Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
  • G01N33/56988—HIV or HTLV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/557—Immunoassay; Biospecific binding assay; Materials therefor using kinetic measurement, i.e. time rate of progress of an antigen-antibody interaction
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/08—RNA viruses
  • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
  • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
  • G01N2333/16—HIV-1, HIV-2
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
  • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

• the present invention relates to the use of an assay to determine receptor occupancy as a means to identify ligands predicted to be clinically efficacious.
• the present invention relates to the identification of ligands of the
• CCR5 receptor using a long receptor residency time as an indicator of anti-viral, specifically anti-HIV, activity.
• Costs up to clinical phase can be around $20 million (almost £13 million). For every ten to fifteen 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 rise sharply.
• HIV-induced AIDS (Acquired Immunodeficiency Syndrome) was first described almost two decades a go, it continues to be a disease of enormous proportions. It is estimated that there were more than 40 million HTV-infected people worldwide by the end of 2002 (UNAIDS/WHO. AIDS epidemic update: Dec 2002 http://www.unaids.org/worldaidsday/2002/press/epupdate.html).
• HTV-1 Human Immunodeficiency Virus Type 1 attacks cells in the immune system, causing the body to lose the ability to fight off infections and diseases. During the course of infection, CD4 T-cells (white blood cells that fight infection) are disabled and killed as their numbers decline. Infection with HIV-1 leads, in the vast majority of cases, to progressive disease and ultimately, AIDS and death.
• HTV-1 is one of the most rapidly mutating viruses ever encountered.
• HAART highly active antiretroviral therapy
• HAART currently targets two different stages in the virus life cycle and consists of two or three inhibitors of the virus's reverse transcriptase 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 the progression to AIDS in many patients.
• HTV-1 To invade the human cell, HTV-1 must bind to the cluster of differentiation (CD) CD4 receptor and a chemokine co-receptor (either CCR5 or CXCR4) located on the CD4 T cell surface. Binding to the CD4 receptor alone is not sufficient to render cells susceptible to infection by HIV-1. Macrophage tropic (M-tropic) HIV isolates, irrespective of subtype, predominantly use the ⁇ -chemokine receptor CCR5, and are referred to as R5 viruses. R5 viruses are preferentially transmitted and predominate in early asymptomatic disease. A switch in chemokine receptor utilization from CCR5 to CXCR4 may indicate the transition from asymptomatic infection to AIDS. It is not known whether the co-receptor switch contributes to or results from disease progression.
• CD cluster of differentiation
• CXCR4 chemokine co-receptor
• CCR5 The inhibition via CCR5 is a particularly attractive drug target for two reasons. Firstly, as mentioned above, the viruses that establish a new infection are generally CCR5-tropic. Thus, successful inhibition of infection via CCR5 might significantly decrease the probability of transmission. Secondly, individuals homozygous for a naturally occurring 32 base-pair deletion in the CCR5 gene (CCR5 ⁇ 32) and thus lacking this receptor are apparently immunologically normal. These individuals are also resistant to infection by R5 strains of HIV and people heterozygous for this CCR5 defect exhibit a slower progression to AIDS and death. The frequency of the different genetic polymorphisms varies between populations. CCR5 ⁇ 32 is most prevalent in northern Europe (allelic frequency of 16%), less common in individuals from southern Europe (4%) and is extremely rare in African populations. Approximately 1% of Caucasians are homozygous for CCR5 ⁇ 32 and effectively represent human equivalents to experimental "knockout" animals.
• Chemokine receptors are seven membrane-spanning molecules found on the surface primarily of cells in the immune system. When these receptor molecules bind their ligands, i.e. chemokines, the end result is to recruit cells of the immune system to the site of tissue damage or disease.
• CCR5 (CC chemokine receptor 5) is the major co-receptor for macrophage-tropic (M-tropic) HIV-1 strains and also appears to be the major co-receptor for microglial tropic HIV-1 primary isolates.
