A population pharmacokinetic-pharmacodynamic analysis of single doses of clenoliximab in patients...

246

T cells have been implicated for inducing or sustain-ing specific pathologic conditions, including autoimmunediseases such as rheumatoid arthritis, transplant rejection,or graft versus host disease. Monoclonal antibodiesdirected against T cells have been studied for their poten-tial to modify these pathogenic immune responses.1-3Oneof the more promising avenues of research has been thestudy of antibodies specific for CD4, a surface proteinexpressed on certain T cell subsets.

In the peripheral immune system, mature CD4molecules are expressed on T cells that interact withantigen-presenting cells bearing class II major histo-

compatibility complex (MHC) molecules, fostering andstabilizing antigen recognition by the T-cell receptor.This interaction likely initiates an intracellular signal,4

which results in an enhancement of the immuneresponse. Antibodies to CD4 thus interfere with eventsmediated by the CD4 antigen. CD4 antibodies inhibitT-cell proliferation and cytokine production in vitro.5,6

Although clinical trials that involve the administra-tion of antibodies against various epitopes on the CD4molecule have shown promise in the treatment ofrheumatoid arthritis,7-9 the precise mechanism of actionof these agents remains elusive. Depletion of CD4+ Tcells could contribute to clinical activity, but CD4 lym-phopenia may cause unnecessary long-term immuno-suppression.

Clenoliximab (IDEC-151, SB-217969) is a chimericmacaque-human (PRIMATIZED) anti-human CD4monoclonal antibody of immunoglobulin G4 (IgG4)isotype. Clenoliximab binds to an epitope on domain 1of CD4, thereby blocking the interaction of CD4 withMHCII on antigen-presenting cells. In this first inves-tigation of clenoliximab in humans, and in earlier stud-ies of the IgG1 isotype of this antibody10 it was evident

A population pharmacokinetic-pharmacodynamic analysis of singledoses of clenoliximab in patients withrheumatoid arthritis

Clenoliximab (IDEC-151) is a macaque-human chimeric monoclonal antibody (immunoglobulin G4) spe-cific for the CD4 molecule on the surface of T lymphocytes. It is being studied in patients with rheuma-toid arthritis in which T cell activation orchestrates inflammation and tissue damage. In this initial studyin humans, the pharmacokinetics and pharmacodynamics of clenoliximab were investigated after singleintravenous infusion. Blood was collected up to 12 weeks after dose administration to measure clenolix-imab concentration, CD4+ T-cell count, CD4 antigen coating, and CD4 cell surface density. Clenoliximabdisplayed nonlinear pharmacokinetic behavior and caused an 80% reduction in CD4 density for up to 3weeks, without depleting T cells. A pharmacokinetic-pharmacodynamic model was developed that describedthe relationship between antibody concentration, antigen coating, and the observed decreases in CD4 cellsurface density. This was used to anticipate the effects of clenoliximab in untested regimens and optimizethe design of future clinical trials. (Clin Pharmacol Ther 1999;66:246-57.)

Diane R. Mould, PhD, Charles B. Davis, PhD, Elisabeth A. Minthorn BS,Deborah C. Kwok, MS, Michael J. Elliott, MD, Michael E. Luggen, MD, andMark C. Totoritis, MD King of Prussia, Pa, Harlow, England, Cincinnati, Ohio, andSan Diego, Calif

From Drug Metabolism and Pharmacokinetics, SmithKline BeechamPharmaceuticals, King of Prussia; Clinical Development, Smith-Kline Beecham Pharmaceuticals, Harlow; University of Cincin-nati College of Medicine, Cincinnati; and Clinical Therapeutics,IDEC Pharmaceuticals, San Diego.

Received for publication Feb 23, 1999; accepted June 8, 1999.Reprint requests: Diane R Mould, PhD, Drug Metabolism and Phar-

macokinetics, SmithKline Beecham Pharmaceuticals, 709 Swede-land Rd (Mail Code UW2730), King of Prussia, PA 19406.

