Scribd Logo
Upload

Welcome to Scribd!

  • MMF

    Uploaded by

    Roby Syah Putra Firmansyah
    14 views15 pages
    jurnal ocan

    Copyright

    © © All Rights Reserved

    Available Formats

    PDF or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    Report this Document
    jurnal ocan

    Copyright:

    © All Rights Reserved
    Download as PDF or read online from Scribd
    Flag for inappropriate content
    14 views15 pages

    MMF

    Uploaded by

    Roby Syah Putra Firmansyah
    jurnal ocan

    Copyright:

    © All Rights Reserved
    Download as PDF or read online from Scribd
    Flag for inappropriate content
    of 15
    ‘Transplant Immunology ELSEVIER ‘Transplant Immunology 10 2002) 1-14 wwwchevierconTocatem Novel assays of multiple lymphocyte functions in whole blood measure New mechanisms of action of mycophenolate mofetil in vivo Markus J. Barten, Teun van Gelder, Jan F. Gummert, Randi Shorthouse, Randall E. Morris* Department of Cardiothoracic Sages, Transplantation Inumusolo Stanford University Medical School, Stanfond, (CA 94305-5407, USA Received 19 February 200; aoceped 17 May 2001 Abstract ‘The antiproliferative effects of MMF are believed to be the mechanism of its immunosuppressive action. We further investigated the mechanisms of action by assessing the pharmacodynamics (PD) of MMF in treated animals using whole blood ‘assays not only of lymphocyte proliferation but also of activation. Invitro, different MPA concentrations were added to rat whole blood. In vivo, Lewis rats were treated with single doses of 5, 10 or 20 mg/kg MME (n= 6 rats /dose group). Blood was obtained before and at different times after drug administration. For both in vitro and in vivo studies, different mitogens with calcium-dependent (TCR) of -independent (co-stimulatory) pathways of lymphocyte activation were added to the blood for stimulation. Proliferation was measured by [ HITGR incorporation and by flow cytometric detection of DNA content. Activation was measured by changes in T cell surface expression of CD25, CD134, CD71, CDIa and CDS4. In vitro and in vivo studies showed a dose-dependent inhibition by MPA and MME, respectively, of lymphocyte proliferation and surface antigen expression, We observed high correlations between MME PD effects over time with both MMF dose and MPA plasma concentrations in vivo, We show that MME, apart from its antiproliferative effect, induced a dose-dependent suppression of calcium-dependent and independent stimulated expression of important lymphocyte cell surface antigens, These data suggest that the ex vivo assessment fof immune function in whole blood can uncover new mechanisms of MMF action. Our results demonstrated that the ‘measurement of the PD is a means to assess the functional effects of MMF after its administration in vivo. © 2002 Elsevier Science B.V. All rights reserved. “Kewonds: Whole blood; Lymphocyte functions; Mycophenolate mofetil; Mechanisms of action 1. Introduction intracellular guanosine levels observed in many in vitro studies with purified lymphocytes in different species After absorption from the gut, MMF is rapidly de- e e a esterified to its active metabolite MPA [1]. MPA in- hibits the key enzyme inosine monophosphate dehydro- genase (IMPDH) in the de novo pathway of guanine nucleotide biosynthesis, which is a requisite comple- ment to the salvage pathway for T and B cells to undergo mitogenic transformation [2,3]. The antiprolif- erative effect of MPA is explained by the reduction of * Cornesponding author, Tel: + 1-650-723-5771; fax: +1-680 3846, E-mail addves:rem@stanford.edu (RE. Mortis). (9653274 /02/$- see front matter © 2012 Elsevier Science BLV. All PI: $0966-3278(01}00081-7 [4-6]. Recently, it was shown that MPA also decreases ATP pools in mitogen-stimulated T lymphocytes [7] Earlier studies have shown antiproliferative effects of MMF or MPA in vivo [8.9 To learn more about the immunosuppressive mecha- nisms of action of MMF administered in vivo, we used whole blood cultures instead of purified. peripheral blood Iymphocytes. The use of whole blood better reflects the environment in which the drug is acting in vivo than using purified peripheral lymphocytes. The use of whole blood overcomes the loss of drug-target ‘binding, the alteration of drug distribution among i reserved, 2 ML. Barten eta. / Transplant Immunol 10 (2002) 1-14 plasma proteins and red blood cells, and the selective Joss of cells, which is caused by isolation of immune cells. Additionally, the whole blood technique is more rapid and requires smaller sample volumes than meth- ‘ods that rely on purified lymphocytes [10,11]. In earlier studies, the PD effect of immunosuppres- sive drugs in Con A-stimulated whole blood was mea- sured by [HITAR incorporation [12-14]. But since the immune function is also determined by direct cell-cell interaction through surface antigens with adhesive, co- stimulatory or inhibitory functions [15], we exploited the specificity, sensitivity of flow cytometry to de- termine PD of MPA over time in MPA treated rats to assess proliferation and the expression of T cell surface antigens, CD25 and CD134. We observed in this study a high correlation between PD and PK [16]. Next, we used these whole blood assays to assess PD of MPA treated heart transplanted rats and found a high corre- lation between PD and histologic severity of rejection un. ‘The whole blood assays used in these studies were fed: (1) blood was stimulated by one mitogen (Con A); 2) only single- or two-color flow cytometry was used; and (3) only two cell surface antigens were de- tected, We optimized our assays to reduce these limita- tions, using mitogens which signal through calcium- dependent (Con A, PMA +IONO) or calcium-inde- pendent (PMA + anti-CD28) pathways, using a thre color flow cytometry technique and measured the ex- pression of many different cell surface receptors (CDila, CD25, CD54, CD71 and CD134) with impor- tant functions in the immune response [18] Although our previous work investigated the PD effects of MPA the present study used MMF since this drug is approved for use in clinical transplantation. We «designed this study to uncover the mechanism of action of MMF and to determine the correlation between MMF PD and PK. To achieve these goals, we used our optimized whole blood assays to measure lymphocyte proliferation by ["HITAR incorporation and flow cyto- metric detection of PCNA expression and DNA con- tent. The cyclin PCNA is an ausiliary protein necessary for DNA polymerase and is maximaily expressed in the phase of the cell cycle [19] We also measured the expression of different T lymphocyte surface antigens with three-color flow cyto- metric detection. CD25, a-chain of the IL-2 receptor, is expressed in the early phase after T-cell activation. The clonal proliferation of activated T cells depends upon the expression of this receptor and resting lymphocytes do not express CD25 [20]. Expression of CD134 (OX40), ‘@ member of