• Putative physiological ligands of the CCR5 receptor are the chemokines Macrophage Inflammatory Protein- 1 (MIP- l ⁇ ), MlP-l ⁇ , and Regulated upon Activation, Normal T Expressed, and Secreted
• RANTES HIV-1 co-receptor activity
• HIV-1 During the M-tropic phase of HIV infection, the virus favours macrophages, which it invades by binding (through its gpl20 protein) to the molecules CD4 and CCR5 on the macrophage surface. Eventually, however, HIV-1 can become dual- tropic. Such strains produce gpl20 molecules capable of recognizing the CXCR4 protein on CD4-bearing T-cells. During this phase HIV-1 may infect both macrophages and T-cells. Still later, the bulk of the viral population may switch its preference to the CXCR4 receptor and become T-tropic. T-tropic viruses readily destroy infected T-cells, contributing to the collapse of the immune system and the onset of AIDS. Alternatively, some viruses, such as certain strains of HIV-2 could attach to CXCR4, leading quickly to AIDS.
• Possibilities include blocking the wild-type expressed receptors with suitably constructed agonists or antagonists to these molecules. The challenge remains to ensure that analogues are potent, non-toxic, exhibit good pharmacologic profiles and can be administered orally.
• the present invention is based on the observation that the offset time, or functional occupancy, of a receptor by a ligand or modulator provides a guide to the in vivo efficacy and pharmacodynamics of the ligand or modulator.
• the Applicant has found that compounds with very similar binding affinity to a receptor show marked differences in their residency time on the receptor, which may translate to improved antiviral potency.
• the aim of the present invention is to provide a method whereby the potential for favourable pharmacodynamics and improved potency, and therefore clinical efficacy of a compound, may be predicted more accurately from pre-clinical data.
• the present invention resides in 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.
• the present invention relates to a method for identifying ligands with high potency and/or clinical efficacy for a disease that responds to modulation of a receptor's natural function which comprises measuring their residence time on the receptor and selecting ligands on the basis of the desired residence time.
• a "ligand" for a receptor means any compound that binds to the receptor and thereby modulates 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.
• the ligand may be an antibody or antibody fragment, or a nucleic acid or nucleic acid derived material.
• “residence time” means the average time a ligand spends bound to its receptor, i.e. the average time between the "on” and “off states of the ligand with respect to its receptor. Expressed in another way, the term refers to the time during which the receptor is in a state that does not allow a natural ligand, such as a chemokine, or invading molecule, such as HIV glycoprotein, to bind as a result of ligand binding.
• Residence time is also referred to as 'receptor occupancy' and may be determined by any method that measures the off-rate, dissociation rate, or off-set rate of a ligand.
• a typical method comprises the steps of incubating a labelled ligand with an appropriate receptor, for example cells expressing the receptor or membrane preparations derived from such cells, until equilibrium is reached. Excess unlabelled ligand is then added and the amount of labelled ligand bound to the receptor is measured at desired time intervals.
• the label may be any detectable tag that does not influence the binding of the ligand to its receptor.
• a radiolabel such as 3 H, 14 C, 32 P or 125 I is incorporated into the ligand, most preferably 3 H.
• the receptor residence time is at least 1 hour, at least 3 hours, at least 6 hours or, more advantageously, at least 9 hours.
• the longer the functional occupancy of a ligand on a receptor the longer the clinical effect of that ligand, thereby reducing the number of doses that would need to be administered to a patient.
• the ligands identified by the present invention may be agonists for the receptor but, preferably, are antagonists.
• the receptor is a G-protein coupled receptor, most preferably the chemokine receptor CCR5.
• the receptor residence time (or clinical efficacy) of a ligand may be a predictor of anti-viral activity, preferably anti-HTV activity.
• the current invention provides a method to select CCR5 antagonists, which will show potent antiviral activity and clinical efficacy, by measuring the residence time of the antagonists on CCR5 and selecting ligands that have a long residence time.
• the present invention relates to a research method comprising measuring the receptor residence time of each of a plurality of ligands for that receptor, and selecting for further research at least one ligand whose residence time if longer than that of at least one other ligand.
• the present invention resides in a research method comprising contacting a plurality of ligands for a given receptor with that receptor, measuring the receptor binding affinity and receptor residence time of each ligand, assigning to each ligand a rank value which is the product of its measure binding affinity and its receptor residence time, and selecting for further investigation one or more ligands having a rank value greater than a chosen cut-off rank value.
• Potency and/or clinical efficacy relates to the functional effect a ligand exhibits while resident on its target receptor.
• This may be any effect an agonist or an antagonist to a receptor may have, for example the inhibition of viral entry into T- cells.
• the effect is an antiviral effect, preferably an anti-HIV effect.
• 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, even more preferably at least six hours, most preferably at least nine hours.
• the invention relates to a ligand selected by the method of the invention.