Copyright © 1999 by Mosby, Inc.0009-9236/99/$8.00 + 013/1/100713

that, in addition to coating the T cell, these antibodiesaffect the density of the CD4 molecule on the surfaceof the T cell. Down-modulation of CD4 on otherwisefunctional T cells may occur through internalization ofthe CD4 protein or by stripping the receptor from thecell surface. Although not yet proven to be related toclinical outcome, these changes in the state of the T celllikely play an important role in the clinical activity ofthe antibody.

In this single-dose study, CD4 antigen coating andCD4 down-modulation were selected as the primarypharmacodynamic end points of interest because theywere likely to be central to the mechanism of action ofthe antibody. A pharmacokinetic-pharmacodynamicmodel was developed to describe the effects of cleno-liximab on the CD4 positive T cell with the expectationthat dosage recommendations may be made for longer-term multiple-dose trials to evaluate safety and efficacy.

METHODSStudy design. This was a double-blind, placebo-

controlled, randomized, single-ascending-dose, parallelstudy performed on 32 adult patients. Six dose groupswere studied: 0.05, 0.2, 1.0, 5.0, 10.0, and 15.0 mg/kg.There were three to five subjects in each active dosegroup, and eight patients received placebo. The studywas approved by the Institutional Review Board at theUniversity of Cincinnati College of Medicine in Cincin-nati, Ohio, and each subject gave written informed con-sent before enrollment. Clenoliximab was administeredin the morning as a single 2-hour intravenous infusionto the cubital vein. Blood samples were obtained bydirect venipuncture in the arm contralateral to the infu-sion site. Blood samples (~10 mL) for the determina-tion of clenoliximab concentration were obtained imme-diately before drug administration (0 hours) and at 1, 2(end of infusion), 3, 4, 24, 48, 72, and 96 hours after thestart of the infusion. Additional blood samples were col-lected at approximately 1, 3, 4, 5, 7, and 12 weeks afterdrug administration. Blood samples (~3 mL) for phar-macodynamic evaluation were obtained at 0, 2, 24, 48,72, and 96 hours and at 2, 4, 6, 8, and 12 weeks afterthe start of drug administration.

Patient population. Patients had moderate to severerheumatoid arthritis (median of 6.7 years from initialdiagnosis). They ranged in age from 31 to 74 years(mean, 51 years) and weighed 51 to 125 kg (mean, 82.4kg). The patients were predominantly women (75%) andwhite (92%). The majority of patients (83%) were tak-ing steroids concomitantly (stable dose of at most 10mg/day prednisone for at least 4 weeks). Baselineabsolute CD4+ T-lymphocyte counts ranged from 294

to 2133 cells/µL (median, 969 cells/µL). More than 80%tested positive for serum rheumatoid factor at baseline.

Clenoliximab assay. Serum concentrations of cleno-liximab were determined with use of an electrochemi-luminescent immunoassay based on the binding of freeclenoliximab to recombinant soluble CD4. In the assay,serum samples were incubated with biotinylated solu-ble CD4, streptavidin-conjugated paramagnetic beads,and a ruthenium-labeled mouse anti-human monoclonalantibody specific for the CH3 domain of human IgG4.11

Electrochemiluminescent response was recorded withan Origen analyzer. Standard curves ranged from 100to 4250 ng/mL clenoliximab. The lower limit of quan-tification for the immunoassay was 100 ng/mL with useof a 10 µL aliquot of serum. Assay precision, bothbetween- and within-run, was within 11.0% over thecalibration range, and average bias was less than 8.0%.A positive response in the assay is indicative of aspecies that contains an antigen-combining site and anepitope of the constant region of the heavy chaindomain 3. The presence of these two features wouldsuggest that the analyte measured was primarily active,intact monoclonal antibody.