the nerve growth factor/tumor necrosis superfamily (NGF/TNP), is restricted to expression on activated CD4 lymphoblasts [21] and is involved in adhesion of activated T cells to vascular endothelial cells [22], Expression of CD71, the transferrin receptor, must be increased to internalize the iron required for DNA synthesis and proliferation [23]. CDIla (LFA-1 chain), is the counter-receptor for CDS4 (ICAM-1) and both are bidirectionally expressed on the cell sur- face (ICAM-1 on APC bind LFA-1 on T cells and LFAc1 on APC bind ICAM-1 on T cells) [24]. LFA-I is the ligand for ICAM-1 and provides a co-stimulatory signal for T-cell receptor-medited activation of resting T cells [25]. ‘The mechanisms of action and effects of immuno- suppressive drugs on immune cells have been primarily defined from in vitro studies. The effects of MPA in vitro on the expression of CD25 are controversial MPA has been reported either to have no effect [26,27] of to inhibit the expression of CD25 in antigen- or mitogen-activated purified lymphocytes [5]. In MLR cultures, MPA inhibits CD25 and CD71 on allostimu- lated T-cell subsets [28]. Earlier studies showed that MPA inhibits expression of adhesion molecules ex- pressed on endothelial cells or monocytes [8,29], but recent studies do not show MPA decreases glycosyla- tion in Con A-stimulated PBL (30). ‘The inhibition of lymphocyte proliferation is pre- sumed to be the most important immunosuppressive effect of MMF. However, our data showed that MMF inhibited the expression of many T cell surface activa- tion antigens after stimulation of whole blood with different mitogens signaling through calcium-depen- dent and -independent pathways of T-cell activation. These new mechanisms of action of MMF may con- tribute significantly to its overall efficacy. In addition, we also showed a significant correlation between PD and PK of MME. 2. Objective Our primary objectives were to exploit improved whole blood assays of T cell functions to investigate the mechanisms of immunosuppressive action of MPA in vitto and the PD effects of MMF administered in vivo. More specifically, we used three-color flow cytometry to quantitate the suppression by MMF of T-cell acti tion caused by three distinct mitogenic stimuli. T-Cell activation was determined by quantitating proliferation and suppression of expression of cell surface growth factor receptors (CD25, CD71) and adhesion and co- stimulatory molecules (CD54, CD134 and CD12). Additionally, our goal was to determine the dose- dependent relationships between PD and PK after rats were treated with MMF. MAL Bare et al / Transplant Immunology 10 (2002) 1-14 3. Materials and methods 3.1. Animals Adult male Lewis (LEW, RT'I/CrIBR, viral anti- body-free, Charles River Laboratories, Wilmington, MA) rats weighing 335-370 g were housed in polycar- bonate microisolation cages. Standard diet and tap water were provided ad libitum and animals were accli- mated under a 12-h light/dark eycle for 2 weeks before the study began. The study was approved by the institu- tional animal care and use committee. The animals received humane care in compliance with the ‘Princi- ples of Laboratory Animal Care’, formulated by the ‘National Society for Medical Research, and the ‘Guide for the Care and Use of Laboratory Animals’, prepared by the National Institutes of Health (NIH Publication No. 80-123, revised 1985). 3.2, Reagents Culture medium (CM) was prepared using RPMI 1640 supplemented with 100 U/ml of penicillin, 100 ug/ml streptomycin and .-glutamine (2 mM), all ob- tained from Sigma Chemical Co. (St. Louis, MO). Con A (Vector Laboratories, Inc., Burlingame, CA) was diluted in CM, sterile filtered (2 um, Applied Scientific, San Francisco, CA) and stored at ~70°C. PMA (Sigma Chemical Co,, St. Louis, MO) was dissolved in dimethyl sulfoxide (DMSO, Sigma Chemical Co,, St. Louis, MO) at a stock solution of 100 ug/ml and IONO (Sigma Chemical Co., St. Louis, MO) was dissolved in DMSO at a stock solution of 500 wg/ml. Both PMA and IONO were stored at ~20°C and working solutions of PMA were freshly prepared in phosphate buffered saline (PBS, Coulter, Miami, FL) and solutions of IONO were freshly prepared in CM. Purified mouse monoclonal antibody (mAb) anti-CD28 (Clone, 43319, Pharmingen, San Diego, CA) was diluted in CM before ‘each experiment, [HITAR with a specific activity of 6.7 Ci/mmol was purchased from New England Nuclear (Boston, MA). Vehicle was prepared by dissolving sodium carboxymethyleellulose (City Chemical Corp., NY, viscosity of 1300-2200 centipoises at 25°C in a 1% deionized water solution) in 0.9% benzyl alcohol, 0.4% polysorbate 80, 0.9% sodium chloride in water to pro- duce a 0.2% solution and was stored at room tempera- PE- and biotin-labelled mAb (anti-CD2: anti-CD71, anti-CD134, anti-Pan-T cell (Ox52), anti CDila, anti-CDS4 and the mouse isotype controls IgG,,, IgG,,, IgG, were purchased from Pharmingen WE con CO PMALCO2E I PMASIONO Ic, ‘ @ = 100 a 5 soo. 2 1000 7 ah hut « PHLTaR GM CD25 CIM CDT Coit Imax » a 2 : I il PHETER SGM CD2s CDI COM Colla cDse Fig. 1. Elect of MPA aced in vitro t9 rat blood on inhibition of Con (A, PMA+ CD28 and PMA-+1ONO-stimulated. lymphocyte proliferation and setivation measured by witle blood assays. Hep srinized rat blood was either untreated or treated with MPA to produce diferent blood concentrations (0.25, 05, 1,5 10 and 100 uM. (048, 0.16, 032, 16, 32 and 32 mg/D) before 1:10 dilution in supplemented RPMI and distribution 10 99-vell microplates. Com A, PMA + CD2S or PMA + IONO were aed to all wells except unst- ‘mulated contol wells Incorporation of [ HITUR was measured du ing the last 16-28 h of 48 h (PMA + CD28 and PMA + IONO says) o7 96h cultures (Con A. assy) and five replicate wells of lunteated controls and each MPA concentration were analyzed Aer 2 days (PMA + CD28 and PMA +IONO assays) oF 3 days (Con A assay) of culture, she contents of si microplates wells were pooled for fow eytometric determination ofthe percentage of PCNA Positive cells in $/G. M-phase ofthe cell el, and for percentage of cells positive for surface antigens (CD25, CDI34 CD71 CDI la and (CD54), All data from three separate experiments for each of the three diferent mitogensstimulted assays are shown and were fited in ta sigmoidal PD model, (San Diego, CA), Streptavidin PECyS and anti-PCNA mAb (clone PC 10) were purchased from Dako Cor- poration (Carpinteria, CA). RNAse, propidium iodide (PD, saponin and sodium azide were purchased from Sigma Chemical (St. Louis, MO), Red blood cell (RBC) Iysis buffer was made daily by dissolving 8.29 g NH,CL, 1g KHCO,, 37.2 mg Nay-EDTA (J.T. Baker Chemical Corp, Phillipsburg, NI) in 1 1 of deionized H,0 (pH 7.2). Permeabilizing buffer contained 1% heat ina vated fetal calf serum (FCS, Hyclone, Logan, UT), 4 MI. Barton ta. / Transplant Immunology 10 (2002) 1-14 MME Dose (nag) | + Smelke 2 lOmgke @ 20meke| 4 o Fig. 2. MPA levels in rat plasma were determined by HPLC. Rats were treated with a single dose