Treatment by Mycophenolate Mofetil of AdvancedGraft Vascular Disease in Non-Human PrimateRecipients of Orthotopic Aortic Allografts

Jochen Kluppa,d,*, Camille Dambrina, KiyoshiHibib, Jorge Lunab, Takeshi Suzukib, BernardHausena, Tudor Birsana, Teun van Geldera,Peter J. Fitzgeraldb, Gerald Berryc andRandall E. Morrisa

aTransplantation Immunology, Department of CardiothoracicSurgery, bDivision of Cardiovascular Medicine, cDepart-ment of Pathology, Stanford University School of Medi-cine, 300 Pasteur Drive, Stanford, CA 94305–5407, USAdDepartment of Surgery, Charite-Virchow, Humboldt-University Berlin, Augustenburger Platz 1, D-13353 Berlin,Germany*Corresponding author: Jochen Klupp, jochen.klupp@charite.de

Failure to control chronic graft dysfunction [e.g. graftvascular disease (GVD)] is the primary cause ofimmunologic graft failure. This is the first study ofmycophenolate mofetil (MMF) for the treatmentof GVD in non-human primate recipients ofaortic allografts. Abdominal aortic allografts wereexchanged between mixed leukocyte reaction (MLR)-mismatched, blood-group-compatible cynomolgusmonkeys. Six control recipients were untreated.Individualized treatment with frequent dose adjust-ments of MMF insured that treatment was close tothe maximum tolerated dose (mean 99.2 mg/kg/day).Immune-mediated injury proceeded unhindered untilday 45, after which MMF treatment began. Changes inintimal volume (IV) were quantified by intravascularultrasound (IVUS) and compared to histology on day105. Serial IVUS measurements of IV (mm3) in controlsshowed progressive GVD. In four out of six animals,MMF was well tolerated, thus enabling optimumtreatment; in all these animals, IV was significantlyless than in the control animals (p =0.02). In the tworemaining animals, high doses were not tolerated; atday 105, there was no significant difference in IVbetween them and the controls. We found a signifi-cant correlation between the mean MMF tolerateddose and the inhibition of progression of IV(r =�0.88, p= 0.015). When high MMF doses weretolerated, MMF slowed progression of GVD.

Key words: Chronic rejection, mycophenolate mofetil,non-human primates, pharmacodynamics, pharmaco-kinetics

Received 15 February 2002, revised and accepted forpublication 4 March 2003

Introduction

Graft vascular disease (GVD) and neointimal thickening are

pathognomonic for chronic rejection after heart, kidney and

other solid organ transplantations. The pathophysiological

mechanisms of GVD development are becoming increas-

ingly well understood, and smooth muscle cell prolifer-

ation plays a central role in the development of intimal

thickening. The mechanism of action of mycophenolic

acid (MPA), the active metabolite of mycophenolate mofe-

til (MMF), makes MMF attractive to investigate for the

treatment and prevention of GVD. As previously well

described (1), MPA inhibits inosine-monophosphate-

dehydrogenase isoenzymes I and II, and thereby de novo

purine synthesis. Proliferating lymphocytes and, to a lesser

extent, smooth muscle cells, fibroblasts and endothelial

cells depend on de novo purine synthesis (2,3). Therefore,

these cells may be targets for the treatment of chronic

rejection by MMF. In vitro inhibition of growth-factor-

stimulated smooth muscle cells by MPA (3,4) led to

several animal studies that investigated the effect of

MPA and MMF on intimal thickening in rodent models of

chronic rejection (5–8) and on re-stenosis after mechanical

arterial injury (9,10). After xeno-heart-transplantation MMF

reduced vascular rejection and graft coronary disease (11).

In addition, early clinical results suggest that MMF

reduces chronic graft dysfunction after heart (12,13),

kidney (14–16) and liver transplantation (17,18).