Flow cytometry. Peripheral blood lymphocyte popula-tions were analyzed with an EPICXL-MCL flow cytome-ter (Coulter Corp). In these assays, cell surface mem-brane-bound proteins in whole blood were stained with arange of different fluorescence-labeled antibody probes(two-, three-, or four-color analysis). After a 20-minuteincubation period, the red blood cells were lysed, leav-ing the antibody-coated white cells intact. Immuno-phenotyping analysis included a determination of theabsolute number of CD4+ cells on CD3+ T lymphocyteswith OKT4 (Ortho Pharmaceuticals), as well as the meanfluorescence intensity from cells identified as CD4+. Thelatter value (mean fluorescence intensity) is proportionalto the CD4 density on a CD4+ T lymphocyte.

The same lots of anti-CD4 reagent were used through-out the study so that the fluorochrome to protein ratiowas the same and so that comparison of each patient’ssample was possible. The flow cytometer was calibrateddaily with fluorochrome calibration beads provided bythe instrument manufacturer to establish color compen-sation and for determination of photomultiplier tube set-ting for detection of various fluorochromes.

The OKT4 reagent used in these analyses binds toan epitope on the CD4 molecule that is distinct fromthe epitope recognized by clenoliximab. These mea-surements were therefore not expected to be affectedby the presence of clenoliximab. This was supportedby a relatively constant OKT4 CD4 lymphocyte countbefore and after drug administration (data not shown).

Mould et al 247CLINICAL PHARMACOLOGY & THERAPEUTICSVOLUME 66, NUMBER 3

The percentage of T-cell coating by clenoliximab wasassessed by counting the CD4+ cells with use of OKT4and a second reagent, Leu3a (Becton-Dickinson),which binds to the same epitope as clenoliximab. Per-centage of T-cell coating was derived from the absolutecell counts (OKT4 and Leu3a) with use of equation 1:

%CD4 Coating = 100 – 1100 · 2 (1)

Data. A substantial number of serum samples col-lected were nonquantifiable, particularly in the low-dose groups but also in the high-dose groups at collec-tion times ≥3 weeks after infusion. Nonquantifiableserum concentration values were not represented in thedatabase. Six quantifiable serum concentrations wereremoved from the data set as outliers because these con-centration values were inconsistent with other data inthe concentration–time profiles. Concentration–timedata from one patient in the lowest dose group were notmeasurable; data for that patient were therefore notincluded in the analysis. There were 18 mean fluores-cence intensity values that were deemed to be outlierson the basis of high weighted residual values (WRES>6) after initial analysis; these were removed from thedatabase. There were 24 coating values that were equalto or close to 0 and were consequently removed; sevenadditional coating values were removed from the data-base (also very small) because of their high weightedresidual values (WRES >6). The substantial noise asso-ciated with these very small values affected the mini-mization. The final population data set consisted of 23patients with 149 serum concentrations, 146 coatingvalues, and 231 mean fluorescence intensity values.

Data were analyzed with nonlinear mixed-effectsmodeling as implemented in the computer programNONMEM (version IV, level 2.1).12 NONMEM wasselected because of the apparent nonlinear nature of thepharmacokinetics and pharmacodynamics. Simultane-ous fits of the pharmacokinetic and pharmacodynamicdata were performed. To explore relationships betweendrug concentration and dynamic markers, withoutmodel assumptions, the area under the CD4 coatingversus time curve and the areas above the CD4 cellcount and mean fluorescence intensity versus timecurve were estimated for the observation period (0-t).These areas were computed by use of a linear trapezoidmethod with appropriate correction for the baselinedynamic value.

Pharmacokinetic analysis. After the completionof the infusion in the high-dose groups, serum con-centrations described a biphasic curve, with a rela-tively short initial phase followed by a longer periodof slow decay. After the decay period, serum concen-trations rapidly fell to nonquantifiable levels. Thisbehavior is indicative of nonlinear pharmacokinetics.A two-compartment open model with Michaelis-Menten elimination (ADVAN 8, TRANS 1)13 wastherefore used to describe serum concentration data.This model is schematically represented in Fig 1,A.