of 5, 10 or 20 mg/kg MME (n = 6/dose group). The MPA concentrations were calculated using cll bration curves and were corrected sing the internal standard 0.1% saponin and 0.1% sodium azide in PBS. Formaldehyde solution was purchased from Fluka Chemie AG, Switzerland, and methanol was purchased from Fischer Scientific, CA. 3.3. Treatment of blood in vitro with MPA Lewis rats were anesthetized with ether (J.T. Bafer, Philishurg, NJ). We have shown that ether anesthesia does not effect Con A stimulated lymphocyte prolifera- tion in rat whole blood assays [31]. ‘After laparotomy, blood was drawn from the abdomi- nal aorta and the animals were killed. The blood was anticoagulated with 100 U/ml sodium heparin (Fujisawa, Deerfield, IL). MPA (Sigma Chemical, St. Louis, MO) was dissolved in undiluted DMSO as a stock solution of 20 mM. All further drug solutions ‘were made in CM. The highest concentration of DMSO. in blood was 0.05%, which did not affect lymphocyte function (data not shown). Ten microliters of diluted MPA solution or 10 jul of CM alone was added to the blood to get the desired blood concentrations of 0, 0.25, 05,1, 5, 10 and 100 wM (0, 0.08, 0.16, 032, 1.6, 3.2 an 32 mg/. These mixtures were incubated for 30 min at 37°C in a 5% CO, — air incubator before processed as described for the whole blood assay. Three consecutive ‘experiments for each mitogen assay with different a oon different days were done under the same 34, Whole blood mitogen-stimulated lymphocyte function assay One hundred and fifty microliters of diluted blood were added into wells of flat bottomed 96-well tissue culture microtiter plates (Becton Dickenson Labware, Franklin Lakes, NJ). To each wel, 50 yl of Con A, or 25 yl of PMA plus 25 yal of anti-CD28, 25 wl of PMA plus 25 il of IONO or 50 yl of CM (unstimulated) were added to reach the final volume of 200 pl. The final ditution of blood in the well was 1:10. The final concentration in 200-1 blood cultures for Con A was 15 ug/ml, for PMA 50 ng/ml, for anti-CD28 10 jug /ml and for JONO 125 ng/ml. Cultures were analyzed by CHITAR incorporation on day 4 or by flow cytometry on day 3 after Con A-stimulation. The PMA + CD28 or PMA+IONO assay cultures were analyzed after 2 days by PHITAR incorporation or flow cytometry. All cultures were incubated for 2~4 days at 37°C in a humidified 5% CO, air-water jacketed incubator. 35. Assessment of mphocyte proliferation by [*H/TAR incorporation ‘Ten microliters of PHITAR (1 Ci) diluted in REMI were added to each of five replicate wells during the last 16-24 h of incubation. Cells were collected onto glass fiber filters and washed with PBS using a TOMTEC multichannel cell harvester (Wallac Oy, ‘Turku, Finland). Methanol was used in the last wash to bleach the sample to reduce the color quenching effect (32]. Bach filtermat was placed in a sample bag and filled with 4.5 ml OptiScint “HiSafe’ scintillation fuid (FSA Laboratory Supplies, Loughborough, England). Beta emissions from each fitermat were quantified as counts per minute (CPM) in a Wallac 1450 Micto Beta Plus liquid scintillation counter (Wallac Oy, Turku, Finland). The background activity defined as CPM in ‘empty wells was below 100 CPM. The mean CPM of five replicates was calculated and the coefficient of variation was not greater than 20%. 3.6. Analysis of lymphocyte function by flow cytometry After different durations of incubation, the contents from seven wells were pooled (1400 yl). Eight hundred microliters were used for detection of DNA content and 600 jul used to detect surface antigens. All samples were analyzed with an Epics XL-MCL flow cytometer equipped with an air-cooled argon laser (488 nm) using system TI coulter software (Coulter Corporation, Mi- ami, FL), Emitted light of the fluorochromes was col- lected with bandpass filters through 525 nm (FITO), 575 nm (PE) and 675 nm (PECyS and PD. Unstimu- lated blood cultures were used as a negative control Specifity controls included replacement of primary ‘mAbs with isotype mouse immunoglobulins. 3.6.1. PCNA / DNA content We performed a simultaneous analysis expression with DNA content, the combination of FITC-labeled anti-PCNA mAb and PI to determine cells, which were in S + G;/M-phase of the cell cycle. For simplicity, these cells are denoted as S/G,M posi- tive, Bight milliliters of lysis buffer was mixed with 800 ML. Bartn ta. / Transplant Irumunology 10 (2002) 1-14 5 iil cultured blood and RBCs were lysed for 10 min at room temperature. Leukocytes were pelleted (200g, 10 min) and again washed with 2 ml PBS. Leukocytes were fixed by adding 1 ml PBS containing 1% (v/v) formaldehyde and then kept on ice for 5 min, After washing with 2 ml PBS (200 xg, 5 min), cells were resuspended with 2-ml ice cold 100% methanol and stored for at least 10 min at 4°C. Prior to staining, cells were washed with 2 ml PBS (200 xg, 5 min) to remove methanol. The cell pellet was then resuspended in a staining mixture containing 107 jul permeabilizing buf- fer, 10 ul RNAse A (stock solution 100 mg/ml in 1,0), 5 ul PI (stock solution 1 mg/ml in HO and 2.5 ConA pl anti-PCNA FITC mAb. Cells were incubated in the staining mixture overnight at room temperature. After ‘washing with 2 ml PBS, leukocytes were pelleted (200 Xg, 5 min) and resuspended in 500 jul PBS containi 10 ug/ml PI. Total lymphocytes were collected using forward and side scatter and displayed as two-color DNA/PCNA dot plots as PCNA-positive cells with S/G,M-phase DNA content. Five thousand events were analyzed per saraple. 3.6.2. Expression of mitogen-simulated cell surface antigens We performed a two- and three-color analysis for PMA+cD28 wo, cos PMA+CD28 Fig. 3. Correlations between MPA plasma conceat tos in rats and init and activation measured by whole blood assays. Rats were treated wit sof Con A- or PMA + CD28timulated bmaphoeyte protiferation 10 or 20 mg/kg of MMF (n = 6/dose group). Blood was sampled before treatment (pretreatment value) and 6, 12, 24, 48 and 60 h after drug administration. MPA olasma concentrations were determined by HPLC, and each concentration is shown. All PD resls ae expressed as percent inhibitions normalized ta the pre-treatment value. Reslts were fitted in 8 oidal PD model and examples for CD25 (ab), CD7 (ed), CD11 Ce.) and CDS4 (gare shown. 