healthy volunteers showed that even at a dose of 25 mg, where the concentrations; of the drug rapidly decrease well below the IC9o or antiviral, essentially complete saturation of the receptor was achieved for more than 24 hours after the dose. The main reason for this study was, therefore, among other things, to determine if saturation of the CCR5 receptor correlates with antiviral effects in vivo.

Methods Twenty-four asymptomatic HIV seropositive male subjects with CD4 T cell count > 250 cells / mm3 and plasma viral load > 5000 copies / ml, received compound A 25 mg every day, 100 mg twice daily or placebo for 10 days. The main exclusion criteria were abnormalities of liver enzymes, CD4 T cell count < 250 cells / mm3, viral load < 5000 copies / ml, and the presence of CXCR4 or double tropic HIV.

Saturation of the receiver The saturation of the receptor was evaluated using the CCR5 / MIP-i p incorporation test described above, at the following time points: Day 1: 0 (before the morning dose) and 4 hours after the Dosage Day 5: before the morning dose Day 10: before the dose and 12 hours after the dose Days 1 1, 12, 13, 15, 19 and 40.

Viral load The viral load was determined using an RT-PCR test (Roche Amplicor v1.5) with a lower detection limit of 400 copies / ml as standard. For samples with a reading < 400 copies / ml, the ultrasensitive method with a lower detection limit of 50 copies / ml was used. Samples were taken at the following time points: Day 1: O (before the morning dose) and 4, 12 and 18 hours after the dose Days 2-10: 0 (before the morning dose) Days 1 1, 12, 13, 15, 19 , 22, 25 and 40.

Results The mean saturation of the CCR5 receptor in patients who received 100 mg twice daily was greater than 90% throughout the dosing period, but in subjects who received 25 mg every day, the average saturation of the receptor decreased to less 80% around day 10 before the dose (steady state). Figure 9 shows the saturation of the receptor over time (in hours after the first dose) per dose group. Seven of eight patients who received 00 mg twice daily of compound A, showed a good response to viral load with a decrease in viral load >; 1 logi0. The average decrease in viral load, from the reference line to day 1 1 in this group, was 1,200 log. A subject in the 100 mg dose group twice daily, in whom co-existence of the viruses was demonstrated using CCR5 and / or CXCR4 as their co-recipient of admission on day 1, had no response to compound A. If this individual is excluded from the analysis, the decrease in viral load in the 100 mg group is 1,419 log-i0. In the 25 mg dose group every day, a mean viral load decrease of 0.425 logi0 was demonstrated (Table 2).

After the end of dosing (day 11 onwards), viral load returned to the baseline over time, and around day 40, most patients had returned to the baseline.