Leu3a—OKT4

248 Mould et alCLINICAL PHARMACOLOGY & THERAPEUTICS

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Fig 1. Schematic representation of cascade pharmacokinetic-pharmacodynamic model of clenoliximab in patients. A, Two-compartment pharmacokinetic model with nonlinear elimi-nation from the central compartment. B, Pharmacodynamicmodel for CD4 coating. C, Pharmacodynamic model describ-ing the relationship between CD4 coating and CD4 modula-tion. MFI, Mean fluorescence intensity. Other parameters aredefined in the text.

A

B

C

The differential equations for this model are given inequation 2:

= k21 · A(2) – 1 2 – k12 · A(1) (2)

= k12 · A(1) – k21 · A(2)

In the above set of equations, A(1) represents theamount of clenoliximab in the central compartment,A(2) represents the amount of clenoliximab in theperipheral compartment, Vmax represents the maximalelimination rate, Km is the Michaelis-Menten constant,Cs is the predicted serum concentration of clenolix-imab, and k12 and k21 are the intercompartmental rateconstants. Cs is equal to A(1)/Vc, in which Vc is the vol-ume of the central compartment.

During each step in the model building process,improvements to the model were assessed by evalua-tion of the agreement between the observed and pre-dicted data, improvement in plots of the weightedresiduals versus the predicted values, and increases inthe precision of the estimated parameters. Reduction ininterindividual and random residual variability esti-mates was also considered. Reduction in the objectivefunction values was assessed with use of the log-likelihood ratio test.

Pharmacodynamic analysis. The percentage of CD4coating was determined during a 12-week period afterdrug infusion. Complete antigen coating (100%)occurred 2 hours after administration (the earliest sam-pling time). This was followed by a dose-dependentperiod of saturation of the receptor sites. This behaviorwas described by the binding equation (equation 3)given below:

= kon · (100 – bound) · Cs – koff · bound (3)

In equation 3, “bound” is the percentage of boundreceptor, kon is the association rate constant for bind-ing, Cs is the predicted concentration of clenoliximab,and koff is the dissociation rate constant. This model isschematically represented in Fig 1,B. Stoichiometrywas assumed to be 1:1. The equilibrium dissociationconstant (kd) was estimated from the koff/kon ratio anda molecular weight of 150,000 g/mol for clenoliximab.

The percentage of change in mean fluorescenceintensity after administration of clenoliximab was char-acterized by delayed onset and dose-dependent increasein the duration of response. Percentage of mean fluo-rescence intensity values decreased gradually duringthe first 24 hours. For modeling, the reduction in meanfluorescence intensity was assumed to occur sequen-

dbound—dt

dA(2)—

dt

Vmax · Cs——Km + Cs

dA(1)—

dt

tially as a result of antibody coating (ie, the effect wasdriven by CD4-bound antibody). This response couldbe attributed either to a stimulation of the degradationrate constant or to an inhibition of the effect synthesisrate constant for mean fluorescence intensity.14 In thisstudy, down-modulation was described with use of thephysiologic indirect-response inhibitory model14 withthe addition of the parameter Imax

15 and a Hill constant(fixed at γ = 2) to account for the steep relationshipbetween CD4 coating and CD4 modulation. This modelis schematically represented in Fig 1,C. The differen-tial equation (equation 4) used to describe this model isshown below:

= ksyn · 31.0 – 4 – kdeg· E(t) (4)

In this equation, E is the effect, or percentage of changefrom baseline mean fluorescence intensity, ksyn is thezero-order rate constant for the increase in receptor den-sity (effect synthesis rate constant), Imax is the maxi-mum attainable inhibition of ksyn by the binding ofclenoliximab, IC50 is the percentage of binding of CD4that results in 50% of the maximum attainable inhibi-tion of ksyn, “bound” is the predicted fraction of CD4receptors bound by clenoliximab (percentage of coat-ing), and kdeg is the first-order rate constant for thedecrease of CD4 density on the T-cell surface. Initialparameter estimates were obtained with the methodsdescribed by Dayneka et al.14 The Hill constant couldnot be estimated in the simultaneous model. Severaldifferent values for the Hill constant were tested and γwas fixed to the value that gave the best agreementbetween observed and predicted data (γ = 2).