These previous studies encouraged us to investigate the

effect of MMF on GVD after allogenic aortic transplant-

ation in non-human primates. This procedure (19,20) is

the first – and so far only one – reported for the study of

GVD in non-human primates. Using this procedure, the

effect of immunosuppressive drugs can be tested for

their ability to impede progression of pre-existing graft

vascular disease. With serial intravascular ultrasound

(IVUS) morphometry, the time-course of development of

intimal thickening can be monitored in vivo in individual

monkeys. Furthermore, the effects on the immune

system and the pharmacokinetics, pharmacodynamics

and toxicology of MMF in non-human primates are similar

to those in humans. Most important, however, the intimal

lesions in aortic grafts are histologically indistinguishable

from human allograft vasculopathy (21).

Our primary objective was to test whether MMF could

halt or impede the progression of advanced intimal

thickening in an individualized treatment regimen in this

American Journal of Transplantation 2003; 3: 817–829 Copyright # Blackwell Munksgaard 2003Blackwell Munksgaard

ISSN 1600-6135

817

demanding non-human primate model of graft vascular

disease.

Materials and Methods

Experimental design

To optimize MMF tolerability and efficacy, we performed phase I dose-

finding pre-studies with extensive pharmacokinetic and pharmacodynamic

assays in four nontransplanted cynomolgus monkeys.

In the phase II transplant study, six cynomolgus monkeys received MLR-

mismatched infrarenal artery aortic interposition allografts. To allow for the

formation of substantial GVD, the recipients received no immunosuppres-

sive treatment through day 44 and animals in the MMF group were treated

with MMF from day 45 until day 103 after transplantation using individual

treatment to ensure MMF exposures were close to the maximal tolerated

dose. The treatment group was compared to an untreated control group of

six transplanted animals. In all animals, serial IVUS was performed on

postoperative days (POD) 21, 42, 63, 84 and 105, when the study was

terminated and the animals were euthanized. Grafts were then removed

for histological examination.

Animals

Sixteen healthy male Macaca fascicularis monkeys (5.9–8.3 kg), screened

to be disease free, were purchased from Charles River BRF Inc. (Houston,

TX, USA), and kept in quarantine at Stanford for 6 weeks before release for

scientific use. Four animals were used for the phase I dose-finding study,

and from the 12 animals in the phase II transplant groups, each animal

served as both a donor and a recipient for aortic transplantation. Pairs were

ABO matched, and the mixed leukocyte reaction was used to ensure histo-

incompatibility [Stimulation Index (SI) 2.7–38.3].

Husbandry

Before each procedure, the animals were acclimated for at least 1 week in

controlled conditions of temperature and humidity in the room where they

were housed during the study. Water and primate chow were provided ad

libitum, and the monkeys were cared for according to the standards of the

US Public Health Policy of the Humane Care and Use of Laboratory Animals

(PHS Manual, Ch. 143) and the guide for the Care and Use of Laboratory

Animals (NIH Publication no. 8523, revised 1985). The study protocol was

approved by the Institutional Laboratory Animal Committee.

Anesthesia and postoperative care

For surgical procedures, the animals were anesthetized with ketamine

hydrochloride (Ketaset1) 10 mg/kg s.c., atropine sulfate 0.04 mg/kg i.v.

and buprenorphine hydrochloride (Buprenex1) 0.01 mg/kg i.m. After endo-

tracheal intubation, anesthesia was maintained with continuous propofol

(2.5 mg/mL; 0.1 mg/kg/min) and midazolam infusion (1 mg/mL; 0.35 mg/kg/

min). Until extubation in stable conditions, ECG, body temperature, oxygen

saturation, respiration rate and end-expiratory CO2 concentrations were

monitored continuously. Blood pressure was measured at 3-min intervals,

and all vital signs were recorded at 15-min intervals. Postoperatively, the

animals were transferred to special ICU units with individually controlled

cage temperatures and oxygen concentrations for 24 h. To control for

postoperative pain, 0.03 mg/kg buprenorphine hydrochloride (Buprenex1)

was administered i.m. Cefazolin (25 mg/kg i.m.) and gentamycin (5 mg/kg

s.c.) were used for perioperative antibiotic prophylaxis.