• the term 'receptor' is to be understood broadly and may relate to any molecule comprising a binding site for a ligand as defined above, including but not limited to receptors, enzymes, ion channels, adhesion molecules, antibodies.
• the receptor is a receptor found in or on a cell, preferably a mammalian cell, even more preferably a human cell.
• 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 CCR5 chemokine receptor.
• binding affinity' relates to the binding of a ligand that may be determined by traditional binding assays well known in the art, such as a competition assay.
• a membrane preparation from cells expressing the receptor of interest will be incubated with a known, labelled ligand for this receptor, using a concentration of the labelled ligand that gives about 50% of the total possible binding.
• an unlabelled, competing test ligand is included in varying amounts, in order to measure the ability of the test compound to compete for the binding with the labelled ligand.
• Competition curves are then generated, plotting the concentration of the test ligand used along the x- axis, and the amount of label bound along the y-axis.
• the K D , the IC 50 (i.e. the concentration of competing ligand which displaces 50% of the labelled ligand), or the IC 90 (i.e. the concentration of competing ligand which displaces 90% of the labelled ligand) value may be calculated.
• the concentration of competing ligand which displaces 50% of the labelled ligand i.e. the concentration of competing ligand which displaces 50% of the labelled ligand
• the IC 90 i.e. the concentration of competing ligand which displaces 90% of the labelled ligand
• 'Chemokine receptor' refers to receptors for a large family of proteins which are chemotactic cytokines, i.e have the ability to attract leukocytes as leukocyte chemotactic factors Chemokines share certain important structural features, and bind to families of receptors most of which belong to the G-protein coupled receptor superfamily Chemokines and their receptors are central to the pathophysiology of inflammatory and infectious diseases and agents that are active in modulating, preferably antagonising, the activity of chemokines and their receptors, are useful in the treatment of such inflammatory and infectious diseases
• CCR5' refers to the chemokine receptor which is the cellular receptor for the ⁇ - chemokines RANTES, MlP-l ⁇ and MlP-l ⁇ .
• CCR5 has also been identified as being an important receptor in HIV infection, binding the HIV envelope glycoprotein gpl20 Background teachings on CCR5 may be found in WO 97/32019, the disclosure of which is incorporated herein by reference.
• HTV' refers to human immunodeficiency virus, presumed to be the agent causing AIDS
• Figure 1 shows the FACS fluorescent overlays for RANTES -mediated CCR5 lnternahsation in 300 19 cells and the inhibition of mternahsation 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 lnternahsation is depicted by trace D, and the vehicle control (no compound or RANTES I e. total
• FIG. 1 Isotype control is depicted by trace E
• FIG. 1 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
• the vehicle control no compound or RANTES i.e. total CCR5
• Isotype control is depicted by trace F;
• Figure 3 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).
• Figure 4 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;
• Figure 5 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 bid oral doses 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.
• Figure 9 is a graph showing CCR5 receptor saturation over time in asymptomatic HIV seropositive male subjects administered with Compound A 25mg, lOOmg or placebo.
• Cell Preparation Cells from each 75cm 2 flask used were centrifuged at 1500rpm 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 5xl0°/ml by dilution in the same medium.
• CCR5 antagonists were dissolved in 100% dimethyl sulphoxide (DMSO) to a concentration of ImM and diluted in RPMI (10% FBS) cell culture medium to enable testing in the FACS assays at concentrations from lOnM to lOOOnM in situ.
• DMSO dimethyl sulphoxide
• 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.
• washed samples Half of the washed samples were resuspended in lOOO ⁇ l 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 (lO ⁇ l) was added to the samples followed by incubation at 37°C for 45 minutes to provoke CCR5 internalisation.
• the samples were centrifuged (1500rpm in a bench top centrifuge) and washed twice in 0.5%BSA/PBS. Washed samples were resuspended in 40 ⁇ l of the same buffer. 2D7 antibody or isotype control (lO ⁇ l) was then added to the samples followed by incubation for 45 minutes at 4°C to enable antibody binding to CCR5.
• Culture medium lx 500ml RPMI- 1640 medium with NaHCO 3 without L- glutamine - Gibco BRL (cat no:31870-025); 5ml 200mM L-Glutamine - Gibco
• 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's phosphate buffered saline (PBS) without Ca 2+ and Mg 2+ - Gibco BRL (cat no: 14190-094).