Because of limitations in the number of terms avail-able for describing variance, a series of different mod-els for interindividual and random residual variabilitywere tested to optimize the final model. Interindividualvariability was assumed to be log-normally distributedand was fitted by use of an exponential model. Eachpiece of the model was also fitted separately to furtherinvestigate the variability in the data and the parameterstability. Several models for random residual variancewere tested for each piece, with the best variance modelbeing carried forward to the simultaneous model. Thesimultaneous model resulted in parameter estimatesthat were essentially unchanged from those obtainedduring the separate piecewise fits.

The first-order estimation method and the first-orderwith conditional estimation method both with and with-out interaction were assessed during the model devel-opment process. Models run with the first-order withconditional estimation method both with and without

Imax · bound2——(IC50

2 + bound2)dE—dt


interaction did not improve parameter precision or plotsof observed versus predicted data. Therefore the sim-pler first-order method was used for the final model.

RESULTSPharmacokinetics. The serum concentration–time pro-

file of clenoliximab was described by use of a two-compartment model with Michaelis-Menten elimination

from the central compartment. Residual variance was bestdescribed by a proportional error model. Fig 2,A, depictsan overlay plot of the typical predicted concentration val-ues over the observed data for patients in the 1 and 10mg/kg groups. Fig 3 includes log-typical predicted versuslog-observed concentration data for all patients. These datawere plotted on a log scale for visualization purposes.Generally, there was good agreement between the typical


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Fig 2. Overlay plots comparing typical predicted (open symbols)and observed (solid symbols)pharmacokinetic-pharmacodynamic–time profiles from patients who received doses of 1 mg/kg(triangles) or 10 mg/kg (circles) clenoliximab. A, Clenoliximab concentration versus time.B, CD4 coating versus time. C, CD4 modulation versus time.

A

B

C

predicted concentrations and the observed concentrationsof clenoliximab. A representative plot of observed andindividual predicted serum concentration–time data forclenoliximab from two patients (given 1 or 10 mg/kg) isshown in Fig 4,A, again confirming that these data arewell described by the proposed model.

The estimated pharmacokinetic model parameters arepresented in Table I. The Vmax in the group of patientswith rheumatoid arthritis was 1490 µg/h and the Km was1290 µg/L. The Vc (3.58 L) was approximately equal toplasma volume and was consistent with values reported

for other proteins (eg, Vincenti et al16). The pharmaco-kinetic parameters for clenoliximab were determinedwith good precision, with standard errors of the param-eter estimates <24%. Interindividual variability in theparameter estimates was fairly high, especially for Vmax(47.1%), although the terms for between-patient vari-ance might have been underestimated because of thesmall number of patients in the database. Attempts toexplain this variability by the addition of covariates tothe pharmacokinetic model were not successful becausethe number of patients studied was small.


Fig 3. Typical predicted versus observed pharmacokinetic and pharmacodynamic data from thefinal pharmacokinetic-pharmacodynamic model. Solid circles,Pharmacokinetic data (in nanogramsper milliliter); open circles,modulation data (in percent); triangles,CD4 coating data (in percent).Data were reasonably well distributed about the line of unity.

Table I. Final pharmacokinetic parameters from the simultaneous pharmacokinetic-pharmacodynamic model

Population mean (% SE)* Interindividual variability (%)†

Vmax (µg/h) 1490 (4.2) 47.1 Km (µg/L) 1290 (23.3) NEVc (L) 3.58 (8.5) 36.6 k12 (1/h) 0.0102 (5.2) 11.9 k21 (1/h) 0.00682 (20.8) 27.5 Random residual CCV variability as % CV (% SE) 8.8 (28.6)

Vmax, Maximal elimination rate; Km, Michaelis-Menten constant; NE, not evaluable; Vc, volume of the central compartment; k12 and k21, intercompartmental rateconstants; CCV, constant coefficient of variability.

*Percent standard error of the parameter estimate.†Expressed as percent coefficient of variation.