6 MA Barer etal. / Transplant lmmuumolgy 10 (2002) 1-14 detection of CD25, CD134, CD71, CDila and CDS4 on T lymphocytes. To each 200 yl of diluted, incubated blood from pooled cultures, 100 jul of mAb (1:100 diluted in 3% FCS, 0.1% NaN, in PBS) was added. After incubation for 30 min in the dark at room tem- perature, samples containing biotin labeled mAb were washed with 2 ml PBS (200xg, 5 min). To these samples 20 yl of streptavidin PECyS (1:20 dilution in 3% FCS, 0.1% NaN; in PBS) was added and then incubated for 15 min in the dark at room temperature. Following staining, 4 m! lysis buffer was added to each tube and RBCs were lysed at room temperature for 10 min. Leukocytes were washed with 2 ml PBS and then pelleted (200 xg, $ min). Prior to analysis, leukocytes ‘were resuspended in 500 jl PBS containing 0.5% (v/v) formaldehyde. Forward and side scatter was used to differentiate viable lymphocytes from debris, dead cells and other leukocytes as previously described [33], Data were displayed as histograms and as dual color dot plots to measure percent positive cells. Five thousand events of each simple were analyzed 3.7. MPA pharmacokinetics Drug quantification in rat plasma was done by gradi- ent HPLC as described previously [34]. To 50 pl of plasma, phenolphtalein glucuronic acid (Sigma Chemi cals, St. Louis, MO) in acetonitrile (single-step extrac- tion) was added as an internal standard. The mobile phase was comprised of acetonitrile and sulfuric acid adjusted to pH 40. A sphereclone 5 um ODS (2), 200 x 4.6 mm column from Phenomenex. (Torrance, CA) was used. Samples were analyzed on a binary HPLC-system equipped with an autosampler (Shimadzu Kyoto, Japan). The MPA concentrations were calcu- lated using calibration curves and were corrected using the internal standard. The area under the plasma con- centration-time curve (AUC), ,) was calculated by the trapezoidal rule using the MPA plasma concentra- tions 6, 12 and 24 h after dosing. PK data from studies with MPA in rats (34] showed that this ‘abbreviated’ AUC js, on average, 85% lower than the AUC calcu lated from MPA plasma concentrations analyzed after 05, 1,2, 6, 12 and 24 b. 3.8 Treatment of rats with MMF in vivo MMF was suspended in vehicle and administered by gavage. Eight hundred microliters of blood was obtained at scheduled timepoints by retro-orbital bleeding under ether anesthesia, The blood was antico- agulated with 100 U sodium heparin/ml and hema- tocrits and white blood cell counts determined with a Coulter Microdiff 11 cell counter (Software 4C® plus, Coulter, Miami, FL), The blood was cultured according con A Clpmaveo2s 4 IC @ i Bo ey | | j eo oo oe oe 7 PHETéR SiGM —CN2s CDI «CDT CDs CDs4 190 Inne ® Tl PHETERSGM Cb2s CDI CDT CDlla COs Fig. 4. Fity peroeot inhibition (IC, a) and maximal effect Uys) of MME on ex vito Con A- or PMA + CD2§timulated rat blood afer treatment of rats with a single dase of 5, 10 or 20 mg/kg MMF: Blood was sampled before dosing (0'h) and 6, 12,23, 48 and 60 hk ater dosing and further processed as desribed in Fig. 1. Results were ited in sigmoidal PD mode to the protocol for the whole blood mitogen-stimulated lymphocyte function assay. The remaining plasma was frozen for HPLC-analysis, For each timepoint, (HJTAR incorporation and expression of cell surface antigens (CD25, CD134, CD71, CDila and CD54) and DNA. content were determined. Each dose level group and vehicle control group consisted of six animals. Rats were administered a single dose of 5, 10 or 20 mg/kg MMF. The animals were bled at time 0 (before dosing) and 6, 12, 24, 48 and 60 h after dosing, 3.9. Calculation of MPA pharmacodynamics The inhibitory effect of MPA was expressed as per cent inhibition of [HITAR incorporation or inhibition Of expression of lymphocyte function analyzed by flow cytometry and calculated as follows: 1 —(reatment /pre-treatment)) 100, ML Barer eta, / Transplant Immunology 10 2002) 1-14 “Pre-treatment” represents the results obtained from stimulated blood without addition of drugs. “Treatment” represents the results from stimulated blood containing MPA or blood from rats treated with MMF. MPA. blood concentrations (in vitro experiments) or MPA. plasma concentrations (in vivo experiments), which produced @ 50% inhibition (IC) of the maximum inhibition, were calculated after fitting the concentra- tion-effect curves in an Jyq, sigmoidal pharmacody- namic model using WinNonlin Software version 1.1 (Scientific Consulting, Inc., Cary, NC). Analogous to the calculation of the AUCy.., , the area under the pharmacodynamic effect time-curve (UE. ) Was calculated using the trapezdoidal rule: AUE 24) =[(Eqy +o n)/2X 6] F1CE,y + Bi2y)/2 61 + [CE ys + Ena y)/2 12) ‘and expressed the inhibitory effect of MPA on Iympho- cyte function over 24h after dosing. MMF Dose | Vehicle * Smgke S10 mgikg © 20 marx 3.10, Statistics All data are expressed as mean + standard error of mean (S.E.M). For statistical analysis SPSS version 8.0 (SPSS Corp., Birmingham, AL) and SigmaStat version 2.0 Uandel Scientific) were used. The two-tailed Stu- dent's t-test was used to estimate the levels of signifi cance for the differences between two groups with ‘equal variance. The Kruskal-Wallis one way analysis of variance on ranks (ANOVA on ranks) with the Stu- dent-Newman-Keuls (SNK) post-hoc test was used to estimate the levels of significance for the differences between the two groups. A P-value of tess than 0.05 ‘was considered significant. 4. Results, 4.1. MPA potency and inhibition of lmphocyte functions in vitro MPA was added to whole blood to produce six "MMF Doxe OVehicle * Smake A 10mgke © 20mpkg | ConA PMA+CD28 ConA PMA+CD28 Pa pra | — ] Bg, te 3) 140 cos 140 5 iol 2-0 a0 3 100] @ Sho eH =|) \ i | & Fo. Hi | #40} \ if 40. a \ A | | zo. ° TEE HR bo Toe wo “ eeeaH “le gl, wom Mo) ye ae Bl tool i aby 3) 0] oppo oof = 3) so 7 || ar 60 i] | wo: VA/ \ Ho j, ay 7, | Ea a Yd . | go ° o 8 BMH Hy ime ater rome (8) time ater ween) cteaaw 24 48 60 cree Alp 9 6 2 ‘ie ae renter) Time afer oust h) Fig, MMF PD values aftr rats were treated with a single dose 5, 10 o 20 mg/kg of MMF. For each dose level group, blood samples were en before dosing (pre-treatment) and 6, 12,24, 48 and 60 after dosing, PD values were derived from assys in which heparinized, diluted G10) whole blood was stimulated with 15 ug/ml Con A and 50 ng/ml PMA plus 10 ng/ml CD28. Blood sas cultured for 2 days after PMA+CD2stimuletion end 3 days (ow cytometric detection) or 4 days (HITUR incorporation) when Coa A was used as mitogen. Incorporation of [ H]TAR was measured of fhe replicate wells for each tine point and for each rat (a). The contents of six microplate wells cach timepoint for each rat were pooed fr flow cytometric determination ofthe percentage of PCNA positive ells with S/G.M DNA (S/G;M) {Gad and percentage of cells positive for cll sue antigens CD28, CD134, CD71, CDI La and CDS4 (e-n). All PD resis are expressed as percent inhibitions ofeach rat's pre-treatment assy values, 8 MI. Bart eta. /Transplansirmunolegy 10 (2002) 1-14 MMF Dose OVehicle * Smelke A 10meke @ 20meke Coma PMA+CD28 3 vo wo bi | 4 bs is fr Oy, i [3] \/ ; a | a1 ¥ ° ° Bs = Peas CoRHeE mo 3] 120 “i} 100. 1 2 6 of preven va Fig 5. (Comte, different blood concentrations: 0.25; 0.5; 1; 5; 10; and 100 4M (0.08, 0.16, 0.32, 1.6, 3.2 and 32 mg/)) and allowed to equilibrate for 30 min at 37°C. Blood was then diuted and stimulated with Con A, PMA + CD28 or PMA + IONO. We did not measure CDila or CD54 expression after PMA + IONO-stimulation, because of the lack of adhesion molecule expression following stimulation with this mitogen (35) There were clear concentration-lependent inhibi- tions of [HJTAR incorporation, inhibition of percent S/G,M lymphocytes and percent lymphocytes express- ing surface antigens CD25, CD134, CD71, CDila and (CD54 after Con A- (7? = 0.97-1.0), PMA + CD28 (7? 