• Micro centrifuge tubes - Costar (cat no: 3621); FACS tubes: Falcon tubes - Becton Dickinson (cat no: 35 2054); Labsystems Finn pipettes Thermo Life Sciences (cat nos: 4500/090/050); 1ml Pipette Tips - Thermo Life Sciences (cat no: 9401103); 250 ⁇ l 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's phosphate buffered saline (PBS) without Ca 2+ and Mg 2+ - Gibco BRL (cat no: 14190-094); EC Micromax RF centrifuge; RANTES - R & D Systems (cat no: 278-RN-010); Mouse anti-human CCR5 monoclonal antibodies:
• 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.
• RANTES-mediated CCR5 internalisation was apparent as observed by the shift in fluorescence upon following incubation of 300.19 cells with chemokine.
• compound B dissociates slowly in these experiments, whereas compound C dissociates more rapidly.
• compound C does not inhibit receptor update completely even at concentrations well in excess of its receptor IC 5 o. This is consistent with offset from CCR5 as measured by radioligand dissociation studies (see Example 3).
• 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.
• MlP-l ⁇ mediated CCR5 internalisation 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 MlP-l ⁇ 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- l ⁇ 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.
• 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:-
• Cohort 1 lOOmg b.d. Compound A or Placebo
• Cohort 4 25mg b.d. Compound A or Placebo
• Cohort 6 3mg b.d. or lOmg b.d. Compound A or Placebo.
• 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.
• Subjects were discharged at 24 hours post last dose but returned for a follow-up physical examination 7-10 days following the final dose.
• 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 lOmg bid 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 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.
• 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.
• Reagents 10X concentrated PBS; 10% Paraformaldehyde; 10% Sodium Azide solution; lyophilized BSA; MlP-l ⁇ working solution (aliquots stored frozen at -
• CCR5 Stabilising Solution contains 600nM Compound A in PBS
• Stabilising Control Solution PBS
• CCR5 and IgG Isotope Control Antibody cocktails Store at 2-8°C
• the control stabilising solution is PBS. 1.
• 600nM drug solution (Stabilizing Solution) should be added 1 6 m plasma to achieve a final concentration of lOOnM
• stabilizing solution should be added at 50 ⁇ L to 250 ⁇ L 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 p ⁇ or to use to ensure that the drug is in solution.
• Lymphocyte ⁇ ch plasma was isolated by cent ⁇ fugation at 1550g for 25 nuns
• Antibody reagents 50 ⁇ l were added in the following manner: To Tube 1 - MsIgG R-phycoeryth ⁇ n (PE) and CD4-Fluoresce ⁇ n (FTTC) (Control) was added
• CCR5 occupancy was related to dose. Volunteers receiving lOOmg b.i.d. demonstrated CCR5 receptor occupancy in excess of 90% throughout the dosing period, while in those subjects receiving 3mg, mean receptor saturation was ⁇ 80%.
• 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.
• HEK-293 cells stably expressing CCR5 were grown in 225cm 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 225cm 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.
• PBS ml phosphate buffered saline
• the supernatant was removed and the cells were dislodged by rapping the side of the culture flask in the presence of 10ml binding buffer at room temperature (see Materials).
• the cells were placed into a 50ml centrifuge tube.
• a further 10-20ml 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 350g for 10 minutes at 20°C.
• the cells were resuspended in 3ml binding buffer, counted in a modified Neubauer haemocytometer and resuspended to a density of 2 l0 6 cells/ml.
• Membrane preparation Each 225cm 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 5-10ml PBS (room temperature). The cells were transferred into a 50ml centrifuge tube. A further 10-20ml 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 350g for 10 minutes at
• the cells were resuspended in 15ml lysis buffer (see Materials) at room temperature, and homogenised with a Polytron hand-held 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 25000rpm (40,000g) 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 0.25mg/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 l-2mg Compound A, B or C was dissolved in 100% dimethyl sulphoxide (DMSO) to a concentration of lOmM and diluted in binding buffer to enable compound addition to the binding assays over a concentration range of 0.41nM to 50 ⁇ M in situ.
• 20 ⁇ l of [ 3 H] Compound A (62.5 ⁇ M, specific activity 16 Ci /mmol and radioactive concentration of lmCi/ml) was diluted in binding buffer to enable radioligand addition to the binding assay over a concentration range of 0.41 - lOOnM in situ. Similar dilutions were performed with [ 3 H] -labelled Compounds B and C.
• Binding buffer 25 ⁇ l was added to wells of a 96 well assay plate, followed by 50 ⁇ l cells or cell membrane preparation.