Pharmacodynamics. A reversible binding modelwas used to describe the interaction of clenoliximabwith the CD4 molecule. Random residual variabilitywas best described by an additive error model. Fig 2,B, is an overlay plot of the observed and predicted CD4coating by clenoliximab for patients in the 1 and 10mg/kg dose groups. Fig 3 includes a plot of the log-

typical predicted versus log-observed coating data forall patients. As with the concentration data, the coatingdata were generally uniformly distributed about the lineof identity although there was scatter. In general, themodel represented the coating data well, though therewas limited information at intermediate coating values.Fig 4,B, shows representative individual coating data


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Fig 4. Individual pharmacokinetic-pharmacodynamic profiles from two patients who received adose of 1 mg/kg (solid circles)or 10 mg/kg (open circles). Symbolsrepresent observed data; solidlinesrepresent individual predicted values from the final pharmacokinetic-pharmacodynamic model.A, Clenoliximab concentration versus time. B, CD4 coating versus time. C, CD4 modulation ver-sus time. D, CD4 count versus time. The lack of a trend is indicated by a dotted line through themean CD4 count.

A

B

C

D

with the individual predicted coating profiles for twopatients given 1 or 10 mg/kg clenoliximab. The esti-mated parameters for the binding portion of the phar-macodynamic model are given in Table II. Bindingparameters were determined with good precision, withthe highest standard error being 39.4%. Interindividualvariability was estimated for kon (27.7%) although,again, this value may be underestimated.

For down-modulation (change from baseline meanfluorescence intensity), an indirect-effect inhibitorymodel was used to describe the data. Random residualvariability was best described by a combined additiveand proportional error model. Fig 2,C, depicts an over-lay plot of the typical values of the predicted mean fluo-rescence intensity over the observed data for patientsin the 1 and 10 mg/kg dose groups. Fig 3 shows the log-typical predicted versus log-observed modulation datafor all patients. These data were uniformly distributedabout the line of unity. A representative plot of observedand individual predicted mean fluorescence intensityfrom two patients given 1 or 10 mg/kg is shown inFig 4, C, along with individual pharmacokinetic andcoating data for comparison.

The estimated pharmacodynamic model parametersare presented in Table III. The pharmacodynamic param-eters for clenoliximab were determined with good pre-cision. Standard errors of the parameter estimates were

<13%, with the exception of the IC50, for which thestandard error was 44%. This higher standard error waslikely a reflection of the limited information at inter-mediate coating values. Interindividual variability forImax was 2.60%. Because modulation may be causedby a number of mechanisms, both stimulatory andinhibitory in nature, an indirect-effect model that useda stimulatory effect on kout was also tested. This modelperformed equally as well as the inhibitory model (datanot shown).

DISCUSSIONIn this first trial of clenoliximab, pharmacokinetics

and pharmacodynamics were investigated after a sin-gle intravenous infusion (50 µg/kg to 15 mg/kg) topatients with moderate to severe rheumatoid arthritis.Plasma concentration–time profiles were convex, withconcentrations falling precipitously after a duration thatwas dependent on the dose administered. In the twopatients depicted in Fig 4,A, concentrations of cleno-liximab at 72 hours differed by >500-fold, although thedifference in the administered dose was tenfold. Otherproteins have been shown to display subtler nonlinear-ity as a result of saturation of a degradation mechanismor saturable binding to a pharmacologic receptor.17 Themarked nonlinearity of the pharmacokinetics of cleno-liximab may be attributed to saturable binding of the


Table II. Final CD4 coating parameters from the simultaneous pharmacokinetic-pharmacodynamic model


kon (h · µg/L)-1 0.00015 (39.4) 27.7 koff (h)-1 0.00454 (17.7) NERandom residual additive variability (%bound) (% SE) 41.1 (20.2)

kon, Association rate constant for binding; koff, dissociation rate constant; NE, not evaluable.*Percent standard error of the parameter estimate†Expressed as percent coefficient of variation.