Aortic transplantation

After deep anesthesia, a midline laparotomy was performed. The abdominal

aorta was exposed. After heparinization (70 IU/kg) and aortic clamping, a

3.0-cm segment of the abdominal aorta distally to the inferior mesenteric

artery was removed, flushed with cold saline and anastomosed (beveled to

avoid anastomotic strictures) to the recipients aorta using 7–0 Prolene1.

After homeostasis, the retro-peritoneum was sutured and the abdominal

wall was closed.

Intravascular ultrasound methodology

Intravascular ultrasound methodology was performed on POD 21, 42, 63,

84, and 105. After deep anesthesia, the femoral artery was exposed, and a 5

French sheath was introduced into the vessel under heparinization (70 IU/kg).

After injecting 20mg nitroglycerin into the catheter, intra-aortic pressures

were recorded with a 5-French Swan-Ganz catheter (Baxter Healthcare

Corp., Deerfield, IL, USA), and the peak-to-peak gradient over each anasto-

mosis was assessed by a pullback technique. Sequential IVUS imaging was

performed using an intravascular ultrasound system (CardioVascular

Imaging Systems/Boston Scientific, Sunnyvale, CA, USA) with a 40-MHz,

2.6 French ultrasound catheter (SciMed/Boston Scientific, Sunnyvale, CA,

USA). The imaging probe was positioned proximal to the graft, and with-

drawn at a constant speed of 0.5 mm/ s. The IVUS images were recorded

for later analyses. After completion of the IVUS measurements, the sheath

was removed, and the femoral arteriotomy was repaired with 7–0 Prolene1

sutures.

Off-line 3-D reconstruction of the IVUS data was performed with Echo-

Plaque1 software (Indec Systems, Inc., Mountain View, CA, USA) using a

digitization frame rate of 15 images/ s, resulting in the corresponding planar

3-D image of each studied segment. Contour drawings of intima and lumen

outer borders were performed manually at 1-mm intervals, and the inter-

polated contours for the remaining frames were automatically generated

by the system (Figure 1). Areas and diameters for all lumen and intima

contours were measured, and volumes were calculated automatically

using the following formula:

Volume ¼ nX

i¼1:n

ðAi � HÞ

where Ai is the measured area of segment, i; H is the thickness of the

segment and n is the number of IVUS images in the 3-D data set. Volu-

metric [intimal-lumen volume (VV), intimal volume (IV), lumen volume (LV)]

and cross-sectional values [cross-sectional intimal-lumen area (VA), cross-

sectional intimal area (IA), cross-sectional lumen area (LA)] were assessed

for segments in the middle 10 mm of the graft segments. The minimal

lumen cross-sectional area within each segment was selected and meas-

ured accordingly.

Mycophenolate mofetil (MMF; CellCept�) treatment

In phase I studies, MMF powder (a gift from Roche Bioscience, Palo Alto,

CA, USA) was dissolved in vehicle and was administered by oral gavage

under ketamine sedation (Ketaset1, 5 mg/kg s.c.). The vehicle was pre-

pared by dissolving sodium carboxymethylcellulose (CMC, City Chemical

Corp., New York, NY, USA; viscosity of 1300–2200 centipoises at 25 �C in a

1% weight/volume in deionized water solution) in 0.9% benzyl alcohol

(volume/volume), 0.4% polysorbate 80 (weight/volume) and 0.9% sodium

chloride (weight/volume) in water to produce a 0.2% CMC solution, which

was stored at room temperature. In the phase II transplant study, MMF

treatment started on day 45 after transplantation. Initially, 50 mg/kg MMF

hydrochloride solution (CellCept1IV, a gift from Roche Laboratories Inc.,

Nutley, NJ, USA) was administered twice daily (b.i.d.) subcutaneously for

7 days and subsequently administered by oral gavage BID. MMF was

dosed individually according to maximal tolerability: total weight loss

exceeding 10% or a 3% weight loss in 3 days, substantial diarrhea or

vomiting prompted dose reduction. In case of toxicity, food was supple-

mented with high calorie gruel gavage once or twice daily to a maximal

gruel volume of 30 mL/kg. Dehydration was treated with subcutaneous

Klupp et al.