• [ 3 H] Compound A, B or C dilutions 25 ⁇ l were added to designated wells to enable radioligand association.
• Non-labelled Compound (lOOx [ H] Compound concentration) was added designated wells (25 ⁇ l) in place of the binding buffer to determine nonspecific 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 [ H] Compound. This allowed radioligand binding to CCR5 to reach equilibrium. After this incubation, 25 ⁇ l 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.
• the Unifilter plates were blocked by applying 25ml blocking agent (room temperature) (see Materials) for 30 minutes.
• 25ml blocking agent room temperature
• PBS Dulbecco's phosphate buffered saline
• Binding Buffer 50mM HEPES in distilled water - Sigma (cat no: H-0763). ImM CaCl 2 - Sigma (cat no : C-3881). 5mM MgCl 2 - Sigma (cat no: M-1028). 0.5% Bovine Serum Albumin (BSA) - Sigma (cat no: A-4503). Buffer adjusted to pH
• Lysis Buffer 20 mM HEPES in distilled water - Sigma (cat no: H-0763). ImM CaCl 2 - Sigma (cat no: C-3881). 1 tablet COMPLETETM protease inhibitors per 50ml lysis buffer - Boehringer Mannheim (cat no: 1 697 498). Buffer adjusted to pH 7.4 (2M HC1) and 0.2 ⁇ m filtered.
• Multichannel Pipettes - Labsystems Finnpipette (cat no: 4510- 000/020/030/040/050). 1ml Pipette Tips - Sigma (cat no: P7174). 250 ⁇ l 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.
• PBLs peripheral blood lymphocytes
• RT supernatant reverse transcriptase
• 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 1ml vials at -80°C.
• For expansion of HTV-Ba-L virus stock (1ml) was removed from -80°C storage, and rapidly thawed in a 37°C incubator for 10 minutes.
• Virus stock (0.5ml) was placed in a labelled well of a 24 well plate with 1ml of fresh PBLs (pre-stimulated 3 days with phytohaemagglutinin (PHA) at a final concentration of 1.5 ⁇ g/ml) at a cell density of 1.0 x 10 7 /ml in complete RPMI 1640 growth medium (supplemented with 10% FCS, 2mM L-glutamine, and lU/ml penicillin, O.lmg/ml streptomycin).
• PBLs phytohaemagglutinin
• PHA phytohaemagglutinin
• the 24-well plate was incubated at 37°C in a humidified 5%(v/v) CO 2 incubator for 1 hour to allow infection.
• the cells and supernatant from this plate were centrifuged at 225g for 10 minutes at 25°C, and the supernatant discarded.
• the cells were suspended in 10ml of RPMI 1640 growth medium at room temperature and transferred to a 25cm 2 tissue culture flask.
• IL-2 was added to a final concentration of lOng/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, 5mls of fresh RPMI 1640 growth medium were added together with IL-2 at a final concentration of lOng/ml.
• Viral expansion was measured by reverse transcriptase (RT assay), and counts of
• Cells were expanded further by dividing the infected culture in to fresh 80cm 2 tissue culture flasks, followed by the addition of 13ml of fresh IL-2-stimulated PBLs (1.0 x 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, 20ml of RPMI 1640 growth medium (containing BL-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 ⁇ l) were > 5000cpm at day 23. These supernatants were clarified by centrifugation at 850g for 10 minutes at 25°C, and the resultant supernatant mixed, and aliquoted into 4ml and 1ml cryotubes for storage at -80°C.
• tissue culture infectious dose 50 (TCID 50 - assumed number of viral particles required to infect 50% of cells) was calculated for the HTV Ba-L stock (harvested 17/12/99), by the Reed and Muench equation ((1938) Am. J. Hyg. 27, 493-497), (using RT as a positive marker for -HTV infection). Titration of virus stock HTV-1
• Ba-L (harvest 17/12/99) produced a TCID 50 5.76 x 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 500g and 25°C, checked for viability by removing lOO ⁇ l of cells, followed by the addition of lOO ⁇ l of cell dissociation solution (see Materials) and lOO ⁇ l of 0.4%(v/v) Trypan Blue. Viable (uncoloured) cells were counted in a Kova counting chamber (see Materials). Only cell suspensions showing >95% viability progressed in assays.
• Viable cells were resuspended to a density of 2.0 x 10 6 cells/ml in RPMI growth medium (lOng/ml IL-2 added for PBL cultures). Expanded frozen HIV Ba-L stock (2 x 1ml) 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.