Table III. Final CD4 modulation parameters from the simultaneous pharmacokinetic-pharmacodynamic model


Imax 0.830 (12.7) 2.60IC50 (%bound) 60 (44.0) NEksyn (%MFI/h) 7.31 (7.47) NEkdeg(1/h) 0.0775 (7.55) NEγ 2 (FIXED) NERandom residual CCV variability as %CV (% SE) 15.6 (19.9)Random residual additive variability %MFI (% SE) 5.64 (23.0)

Imax, Maximum attainable inhibition of ksyn by the binding of clenoliximab; IC50, percentage of binding of CD4 that results in 50% of the maximum attainable inhi-bition of ksyn; ksyn, zero-order rate constant for the increase in receptor density (effect synthesis rate constant); kdeg, first-order rate constant for the decrease of CD4density on the T-cell surface; γ, Hill constant; NE, not evaluable.

*Percent standard error of the parameter estimate.†Expressed as percent coefficient of variation.

antibody to the CD4 molecule and the fact that there isa very large amount of CD4 outside the blood compart-ment in well-perfused tissues. This is supported bystudies of this antibody and its IgG1 version in CD4knockout and human CD4 transgenic mice in which the

pharmacokinetics were nonlinear in the presence of theantigen and saturable uptake of the antibody in thespleen was observed.18

Clenoliximab does not affect the number of circulat-ing CD4+ T cells but rather coats and down-modulates


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Fig 5. Noncompartmental analysis of the relationships between clenoliximab exposure and variouseffects on T lymphocytes. A, Area above the CD4 count–time curve (AAC) versus the area underthe clenoliximab concentration–time curve (AUC). B, Area under the CD4 coating-time curve versusclenoliximab AUC. C, Area above the mean fluorescence intensity (MFI)–time curve versus cleno-liximab AUC. D, Area above the MFI–time curve versus the area under the CD4 coating–time curve.

A

B

C

D

the CD4 molecule in a dose- and concentration-dependent manner. Although CD4+ T cells in the cleno-liximab treatment groups were statistically significantlylower 24 hours after infusion compared with the placebogroup, they recovered by 48 hours and were not signifi-cantly different at any other time point in the study.Fig 4,D, depicts the individual CD4 counts over time fortwo individuals given 1 or 10 mg/kg clenoliximab. Fig 5,A, depicts the area above the CD4 count–time curve ver-sus the area under the serum concentration–time curve for

clenoliximab for all patients studied. No trend betweendepression of CD4 counts and drug exposure was evident.In contrast, in similar noncompartmental analyses, therelationships between coating or modulation and drugexposure, as well as between CD4 coating and CD4 mod-ulation, were clearly evident (Fig 5,B, C,and D).

Coating of the CD4 antigen by clenoliximab was evi-dent in all active treatment groups studied. The durationof saturated (100%) coating increased dramatically withincreasing doses. At 0.2 mg/kg, coating was 100% at the


Fig 6. Simulated clenoliximab serum concentrations versus time (solid lines)and CD4 modulationversus time (broken lines)for proposed multiple-dose regimens. A, 700 mg every 2 weeks for 4weeks. B, 350 mg every week for 4 weeks. C, 150 mg every week for 4 weeks. Administration ofhigher doses less frequently is expected to have effects on CD4 modulation similar to administra-tion of lower doses more frequently.

A

B

C

end of the infusion (2 hours) but not observed at 24 hoursor beyond. At doses of 5 mg/kg, coating persisted for atleast 1 week in all patients. There was a trend towardlonger CD4 coating duration as the dose increased fur-ther. Coating returned to baseline in a time frame thatwas similar to the time that serum clenoliximab concen-trations became nonquantifiable. As expected from thehigh affinity of the anti-CD4 antibody and the smallamount of CD4 in peripheral blood, very low concentra-tions of clenoliximab caused complete antigen coating.