818 American Journal of Transplantation 2003; 3: 817–829

fluid administration (10–20 mL/kg lactate-Ringer solution). Diarrhea was

treated with loperamide (25–50 mg p.o. daily).

In addition to the assessment of toxicity, MMF pharmacokinetics and

pharmacodynamics were considered for MMF dosing. We defined MPA

plasma levels below 2.0 mg/L and more than 5 abs% (absolute percentage)

proliferating cells in the peripheral blood (see methods below) as subthera-

peutic immunosuppression.

Pharmacokinetic (PK) and pharmacodynamic (PD) monitoring

In phase I studies, in vitro assays evaluating the effect of MPA on immune

functions in cynomolgus blood and complete in vivo pharmacokinetic (PK)

and pharmacodynamic (PD) profiles were performed. Blood was collected

after ketamine hydrochloride (Ketaset1, 5 mg/kg s.c.) sedation. For in vitro

assays, blood was incubated with different concentrations of mycophen-

olic acid for 30 min before the mitogen stimulation. For in vivo studies

blood was collected before drug treatment in the morning, and 2, 10 and

24 h thereafter. The plasma MPA PK area under the curve (AUC) and blood

MPA PD area under effect (AUE) were calculated using the trapezoidal

rule. In the phase II transplant study, PK and PD assays were done twice

weekly, just before the morning MMF dose (trough levels).

Mycophenolic acid (MPA) concentrations were measured by gradient

HPLC in heparinized plasma (50 mL). A method described previously for

rats (22) was modified and validated for monkey plasma: mean intraday

precision was 2.5%, mean interday precision was 9.2%, and linearity was

better than 0.99.

Lymphocyte proliferation after mitogen stimulation was measured by flow

cytometry to assess the pharmacodynamic effects of the MMF treatment.

The method was modified from our previous publications (23–27). Eighty

microliters of whole, heparinized (100 IU/mL) blood, diluted 1 : 10 in culture

Figure 1: Digitized 3-D intravascular ultrasound image using Echoplaque� Software. Contour drawings of both intima and lumen

outer borders were performed manually at 1-mm intervals, and the interpolated contours for the remaining frames were automatically

generated by the system.

Treatment of Graft Vascular Disease by MMF

American Journal of Transplantation 2003; 3: 817–829 819

medium (RPMI 1640 supplemented with penicillin, streptomycin and

glutamine; all reagents obtained from Life technologies, Inc., Grand Island

NY, USA), was incubated for 72 h at 37 �C with 7.5 mg/mL concanavalin

A (Con A) (Vector Laboratories, Burlingame CA, USA). After lysing the

erythrocytes, leukocytes were washed, fixed with 1% formaldehyde and

resuspended in 2 mL of methanol for 10 min at 4 �C. After washing, cells

were treated for 25 min at 37 �C with permeabilizing buffer (1% heat-

inactivated fetal calf serum (Hyclone, Logan, UT, USA), 0.1% saponin

(Calbiochem, La Jolla, CA, USA), 0.1% sodium azide (Sigma Chemical

Co., St. Louis, MO, USA) in (PBS), RNAse A (100 mg/mL in water; Sigma

Chemical Co., St. Louis, MO, USA), propidium iodine (PI) and anti-

proliferating cell nuclear antigen (PCNA)-FITC mAb (Dako Corp., Carpinteria,

CA, USA). The washed cells were then resuspended in PBS containing PI.

All samples were assessed by two-color analysis (Epics XL-MCL/498 nm

air-cooled argon laser, Beckman-Coulter, Fullerton, CA, USA) within 6 h of

preparation. Emitted fluorescence was collected with bandpass filters

through 525 nm (– 20 nm; FITC) and 675 nm (660–700 nm; PI). Forward-

sideward scatter gates differentiated cells of interest from debris, and

5000 light scatter-gated cells were analyzed per sample. Cell proliferation

was determined as percentage of cells in the S/G2M phase of the cell cycle

expressing PCNA. As controls, mAbs were added to unstimulated blood

identically processed as stimulated samples and inactive immunoglobulin

isotype controls were added to cultures of stimulated cells.