95-10) or PMA + 1ONO-stimulation (7? = 0.93-0.99), MPA blood concentrations producing 50% of the ‘maximum inhibition (IC), mean + S.E.M) of Iympho- cyte function were higher after Con A-stimulation com- pared to the potencies after PMA + CD28- or PMA + IONO-stimulation; except for the potency of MPA on inhibition of CD71 expression, which was similar re- gardless of the mitogen used (Fig. 1a). Lymphocyte Proliferation measured by [HITAR incorporation showed lower ICs) values than lymphocyte prolifera- tion measured by S/G,M cells in each mitogen assay (Fig. 1a). In the Con A assay, the ICq’s of MPA for flow cytometric detection of S/G,M positive Iympho- oytes and expression of cell surface antigens were simi- lar, Higher IC values were observed for CD54 expres- sion after PMA + CD28-stimulation and for CD25 ex- pression after PMA + IONO-stimulation compared to the other IC, values of lymphocyte function in each assay (Fig. 1a), There was a high correlation between MPA concentrations and the magnitudes of inhibition of all lymphocyte functions for different mitogens (7? > 0.93). ‘The calculated maximal inhibition (Iy,,, mean + S.E.M) of lymphocyte proliferation by MPA measured by [HITAR incorporation was 100% regardless of the mitogen used. Inhibition of lymphocyte proliferation, as measured by S/G:M positive cells, was similar in each mitogen assay, but slightly less complete than using [HITR (Fig. 1b). Apart from its antiprolifera- tive effect, MPA inhibited cell surface antigen expres- sion for all surface antigens studied, but to a varying degree depending on the mitogen used (Fig. 1b) 4.2, Relationships among PK different MMF dose levels Within each dose group, inter-rat variations in MPA plasma concentrations were small. The PK profiles were distinct for each dose group demonstrating a close relationship between dose level and MPA plasma con- centration (Fig. 2). Twenty-four hours after treatment with 5 mg/kg, the MPA plasma concentration was below the detection limit, whereas MPA was still de- tectable in plasma 60 h after dosing with 20 mg/kg MMF. The area under the plasma MPA concentration-time curves (AUCy. > ,) after adminis- tration of a single dose MMF was dose related and for 5 mg/kg = 1442 mg h/l, 10 mg/kg = 28 +2 mg h/I and 20 mg/kg = 55 4 mg h/I. The AUC values were statistically different among all dose levels (ANOVA, SNK, P< 0.05). 4.3. MPA potency and maximal inhibition of lymphocyte function in vivo After showing that MPA, added to whole blood in vitro, inhibited both lymphocyte proliferation and ex- pression of cell surface antigens, we investigated the pharmacodynamic effects of single oral treatment of ifferent dose levels of MMF administered to normal M, Barten eal. / Transplant Immunology 10 (2002) 1-14 ° wn 68% j 25% h Ww Le Relative number of cells cD25 Fig. 6, Histograms show analysis of Con A-induced CD25 expression before treatment (0h) and 6 12,24 48 and 60 after treatment with 20 mg/kg of MMF. At cach timepoint blood was drawo, diluted and stimulated. Cultured blood (200 yD was stained with mAb to perform a three-color flow eytomettie analysis. Aer 30 min of incubation RBCs were bsed, lymphocytes washed and pelleted. Before analysis cells were resuspended in 05% formaldehyde, Forward and side Satter was used to detect able cell and 5000 events of each sample were analyzed LEW rats in vivo. Blood was sampled before drug clear concentration-dependent inhibitions of percent treatment (pre-treatment) and at five successive times expression of CD25, CD71, CDi1a and CD54 after after treatment. We used Con A and PMA + CD28 as Con A- or PMA + CD28-stimulation (Fig. 3a~h). Simi- stimuli to assess lymphocyte function. Fig. 3 shows lar results were observed for inhibition of ('HITER on oh 2h ip z 10% m7 = = 4 Pe. a 50% 19% s 7 8 24h 48h 60h a=] ee 3] “pS 774 tara ae ore Pan-T cell Fig. 7. Dot plots show analysis of Con A-induced CD 1a expresion before restment (0h) and 6, 12, 24, 48 and 60 h after treatment with 20 mg/kg of MMF. AC each timepoint blood was drawn, diluted and stimulated. Cultured blood (300 yD was stained with mAb to perform a thce-olor flow estometeie analysis. After 30 min of incubation RBCs were sed, lymphocytes washed and pelleted, Before analysis ells were resuspended in PBS contsining 0.5% formaldehyde, Forward and tide seater was used to detect viable cells and SOD) events of each sample were analyzed. 0 (ML, Baren etl, / Transplant Inmsaology 10 (2002) 1-14 incorporation, $/G,M positive cells and percent cells expressing CD34 (data not shown). ‘The IC values of MPA plasma concentrations for lymphocyte proliferation measured by [HITUR incor- poration were lowest (Fig, 4a). As we observed in our in vitro studies the potency of MPA in the Con A assay ‘was lowest for inhibition of CD54 expression and lowest for CD25 expression in the PMA + CD28 assay com- pared to potencies of MPA on the other cell surface antigens. The [x Values of MPA after in vivo treatment (Fig. 4b) were similar £0 /,., values of MPA obtained after in vitro treatment for both assays (Fig. 1b). Again the antiprotiferative effect of MPA measured by [HITAR incorcorporation was complete, but less (Con A, 91 1% or PMA + CD28, 94 + 3%) when measured by flow cytometric detection of S/G,M cells (Fig. 4b). Inhibi- tion by MME on the expression of surface antigens was incomplete or complete depending on the mitogen used (Fig, 4b). The correlation between the magnitudes ‘of MME concentrations and the magnitudes of inhibi- tion of lymphocyte function after Con A stimulation was 17 = 0,89-0.97 and after PMA + CD28 stimulation was 1? = 073-091 44, Relationships among PD and MMF dose levels Fig. 5 shows a low inter-rat variability for suppres- sion of lymphocyte functions in the Con A and the PMA + CD28 assays. In both mitogen-stimulated as- says, the dynamics of inhibition of lymphocyte prolifer- tion and expression of surface antigens over time were inversely related to MPA plasma concentrations as shown in Fig. 2. Maximal inhibitions of all lymphocyte functions, except for CD25, occurred for each dose level of MMF 6 h after the last dose (Fig. Sa-n), which also showed the highest MPA plasma concentration Fig, 2). All lymphocyte functions recovered to pre- treatment levels within 60 h after the last dose regard of the mitogen used (Fig. 5a-n). Vehicle-treated normal rats were designed to study effects of vehicle treatment and multiple bleeding on Iymphoeyte func tion. As shown in Fig. Sa~n none of these manipula- tions had PD effects on lymphocyte functions after Con A- or PMA + CD28-stimulation (Fig. San). Represen tative histograms and dot plots (20 mg/kg MMF dose) for CD25 and CDI ta analysis after Con A-stimulation are shown in Figs. 6 and 7, respectively 4.5, Relationships among PD and MMF dose levels For analysis of PK, exposures