• TCID 5 o determination For titration, 25 ⁇ l of thawed expanded virus stock, was progressed through 8 x 0.5 log serial dilutions in RPMI growth medium. Each viral dilution (20 ⁇ l) was transferred to a 96-well assay plate together with 180 ⁇ l of PBLs. The assay plate was incubated at 37°C in humidified atmosphere of 5% (v/v) CO 2 for 5 days, after which lOO ⁇ l of supernatant was harvested and tested for RT activity, to enable TCID 5 o determination. The titred virus was deemed to be positive with respect to virus infectivity when RT counts were greater (>2 x standard deviations) than the uninfected cell counts.
• Viral titre was calculated using the method reported by Reed and Muench ((1938) Am. J. Hyg. 27, 493-497). This enables the standardisation of viral input for the antiviral assays and subsequent antiviral potency testing of the compounds.
• the TCID50 for HTV-Ba-L in the antiviral assays in this study was 5.76 x 10 /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 (50ml) were transferred into an 80cm 2 tissue culture flask with an equal volume of sterile phosphate-buffered saline (PBS). Aliquots (25ml) of cell suspension were gently layered onto 25ml Ficoll-Paque in separate 50ml centrifuge tubes. Following centrifugation for 30 min at lOOOg at 25°C, the PBL layer was removed by pipetting from between the erythrocyte and plasma layers.
• PBS sterile phosphate-buffered saline
• the separated PBLs were transferred to fresh centrifuge tubes and washed twice with PBS (4°C) and centrifugation for 10 minutes at 850g at 4°C.
• Contaminating erythrocytes were removed by adding 9ml sterile water to the PBL pellet together with xlO Hanks Balanced Salt Solution (see Materials).
• PBS (4°C) was added to give a final volume of 45 ml.
• the PBLs were centrifuged for lOmin at 500g at 4°C. The pellet was suspended in 30 ml RPMI 1640 growth medium
• Cell viability was checked as described above, and only cell suspensions showing >95% viability were processed for antiviral assay.
• the cell suspension was adjusted to a density of 1 x 10 6 cells per ml by the addition of RPMI 1640 growth medium supplemented 1.5 ⁇ g/ml PHA.
• the cells (50ml) 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 >95% viability were transferred to 80cm 2 culture flasks in a total volume of 50ml and cell density of 1 x 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 500g for 10 min at 25°C. The PBL pellet was adjusted to a density of 1 x 10 6 cells per ml by the addition of fresh RPMI 1640 growth medium containing 1.5 ⁇ g ml
• the cells (50ml) 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 50ml centrifuge tube.
• the cells were pelleted by centrifugation for 10 minutes at 500g at 25°C and resuspended in 50 ml RPMI growth medium. Cell viability was checked as before. Only cell suspensions showing >95% viability were processed for antiviral assay.
• expanded Ba-L stock of known titre was added to the PBL cell pellet, at a ratio of 250 ⁇ l stock per 1.0 x 10 6 cells, TCID 50 5.76 x 10 4 /ml followed by the addition of 125 ⁇ l RPMI growth medium (containing lOng/ml IL-2) per 1.0 x 10 6 cells.
• the cells were then incubated for lh at 37°C in a humidified 5%(v/v) CO incubator, followed by centrifugation at 500g for 10 minutes at 25°C.
• the cell pellet was resuspended in RPMI growth medium (containing lOng/ml IL-2) at a density of 2.0 x 10 5 /ml.
• RPMI growth medium containing lOng/ml IL-2
• 1.8ml aliquots of infected cell suspension were transferred to a 24- well assay plate, containing 200 ⁇ l of compound.
• the final concentration range of compound in the assay plate was 0 - lOOnM (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-HTV inhibitor RANTES tested over a 0 - lOOnM and 0 - 33nM concentration range. Following the 5 day incubation, 200 ⁇ l 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' antiviral potency.
• RT activity was assayed by monitoring [ 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, 1ml of stock [ 3 H] TTP (3700KBq) was diluted in 2ml assay kit buffer, and transferred to an 80cm 2 culture flask together with 5 ml sterile water and 1ml SPA bead from assay kit. This solution was mixed thoroughly and 80 ⁇ l aliquots were added to each well in the RT reaction plate, followed by 20 ⁇ l of the HTV-infected PBL supernatants. The reaction plates were sealed and placed in a 37°C incubator for 1 hour.