Pharmacokinetic and pharmacodynamic analysis

Absolute percentages (abs%) of proliferating cells were normalized at each

time point to the individual pretreatment values for both phase I and phase II

studies as 100 relative per cent (rel%). Analogous to the calculation of the

AUC0�24h, the area under the pharmacodynamic effect time – curve

(AUE0�24h) was calculated using the linear trapezoidal rule. The inhibitory

effect was expressed as per cent inhibition of lymphocyte proliferation,

analyzed by flow cytometry and calculated as follows:

%inhibition ¼ ½1 � ðTreatment=PretreatmentÞ� � 100

MPA plasma concentrations (C), which produced a 50% inhibition (IC50) of

the maximum inhibitory effect (Emax), were calculated after fitting the

concentration-effect curves in an Emax sigmoidal pharmacodynamic

model (28)

E ¼ Emax � ðEmax � E0Þ½CGamma=ðCGamma þ IC50GammaÞ�

using WinNonlin Software version 1.1 (Scientific Consulting, Inc., Cary, NC,

USA).

CBC and blood chemistry

Hemograms, sodium, potassium and chloride were obtained every

3 weeks in untreated monkeys, and once a week during immunosuppres-

sive treatment. Every 3 weeks, blood chemistry screens were performed

for total serum protein, albumin, BUN, creatinine, triglycerides, cholesterol,

liver enzymes, alkaline phosphatase, lactate dehydrogenase (LDH) and

lipase.

Graft removal, histology and immunohistochemistry

After the final IVUS-examination on POD 105, a thoraco-laparotomy was

performed under deep anesthesia. The aorta was mobilized, all visceral

branches ligated and the animal euthanized. The whole infra-diaphragmatic

aorta, including the graft, was flushed with saline and perfused with a total

volume of 250 cc of 10% formaldehyde solution for 15–20 min in situ, with

a pressure of 100 mmHg. Thereafter, the graft was removed and perfused

ex vivo under the same pressure for 18 h with a 10% formaldehyde solu-

tion. From 4-mm thick transverse sections from the paraffin-embedded

graft and the proximal native aorta, hematoxylin & eosin (HE) and elastin

van Gieson staining was performed. Deparaffinized sections prepared at

4mm thickness were stained with anti-a-smooth muscle actin (1 : 4000

dilution, Sigma Corp, St Louis, MO, USA) on a Ventana-Nexus automated

stainer (Ventana Medical Systems, Inc., Tucson, AZ, USA).

Morphometric analysis

Video images of elastin van Gieson-stained aortic sections were recorded

with a Dage MTI-81 high-resolution color camera mounted on a Leica

DMAB microscope and analyzed with a C-imaging 1280 morphometric

system (Compix Inc., Imaging Systems, Cranberry Township, PA, USA).

Lumen area (LA), intimal area (IA) the area inside the internal elastic lamina

corresponding to the vessel area assessed (VA) by IVUS, and media area,

were assessed.

Statistics

For statistical analysis, SPSS version 10.0 (SPSS Corp., Birmingham, AL,

USA) was used. All data are expressed as mean – standard error of mean

(SEM). The two-tailed Student’s t-test or the Mann–Whitney U-test was

used to estimate the levels of significance for the differences between two

groups, according to its applicability. The general linear model for repetitive

measurements was used to differentiate between the two groups over the

time. For n�n tables chi-square statistics were computed. Correlations

between PD or PK measurements or both and histology scores were

determined using the Pearson correlation factor. A p-value of less than

0.05 was considered significant.

Results

In vitro study to quantitate the effects of mycophenolicacid on monkey whole blood lymphocyte proliferationTo evaluate the in vitro effects of mycophenolic acid

(MPA) on lymphocyte proliferation, blood from four

untreated animals was incubated with different MPA con-

centrations between 0 and 100 mg/L. Fifty per cent inhibi-

tory undiluted blood concentrations (IC50) varied between

0.3 and 1.2 mg/L among the animals. The maximal effects

(Emax) were reached with 7.7 rel% PCNA expression. The

coefficient of correlation between MPA concentration and

inhibition of proliferation was 0.95.