to drugs over time are described by areas under the concentration-time curves (AUG). » 3) Since the dynamics of inhibition of Iym- phocyte functions were also determined over time (Fig. Sa-n), we used these data to caleulate the areas under [MIF Dose Gughg) ms C10 m0) ConA @ PAPTUR SIGM CD25 COI CBT CDlla CDs g PMA+CD28 w AlLanlts NE san ofp eurevteng 38 8 PayTuR SGM cO2s CoIM CON Clie cos Fig. 8, Effect of MME on PD after treatment witha single dose ofS 10 or 20 mg/kg of MMF. PD results were derived from whole blood PD assay results in Fig. 4 and expressed as areas under the PD cffot-time curves during the 24 h after the treatment (AUB. 5) AUE,. was calculated using the trapezoidal rule. (a) Shows eh PD function measured of the Con stimulated asssy and (b) shows cach PD function of the PMA + CD28-stimulated assay. The levels ‘of significance forthe dtferences between groups were calculated by ANOVA. Postsoe analysis (SNK) was dane of Svs. 10 mg/kg and 10, 1s 20 mg/kg, Levels of significance (P-< 0.05) are indicated by (°), the PD effect-time curves during the first 24 h after dosing (AUE,.»,,). In the Con A assay, the AUEy 24, values for all lymphocyte functions differed signifi- cantly among all. dose groups of MMF (ANOVA on ranks, SNK, P< 0.05), except for CD25 and CD134, where AUE,..,,, values for $ and 10 mg/kg for CD25 and CD134 were not different (Fig. 8). The AUEy->¢4 after PMA +CD28-stimulation are shown in 46. Relationships among MMF dose, PK and PD ‘There was a high correlation between MMF dose and MPA AUC,... , (7? =0.97), between MMF dose and 6 h MPA plasma concentration (r? = 0.97) and between MMF dose and 24 h MPA plasma concentra- tion (r? = 0.86), We correlated the PD of MPA over time with MMF dose and with MPA PK. Correlations between AUEy->: , and AUCy.»: , values were sig- ML. Bart ta. / Transplant Immunology 10 (2002) 1-14 n nificantly high for all lymphocyte functions regardless the mitogen-stimulation (ANOVA, SNK, P< 005, Table 1, The correlation between the AUEy->. , Val ues and MMF dose or AUC). , were lower for PHITaR compared to all flow cytometric measurement of lymphocyte function (Table 1). 5. Discussion In this study we showed that MMF suppressed the expression of important surface antigens on T Iympho- cytes after caleium-dependent or -independent stimula- tion of whole blood. We also found high correlations between PD and PK of MMF. The inability to observe effects of immunosuppres- sive drugs on immune cells in vivo has limited drug development, their optimum clinical use and the design and interpretation of clinical trials in transplantation, But now new technologies using small amounts of whole blood and exploiting the specificity, sensitivity and versatility of flow cytometry have been used to "uncover mechanisms of drug action and to measure PD effects of immunosuppressive drugs [10 Optimal use of current immunosuppressive drugs would insure that drug exposure is balanced between over- and under-immunosuppression to minimize fail- ure of drug efficacy and risk of drug side effects. Measurement of blood levels (PK) of immunosuppres- sive drugs can avoid systemic toxicity [36], but these levels do not precisely predict immunosuppressive ef- ficacy as measured by suppression of immune cell func tion [37]. Therefore, knowledge about the immunosuppressive effect (PD) provides a more complete understanding of Table 1 fa drug's in vivo pharmacology than measurements of only its circulating concentrations. ‘The pro-drug MMF is rapidly converted to MPA, a reversible uncompetitive inhibitor of IMPDH, causing depletion of intracellular GTP pools. The resulting inhibition of lymphocyte proliferation is thought to ‘explain the immunosuppressive action of MPA [38] In vitro, we demonstrate here a dose-dependent in- hibition with MPA. on lymphocyte proliferation and surface antigen expression regardless of the mitogen used for stimulation, After short-term stimulation of purified cells, MPA does not suppress cytokine and surface antigen expression, but after long-term stimula- tion, MPA does suppress the expression of both func- tions [28,39]. In our optimized whole blood assays, we defined conditions for measuring lymphocyte prolifera- tion and antigen expression after 3 to 4 days of stimula- tion by Con A and after 2 days of stimulation by PMA + CD28 or PMA + IONO [18]. The duration of stimulation was sufficient for MPA to suppress the expression of T cell surface antigens after calcium-de- pendent and -independent stimulation. The potency of MPA for inhibition of lymphocyte proliferation measured by CHITGR incorporation was higher compared to ‘low cytometric detection of cells in the S/G,M phase. This may be explained by the incorporation of [ HITAR over the last 16 h of incuba- tion, whereas flow cytometric analysis of cellular DNA content detects all proliferating lymphocytes over the entire incubation time, Also, MPA produces a substan- tial increase in cellular dTTP [40], which may reduce the incorporation of extracellular labeled (°HITAR, Thus, competition between endogenous, unlabelled TTP and labelled [*HITAR may lead to an overesti- mation of the poteney of MPA for suppression of Single dose treatment study in normal rats treated once with vehicle or 5,10 or 20 mg/kg MMF, Cortelations between UE). forall 7 PD measurements and: MPA dose levels and AUC, Correlation coeticient AUE) sas! Dose level” Mitosen Goa A i ‘Gand PMA + Coos Paar 07st os on oar S/G.M os" oot! ‘oot Cas sz oz! oss? epi 9s 90! ase" D7 ase ost oo cpl a9 093" og eps asy 0.96 ase" PD data wore derived fom resls in Fig. in which whole blood PD assays were used (Gee legend for Fig. 4) AUE,. ,, was ealelated using the jineae trapezoidal "Done level groups: vehicle, 5,10 and 20 mg/kg MMF. MPA plasma AUC, 24 halter drug ai sing the neat trapezoidal rl. "Pearson pret moment al cortlations were significant (? < 008) 1 Was caleulated from MPA plasma concentrations assayed on timepok before deug administration and 6,12 and 2 MJ. Barton eta. / Transplant bnuaotogy 10 (2002) 1-14 lymphocyte proliferation measured by [°HITAR incor- poration. ‘The maximal antiproliferative effect of MPA measured by [HITAR incorporation in vitro in our study reached 100% regardless of whether whole blood was stimulated by cither the caleium-dependent or -independent pathways for T-cell activation. Furthermore, we demonstrated a strong and dose- dependent suppression by MPA of expression of all T cell surface antigens we measured in vitro. Previous studies, using purified lymphocytes stimulated in the MLR or by Con A or PMA, showed MPA. does not or incompletely suppresses CD25 expression [26-28]. ‘We, however, showed substantial and dose-depen- dent suppression of CDila, CD25, CD54, CD71 and CD134 expression by MPA in vitro and MMF in vivo. Expression of CD25, in contrast to the other cell sur- face antigens, was incompletely and least potently sup- pressed by MPA or MMF. Failure to completely sup- press CD25 expression may be caused