• assay kit stop buffer 2% (w/v) SDS, pH 8) was added.
• the plates were centrifuged at 250g 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 HTV-infected cell samples, to ensure RT levels from the supernatants were in the linear range for the assay.
• 20 ⁇ l of stock recombinant reverse transcriptase (lOU/ ⁇ l - see Materials) was defrosted and diluted with 9980 ⁇ l 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/ ⁇ l) for a standard curve.
• Each enzyme dilution (20 ⁇ l) was sampled in duplicate in the RT assay.
• Cvtotoxicitv 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 colorimetric cell proliferation kit from Promega (see Materials).
• MTS colorimetric cell proliferation kit
• 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 492nm 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 HTV infection.
• the PBLs were suspended in fresh culture medium to a final density of 2 x 10 5 cells/ml.
• the cell suspensions (180 ⁇ l) were transferred to a 96-well plate, in addition to 20 ⁇ l of compound to yield a final concentration range of 0 - lO ⁇ M in situ (0.1%(v/v DMSO).
• the assay plates were incubated at 37°C in a humidified
• 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 o values were determined using the fit curve option of Microsoft Excel for duplicate datapoints at each concentration.
• the IC50 and IC 9 o 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 5 o and IC 9 o values which fell outside the concentration range of compound, or RANTES were not included in further calculation for geometric mean IC50 or IC 90 .
• HTV-1 strain Ba-L a cell free supernatant was obtained from AIDS Reagent Project (NIBSC, Potters Bar, Herts,UK.).
• RANTES Obtained from R & D systems (cat no. 278-RN-010 or 278-RN-050)
• RPMI medium was obtained from Sigma (cat no: RO883), L-glutamine from Life technologies (cat no: 25030-024), FCS from Sigma for PBL cells (cat no: F2524 batch 77H3399), Penicillin & streptomycin from Life
• PBL Culture medium PHA suspended in sterile water to lmg/ml, and used at 1.5 ⁇ g/ml final concentration, Murex - Abbott Laboratories (cat no: HA16).
• IL-2 human recombinant dissolved in 4mM HCl plus 0.1% (v/v) FCS to
• PBL isolation reagents Buffy coats supplied by North London Blood Transfusion Centre (Colindale Centre, Ficoll-Paque solution from Pharmacia Biotech (Cat no:
• Reverse transcriptase assay kit Quan-T-RT assay system from Amersham Life Science (cat no: TRK 1022). Recombinant HIV reverse transcriptase, supplied as
• Compound D has an IC 9 o of InM
• Compound A and Compound B have an IC o in the antiviral assay of around 2nM
• Compound C has a potency in the antiviral assay of >100nM.
• 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.
• Example 6 Effect of short-term monotherapy with Compound A on viral load in HIV-infected patients
• Viral load was determined using an RT-PCR (Roche Amplicor vl.5) assay with a lower limit of detection of 400 copies/ml as standard. For samples with a reading of ⁇ 400copies/ml, the ultrasensitive method with a lower limit of detection of 50 copies/ml was used. Samples were taken at the following timepoints:
• Table 2 Mean viral load change from baseline by dose group.
• Mean CCR5 receptor saturation in patients receiving lOOmg bid was in excess of 90% throughout the dosing period, but in subjects receiving 25mg od, mean receptor saturation fell to ⁇ 80% by daylO pre-dose. Subjects receiving lOOmg bid had a mean decrease in viral load of 1.2001og ⁇ o from baseline to day 11, and there was evidence of a response in subjects receiving 25mg od.
• 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.
• 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.
• CCR5 or CCR5 chemokines include inflammatory bowel disease, including Crohn'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's Disease, anaemia, atherosclerotic plaque, atopic dermatitis, chronic pancreatitis, cancer, such as non-Hodgkin's lymphoma, Kaposi's sarcoma, melanoma and breast cancer, and pain, such as nociceptive pain and neuropathic pain (e.g. peripheral neuropathic pain).
• 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
• 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' Disease, pneumonia, and cytosporidiosis.
• HBV hepatitis B Virus
• HCV hepatitis B Virus
• bubonic hepatitis B Virus
• septicemic hepatitis B Virus
• pneumonic plague pox virus infection, such as smallpox, toxoplasmosis infection, mycobacterium infection, trypanosomal infection such as Chagas' Disease, pneumonia, and cytosporidiosis.

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