TABLE 2 of dose Dosage N Log- ?? of the viral load change from the reference line * to day 1 1 Placebo 8 -0.019 25 mg od 8 -0.425 1 10U0 (J mg twice a day 8 -1,200 * The reference line is calculated as the average of the three measurements of the viral load before the dose Conclusions The average saturation of the CCR5 receptor in patients who received 100 mg twice daily, was greater than 90% throughout the dosing period, but in patients who received 25 mg every day, the mean saturation of the receptor decreased to less than 80% around the day 0 before the dose Subjects who received 100 mg twice daily had an average decrease in viral load of 1 .200 log10 from the baseline on day 1 1, and there was evidence of a response in subjects who received 25 mg every day Clinical data show that compound A exhibited potent antiviral effects when administered as short-term monotherapy, and thus supports in vitro data and its prediction of clinical efficacy. Given the above, it can be seen that the residence time or occupancy time of CCR5 in vitro, can be correlated with receptor occupancy in vivo. Most importantly, the ligands identified by receptor occupancy in vitro show significant decreases in viral load both in vitro and in vivo. In this way, the measurement of the occupancy of a ligand on its in vitro receptor can be used to identify ligands that are predicted to be effective in vivo. Although this theory has been tested and demonstrated in an HIV scenario, it will be appreciated that the prediction of the clinical efficacy of putative ligands from in vitro receptor occupancy can be extended to other medical indications mediated by CCR5, including Respiratory disorders that include 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 has been established with CCR5 or chemokines of CCR5, include inflammatory bowel disease including Crohn's disease and ulcerative colitis, multiple sclerosis, rheumatoid arthritis, rejection of grafts, in particular but not limited to, transplants of solid organs, such as heart, lung, liver, kidney and pancreas transplants (for example, kidney and lung allografts), endometriosis, type I diabetes, kidney diseases such as glomerular disease, fibrosis, such as liver, lung and kidney fibrosis , chronic pancreatitis, inflammatory conditions of the lung, encephalitis, such as HIV encephalitis, chronic heart failure, psoriasis, stroke, obesity, CNS diseases, such as dementias related to AIDS and Alzheimer's disease, anemia, atherosclerotic plaque, atopic dermatitis, chronic pancreatitis, cancer, such as non-Hodgkin's lymphoma, Kaposi's sarcoma, mel anoma and breast cancer, and pain, such as nociceptive pain and neuropathic pain (eg, peripheral neuropathic pain). Infectious diseases where modulation of the CCR5 receptor is involved, include acute and chronic hepatitis B virus (HBV) and HCV infection, bubonic, septicemic and pneumonic plague, poxvirus infection, such as smallpox, toxoplasmosis infection, Mycobacterium infection , Trypanosoma infection, such as Chagas disease, pneumonia and cytosporidiosis.

Claims (10)

NOVELTY OF THE INVENTION CLAIMS

1. - The use of a test that measures the residence time of a receptor ligand on its receptor in vitro, for the identification of a ligand for that receptor that is predicted to be effective in vivo, in the treatment of a disease that responds to the modulation of the natural function of that receptor.

2. - The use as claimed in claim 1, wherein the residence time is at least 1 hour.

3. - The use as claimed in claim 1, wherein the residence time is at least 3 hours.

4. - The use as claimed in claim 1, wherein the residence time is at least 6 hours.

5. - The use as claimed in claim 1, wherein the residence time is at least 9 hours.

6. - The use as claimed in any of claims 1 to 5, wherein the receiver is CCR5.

7. The use as claimed in claim 6, wherein the ligand is a CCR5 antagonist.

8. - The use as claimed in claim 6 or 7, wherein the disease is infection by a virus.

9. - The use as claimed in claim 8, wherein the virus is HIV.

10. - A research method comprising measuring the residence time of the receptor of each of a plurality of ligands for that receptor, and selecting to further investigate at least one ligand whose residence time is longer than that of minus some other ligand. 1. A research method comprising contacting a plurality of ligands for a given receptor with that receptor, measuring the binding affinity of the receptor and the residence time of the receptor of each ligand, assigning each ligand a value of hierarchy that is the product of the union affinity of your measurement and the residence time of your receiver, and selecting to further investigate one 0 more ligands that have a hierarchy value greater than a selected limiting hierarchy value. 12. - A method for identifying ligands with high potency and / or clinical efficacy for a disease that responds to modulation of the natural function of a receptor, comprising measuring its residence time on the receptor, and selecting ligands based on time of desired residence. 13. The method according to claim 10, 11 or 12, further characterized because the residence time is at least 1 hour. 14. The method according to claims 10, 11 or 12, further characterized in that the residence time is at least 3 hours. 15. - The method according to claims 10, 1 1 or 12, further characterized in that the residence time is at least 6 hours. 16. - The method according to claims 10, 1 1 or 12, further characterized in that the residence time is at least 9 hours. 17. - The method according to any of claims 13 to 16, further characterized in that the receiver is CCR5. 18. - The method according to claim 17, further characterized in that the ligand is a CCR5 antagonist. 19. - The method according to claim 12, further characterized in that the clinical efficacy is antiviral activity. 20. The method according to claim 19, further characterized in that the antiviral activity is anti-HIV activity. 21. A ligand selected by the methods according to any of claims 10 to 20.