by the inability of MPA to inhibit Il-2-induced CD25 upregulation [39]. The molecular mechanisms by which MPA may Suppress expression of specific cell surface activation antigens are not known, Previous studies of MPA’s biochemical effects suggest possible explanations. For example, in studies of Con A-stimulated human T lymphocytes it was shown that MPA induces GTP depletion and decreases ATP pools resulting in a lack of pyrimidine synthesis [7]. Proliferating cells depend ‘on both the purine and the pyrimidine pathways to supply ribonucleotides necessary for DNA, RNA and protein synthesis as well as for ribonucleotide interme- diates, which are required for proper lipid and protein lycosylation and membrane synthesis [41,42]. In ad tion, studies in human cell lines show MPA suppresses the synthesis of RNA primed DNA intermediates [43] In other studies, MPA was shown to suppress gua- nine nucleotide proteins (G-proteins) [44], which are required for activation of some T cell surface antigens (45) MPA. also blocks the glycosylation of adhesion ‘molecules [8,29] necessary for proper folding and regu- lation of other T cell surface antigens (29,46,47], Re- cent studies, however, failed to show that MPA de- creases glycosylation on Con A-stimulated lymphocytes Bo). Brequinar, an inhibitor of the de novo pyrimidine synthesis, decreases ATP pools as does MPA in human lymphocytes [41]. Besides its antiproliferative effect, brequinar suppresses IL-2 R expression in mitogei stimulated PBMC in vitro. Inhibition of mRNA synthe- sis and protein translation by brequinar may explain its suppression of IL-2 R expression [48]. Similar mecha- nisms may explain the suppression by MPA of expres- sion of T cell surface antigens in our study. Using our optimized whole blood assays, we mea- sured the PD of MMF after treating normal rats with different. MMF doses. For the first time, we demon- strated a clear dose-dependent inhibition by MMF on both lymphocyte proliferation and surface antigen ex- pression after both calcium-lependent and -indepen- dent stimulation of whole blood. We showed similar maximum inhibitory effects of MMF on lymphocyte function in vivo compared to the maximum inhibitory effect of MPA in our in vitro studies for Con A- or PMA + CD28-stimulation. Our data suggest that an important part of the overall immunosuppressive effect of MMF in vivo might, in fact, be caused by the inhibitory effect of MPA on the ‘expression of lymphocyte surface antigens. Another objective of our study was to determine how immunosuppression by MMF on lymphocyte functions ‘over time (PD) correlates with MPA blood concentra~ tions (PK). Therefore, we simultaneously measured lymphocyte functions and MPA blood concentrations at different times after treating rats with different doses of MMF. After a single dose of MMF, the rapid and complete recovery of lymphocyte function showed that the drug effect was highly reversible, even after high doses. The differences in the kinetics of recovery of Iymphoeyte activity was clearly related to MPA blood plasma concentrations, Studies on the activity of IM- PDH show a trend towards an inverse relationship between enzyme activity and MPA concentrations [49]. Our data showed the reversibility and inverse relation- ship also applied the inhibition of expression of surface antigens by MPA (Fig. 5). We showed that MMF produced nearly complete inhibition 12 h after oral administration, but considerable recovery occurred by 24 h. Our data support the current clinical practice of dosing MMF twice daily to achieve an inhibition of Iymphoeyte function during the dosing interval [50]. The calculation of the area under the time effect curve (AUE)., ,) is a new concept. Just as the area under the time concentration curve (AUC) is more informative than a single drug trough level or maximal concentration, the AUE is a more comprehensive value than the percent inbibition of lymphocyte function at a single time point. The AUE,.», , values in our study correlated highly with the MMF dose just as AUC) 2. correlated with the MME dose. Our whole blood assay may be useful for following the effects of other im- munosuppressive drugs on lymphocyte functions to compare potencies and durations of action in vivo. In conclusion, we demonstrated a dose-dependent suppression by MMF on lymphocyte proliferation and expression of T cell surface antigens after calcium- dependent and independent stimulation of whole blood. These new mechanisms may account for the overall immunosuppression of MMF. Previously, using less extensive PD assays we showed a high correlation between PD and severity of histo- logic rejection in rat heart allograft recipients after (MA. Barto eal / Transplant Immunology 10 (2002) 1-14 B MPA treatment [17]. Recently, using PD assays de- scribed here, we also found that PD correlated highly with rejection severity after treatment with MME [51]. ‘The applicability of our whole blood assay to mea sure PD of MMF combined with clinically relevant immunosuppressants like calcineurin inhibitors will be tested in different species. Measuring PD effects of immunosuppressive drugs may help to design more efficient immunosuppressive protocols for experimental models of transplantation and may increase the efficacy and safety of immunosuppressive regimens in patients. 6. Nomenclature CHT ——Trium lbeled tymidine PBMC Peripheral blood mononuclear cells PCNA Proliferatng cell auclear antigen 12k Interlethin-2 receptor NGF-/TNE-R Nerve growth factor-/tumor necrosis factor recuptor LEAL Leucooyt function antgen-1 ICAM-1 Intercellular adhesion molecule-1 APC ‘Antigen presenting cell MLR Mixed lymphocyte reaction ConA ‘Concanavalin A PMA Phorbol 12-myristate 13-acetate TONO Tonomin MME Mycophenolate mofetil MPA Mycophenole acid PD Pharmacodynamics Pk Pharmacokinetics HPLC High performance liquid chromatography Fire Phuorescein isothiocyanate PE Phycoorthrin PEGS PhycoerythrineyaninS mab Monoclonal antibody Ie Immunoglobuline Ps. Phosphate buttered saline AUC ‘Area under the time concenteation curve AUE ‘Area under the time effect curve IMPDH —_ Inosine S-monophosphate detydrogenase oa (Culture medium DMSO Dimethyl sulfoxide PL Propidium iodide com Counts per minute Fes. Fetal calf serum RBC Red blood cell, LEW Lewis Ie Inhibitory concentration of S0Gé Toes Maximal inhibition TeR Teel receptor SNK ‘Students Newman Keul G-protein Gusnosine nucleotide protein Gre Guznosine triphosphate Acknowledgements ‘This work was supported by Roche pharmaceutical company, NJ, USA and also by the Hedco Foundation and the Ralph and Marian Falk Trust. Markus J. Barten was supported by the Novartis study grant of the European Society of Transplantation. Teun van Gelder was supported by the Foundation “Vereniging Trustfonds Erasmus Universiteit Rotterdam’ in the ‘Netherlands and by the Dutch Kidney Foundation, Jan F, Gummert was supported by Deutsche Forschungs- ‘gemeinschaft grant Gu 472/1-1 References 11) Bullingham RE, Nicholls