Tolerability phase I PK and PD study evaluating 100mg/kg MMF once daily for 28daysOf four cynomolgus monkeys (P1 – P4; Figure 2), the dose

of 100 mg/kg MMF orally once a day had to be reduced

after 14 days of treatment due to toxicity in two animals

(P2, P3). For animal P3, drug withdrawal for 1 day was

necessary. After 21 days, in all animals the dose was

reduced to 50 mg/kg QD due to side-effects.

Toxicity. Significant weight loss occurred, and reached

an average of 11.6% after 21 days of MMF treatment

(range 7.6–19.4%; p< 0.02 vs. pretreatment weight).

Diarrhea was observed in all animals beginning between

days 10 and 21 of treatment. Additionally, two animals

became anorectic, and two animals started vomiting

during treatment. Signs of toxicity were reversible after

MMF dose reduction to 50 mg/kg orally once a day.

Klupp et al.

820 American Journal of Transplantation 2003; 3: 817–829

Pharmacokinetics and pharmacodynamics. After the first

single dose of 100 mg/kg, a high variability in drug PK and

PD was observed: two animals reached their highest drug

levels (Tmax) 2 h after dosing and two animals reached

Tmax 10 h after dosing. Also, the maximal MPA plasma

concentrations varied from 1.88 to 21.68 mg/L and the

inhibition of proliferation varied, with lowest relative

PCNA expressions being between 17.3 and 34.2%.

On day 7 of treatment with 100 mg/kg MMF PO QD,

another PK/PD profile was performed and later compared

with the results on day 28, when all animals had received

a 50-mg/kg MMF dose for at least 7 days (Figure 3). After

multiple doses of 100 mg/kg of MMF QD, mean MPA

trough levels (Figure 3a) were 6.45 mg/L with a range of

2.04–13.46 mg/L, and MPA peak levels were now reached

in all animals after 2 h (32.02– 12.7 mg/L). In contrast, after

multiple days of 50 mg/kg MMF QD treatment, MPA

plasma levels before dosing and 2 h after dosing were

lower (C0: 2.2– 0.6 mg/L; p¼ 0.06, C2: 10.7– 2.7 mg/L;

p¼ 0.06). Pharmacodynamics (Figure 3b) after multiple

doses of 100 mg/kg MMF PO QD showed complete inhib-

ition of proliferation at all times, whereas after a dose of

50 mg/kg MMF daily incomplete inhibition of proliferation

at all times except 2 h after dosing was shown.

Phase I study evaluating tolerability andpharmacokinetics of 50 mg/kg mycophenolate mofetiltreatment given twice a day (b.i.d.) subcutaneously ororallyTreatment with 50 mg/kg MMF given twice daily as a s.c.

solution prepared with CellCept IV1 produced MPA levels

of 11.3 – 0.6 mg/L within 2 h after the first dose. Ten hours

after the first s.c. dose, mean MPA levels were

2.7– 0.9 mg/L, and 24-h trough levels were 3.7–1.1 mg/L.

After 14 daily doses of b.i.d. treatment s.c., trough MPA

plasma levels were 7.0– 0.9 and 2 h after dosing, MPA

plasma levels were 14.2 –0.25 mg/L. Weight loss (5.2–

10.1%) and other side-effects were less frequent compared

to the 100 mg/kg MMF QD phase I study.

To evaluate oral absorption of the CellCept IV1 formulation,

another single-dose study was performed: 2 h after the

initial 50 mg/kg oral dose, MPA plasma levels of 2.9 mg/L

(range 0.4–5.5 mg/L) were reached; the 24 h MPA trough

levels were 3.9– 2.3 mg/L (range 0.3–7.9 mg/L).

Phase II study evaluating the efficacy of mycophenolatemofetil for treatment of advanced graft vascular diseaseafter aortic transplantation in cynomolgus monkeys

Demographics and morbidity of operative procedures.