The pharmacokinetic-pharmacodynamic model usedto describe the coating data included terms for the first-order association and dissociation of a one-to-one com-plex of clenoliximab and CD4. The kd estimated fromthe model parameters was 0.2 nmol/L, a value that isconsistent with the characterization of clenoliximab-CD4 interaction in vitro. Models accounting for recep-tor synthesis and degradation (bound or free) weretested, but parameter precision and model statistics sug-gested these models were unnecessarily complex forthe data available. The coating database consistedlargely of values that were less than 10% and greaterthan 90% bound. In part because of the sparse bloodsampling, these observations were nearly discontinu-ous over time. The population approach used in thisanalysis permitted characterization of the CD4-coatingtime course despite these limitations.

Down-modulation of CD4 was evident in all activetreatment groups studied. Consistent with the indirectnature of this response, despite high serum clenolix-imab concentrations during the infusion, mean fluores-cence intensity did not decrease until at least 24 hoursafter infusion. The duration over which CD4 cell sur-face density was depressed increased with increasingdoses. Mean fluorescence intensity was decreased to20% to 30% of baseline for a duration as little as 24hours at 1 mg/kg to a duration that exceeded 400 hoursat 10 mg/kg. For some patients, even as serum concen-trations approached nonquantifiable levels and CD4coating returned to baseline, effects on mean fluores-cence intensity apparently persisted (data not shown).

The indirect-response inhibitory model used todescribe CD4 modulation suggested that the half-maximal effect on CD4 receptor density occurred at60% coating, although coating values in this range werenot well represented in this data set. The value of ksyn,7.31% mean fluorescence intensity per hour, suggestedthat even after clenoliximab had been cleared and thepercentage of coating had returned to baseline, a sig-nificant interval of time would be required before CD4surface density would recover. The estimated value forImax—0.83—suggests that clenoliximab is capable of

reducing normal CD4 density by 83%. It is interestingthat clenoliximab does not cause decreases in CD4receptor density below this level. It is plausible thatreceptor-receptor interactions are involved in down-modulation and that below 20% residual density the fre-quency of these interactions is reduced.

The pharmacokinetic-pharmacodynamic modeldeveloped for this agent is broadly applicable to anti-bodies that target cell-bound lymphocyte markers andfor which coating and modulation are expected to berelated to clinical response.19,20Despite the dramati-cally nonlinear pharmacokinetics of these agents, thetime course of pharmacodynamic effect can bedescribed and the multiple-dose effects anticipated withuse of this approach.

The pharmacokinetic-pharmacodynamic profile ofclenoliximab supports the hypothesis that less-frequentadministration of higher doses would have effects onCD4 coating and modulation similar to administrationof lower doses more frequently. This is in contrast todosing strategies, proposed for related therapies, thatsuggest that frequent intermittent administration mayoptimize clinical benefit.21 Multiple-dose trials of cleno-liximab are evaluating the pharmacokinetics and phar-macodynamics after administration of 700 mg everyother week, 350 mg once weekly, and 150 mg onceweekly, each for a 1-month duration. Plots of simulatedconcentration–time profiles and CD4 modulation–timeprofiles with these regimens are displayed in Fig 6.These simulations are based on the single-dose data andpharmacokinetic-pharmacodynamic parameter esti-mates from an individual patient in this study. Thehigher dose regimens both result in sustained maximaleffects on CD4 receptor density, whereas the low-dosesimulation suggests that there will likely be periods oftime in which the CD4 cell surface density will recoverat least partially between doses.

Relationships between CD4 coating, down-modula-tion, and clinical response in rheumatoid arthritis war-rant further study. In phase II trials of the IgG1 isotypeof clenoliximab (CE9.1, Levy et al22), twice-weeklydoses of 80 mg for 1 month resulted in clinical responsethat was sustained well beyond the cessation of dos-ing. The rate that patients flared was inversely relatedto dose and the time to flare after 1 month of treatmentexceeded an additional month. An understanding of thetime course of effects that clenoliximab has on the stateof the T cell provides a solid foundation from whichto explore relationships between regimen and induc-tion of long-lasting immunologic tolerance and toassess the impact of tolerance on the treatment ofrheumatoid arthritis.


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