A. Hale M. Pharmacokinetics of mycophenolate mofel (RS6I443): a short review. Transplant Proc 199628325, {2} Franklin TF, Cook JM. The inhibition of nucleic sci synthesis by mycophenoic acid. Biochem J 1960-13351. [3] Makara GM, Keseru GM, KajtarPeredy M, Anderson WK. [Nuclear magnetic resonance and molecular modeling stady on Imycophenolic acid: implications for binding 10 inosine ‘monophosphate dehydrogenase. J Med Chem 1996:3%1236. Is] Ransom IT. Mechanism of action of mycophenolate mofeti “Thet Drug Monit 199517581, [5]. Weaver JL, Pine PS, Aczalos A. Comparison of the in vitro and biophysical effects’ of cyclosporine A, FK-SI6, and_ay- cophenolic acid on fuman peripheral blood Iymphoeytes. In- ‘munopharmacol Immunotoxico 1991:13:563, [61 Zeevi A, Woan M, Yao GZ et al. Comparative in vitro studies ‘nthe immunosuppressive activites of myeoptenoic aed, bee- dinin, FK 506, eeosporine, and rapamycin. Transplant Proc 1091:23.2928 M1 Oiu ¥, Fairbanks LD, Ruckermann K et al. Mycophonolic acibinduced GTP depletion aso affects ATP and pyrimidine ‘ythesis in mitogen-stimulated primary human T lymphocytes. ‘Transplantation 200069:890. Is} Allison AC, Eugui EM. Purine metabolism and immunosep- pressive effects of mycophenolate mofetil (MMF). Cin Trans plant 1996:10-77, {9} Franklin, Moris WP. Pharmacodynamics of the inl of GIP synthesis in vivo by mycophenolic acd. Adv Enzyme Regul 1994:34107 [U0] Dambrin C, Klupp J, Morris RE. Pharmacodynamics of im ‘munosuppressive drugs. Curr Opin Immunol 200012857 U1] De Groote D, Gevaert Y, Lopez M etal. Novel method for the ‘measurement ofeytckine production by a one-stage procedure. J immunol Method 1993:163:258, {12} Silva HIT, Shorthouse R, Moris RE. Sinle- and multiple-dose pharmacokinetics and pharmacodynamics of leflunomide’ ac tive metabolite A77 1726 in normal Lewis rats. Transplant Proe 1996:28:3082 HJ, Cao W, Shorthouse RA, Loffler M, Mortis RE. In vitro and in vio eifects of leflunomide, brequinar, and cyclo- sporine on pyrimidine biosynthesis. Transplant Proc 1997:29:1282, [14] Wiegers Gi, Croiset G, Reul JM, Hokboer F, de Kloet ER. Differential effects of corticosteroids on rat peripheral blood ‘lymphocyte mitogsnesis in vio and in vitro. Am J Physiol 198326826825. 15) Gaines H, Anderson L, Biberfeld G. A new method for reasuing Iymphoproliferation at the single-cell level in whole blood cultures by flow cytometry. J Immunol Methods 1906:19563 16) Gummert JF, Barten MA, Sherwood SW, van Gelder T, Moris RE. Pharmacodynamics of immunosuppression by my: cophenolic acd: inibition of both Iymphocyte proliferation 03) Tu) st bo bo) en al 3) ba bs ea mm bs fro bo, ba 3) ‘MI. Berton ta. / Transplons tmmuology 10 (2002) 1-14 and activation correlates with pharmacokinetics. J Pharmacol Exp Ther 1999291:1100, Gummest JF, Barten MJ, van Gelder, Billingham ME, Mor. ris RE. Pharmacodynamics of mycophenolic aid in heart llo- {graft recipients: correlation of lymphocyte proliferation and sctivation with pharmacokinetics and graft histology. ‘Tran plantation 200:70:1038, Barten MJ, Gummert JF, van Gelder, Shorthouse R, Mortis RE. Flow cytometric quantitation of éakium-dependeat and independent mitogenstimultion of cel funetins in whole Dlcod: inhibition by immunosuppressve drugs in vitro. J Im smunol Methods 2001;2531-2195, Cals JE, Bravo R, Larsen PM, Fey SJ. Cyein: a nuclear protein whose level comelaes diretly with the proliferative Sate of normal as well as transformed cells.” Leuk Res 19848185, ‘Taniguchi T, Minami Y. The 1-2/1L-2 receptor sytem: & current overiew, Cll 1993;73. Mallet S, Fussum §, Neil Barclay A. Characterization of the ‘MRC OX40 antigen of activated CDs positive TIymphoeytes: rolecule related to nerve growth factor receptor. EMBO J 199039:1083 Tite TV, Weinberg AD, Steinkeler CN, Maziarz RT. Expres- son ofthe T-el aetvation antigen, OX-0, identifies alloreae- tive T cells in acute graft vs. host disease. Blood 199738946 Pighetti GM, Eskew ML, Reddy CC, Sordillo LM. Selenium and vitamin E deficiency impair transferrin receptor internal: ination but not IL-2, 1L-2 receptor, oF transferrin receptor expression. J Leukoe Bio! 1998/65:13, “Mackay CR, Imbof BA. Cell adhesion inthe immune system, Immunol Today 199314399. Van Seventer GA, Shimizu Y, Horgan KJ, Shaw S.'The LEA-I ligand ICAM-1 provides an important costimulatory signal for ‘Tell receptor-mediated setvation of esting T cell J Im rninol 199041444579. gui EM, Almguis SJ, Maller CD, Allison AC. Lymphocyte selective cytostatic and. immunosuppressne effects of my ‘ophenolie ac in vito: role of deoxyguanosine mucleotide depletion. Scand J Immunol 19:33:16 Woo J, Zeevi A, Yao G2, Strednak J, Todo S, Thomson AW: Etfecs of FK S06, mycophenoli acid, and bredinin on OKT, PMA, and alloantigen-induced activation molecule expresion ‘on cultured CD4* and CD” human lymphocytes. Transplant Proc 1991:232939, ‘Thomson AW, Woo J, Yao GZ, Todo S, Staral TE, Zeevi A. Effects of combined administration of F506 and the purine biowyihesis inhibitors mizoribine oF mycophenolic acid on lymphocyte DNA synthesis and T-ell activation molecule ex pression in human mited Iymphooyte cultures. Transpl In- rmunol 1993:1:146, [Laurent AF, Dumont S,Poindron P, Muller CD. Mycophenolic acid suppresses protein N-linked glycosylation in hurman mon0- ‘tes and their adhesion to endothelial cells and to some substrates. Exp Hematol 19952459, Jepson S, Brogan H, Stoddart RW, Hutchinson TV. My= ophenolic acid does not inhibit protein elyeosslation in Iymphoeytes. [In process citation}. Transpl Immunol 2008169. Gurnmert JF, Osto G, Basten MI, Morris RE. Effect of anes: thesia on a whole blood lymphocyte proliferation assay in the tat. Immunopharmacol Immunotoxical 1999:2:267 Shitine M, Taslor NI, Rosenblatt LS, Wikon FD. Comparison ‘of whole blood and puried canine Iymphocytes in a Iympho- ‘te-simulation microasay. Am J Vet Res 197832687 ‘Thompson SC, Bowen KM, Burton RC. Sequential monitoring ff peripheral blood lymphocyte subsets in rats, Cytometty 19867186 [54] Gummert JF, Christians U, Barton M, Sta HE, Morris RE. A high-performance liquid chromatographic assay with a simple ‘extaction procedure for sensitive quantification of my cophenolic seid in rat and human plasma. J Chromatogr B Biomed Appl 1999:721-321 [35]. Stewart MP, Cabanas C, Hogg N. cell adhesion to inteoeta- lar adhesion molecle-1 (CAM-4) is controlled by cell spread ing and the activation of intgsin LFA-I. J Immunol 1996; 136810, (36) ‘Tsunoda SM, Awocka FT. The wse of therapeutic drug moni- toring to optimize immunosuppressie therapy. Clin Pharm cokinet 1996;30:107, 37) Yatscoft RW, Aspeset LJ, Gallant HL, Pharmacodynamic ‘monitoring of immunosuppressve drugs. Clin Chem 1998; 6408, [38] Brazeton TR, Morris RE, Molecular mechanisms of action of new xenobiotic immunosuppresive drugs: tacrolimus (FKSDO, sirolimus (rapamycin), mycophenolate mofetil and lefunomide. Cute Opin Immunol 1996:8710. [59] Nagy SE, Andersson IP, Andersson UG. Effect of my-

    You might also like