Demographics of the transplanted male cynomolgus

monkeys are shown in Table 1. No mortality occurred

during the transplant study, including five IVUS proced-

ures per animal. One animal experienced an

intussusception and needed a small bowel resection on

day 8 after transplantation; GVD progression was not

affected.

Out of 60 IVUS procedures, one animal of each group

(3.3%) experienced an intimal dissection caused by the

IVUS probe without residual injuries, as noted in the sub-

sequent IVUS. Additionally, in one animal an IVUS proced-

ure on day 42 had to be terminated prematurely, due to

uncontrollable arterial spasm. However, no temporary or

continuous limb ischemia was noted in any of the monkeys.

Mycophenolate mofetil treatment and its specific toxicity

and side-effects. All animals in the MMF group began

020406080

100120140

0 14 21 28

P1

020406080

100120140

P2

0

5

10

15

20

25P3

0

5

10

15

20

25P4

0 14 21 28

0 14 21 28

0 14 21 28

Wei

ght L

oss

Afte

r S

tart

of T

reat

men

t (%

)

MMF dose

Time After Starting MMF Treatment (Days)

weight loss diarrhea vomiting loss of appetite

7

7

7

7

Tot

al D

aily

MM

F Q

D D

ose

PO

(m

g/kg

/day

)

Figure 2: Pre-study on treated, but

not transplanted animals. Four

cynomolgus monkeys (P1–P4) were

treated with a maximal tolerated dose

(MTD) of mycophenolate mofetil

(MMF) starting with 100 mg/kg

body-weight given orally once daily

(QD). Figures express the individual

tolerability for each monkey and the

resulting MMF dose, which was

adjusted according to animal safety.

Treatment of Graft Vascular Disease by MMF

American Journal of Transplantation 2003; 3: 817–829 821

treatment on postoperative day 45 with 50 mg CellCept

IV1 twice daily administered s.c. and switched to oral

administration from treatment day 8 on. Absorption and

efficacy of drug treatment were monitored closely (see

below). After 5 days of treatment (POD 50), the MMF

dose was adjusted for each animal based on individual

toxicity, PK and PD values (Figure 4). The mean total

daily dose for the whole treatment period of 58 days

was 99.2 – 4.2 mg/kg/day. In none of the animals MMF

was withdrawn completely, but in two animals (Rx4,

Rx5) temporary dose reduction below 50 mg/kg/day was

necessary.

As shown in phase I studies, the major sign of MMF

toxicity was the decrease in body weight. Through POD

42, there was no difference in body weight changes

between the animals assigned to the control and MMF-

treated groups (Figure 5a). The individual response to the

drug showed a high variation (Figure 5b), and maximal

weight loss differed between 2.5% and 14.2% among

animals treated with MMF. The general behavior of all

animals was unimpaired during the study period. During

the time of treatment (58 days), the appetite of three

animals was reduced for 4–29 days. These animals

stopped eating for a range of 1–8 days. Gruel feeding

was initiated after 4 days of anorexia. Diarrhea occurred

in four animals for a period of 1–15 days.

No other adverse events were noted during the study or

revealed by autopsy on day 105. Changes in hemograms

and in blood chemistries are shown in Table 2.

Pharmacokinetics and pharmacodynamics during myco-

phenolate mofetil treatment in transplanted animals. Before

treatment started, in vitro analysis of immune functions was

performed in all animals. The calculated MPA IC50 of lympho-

cyte proliferation varied between 0.3 mg/L and 0.61mg/L.

The variation for Emax (0–18.75 abs% expression of PCNA)

was higher than in the phase I studies, and the coefficients of

correlation between inhibition of proliferation and MPA

plasma concentration ranged from 0.96 to 0.99.

After the first dose of MMF on POD 45, mean trough

MPA plasma levels were 2.3 mg/L (range 1.8–2.8 mg/L).

A corresponding PCNA expression of 2.1– 0.3 abs% was

noted. Throughout the entire study, trough MPA plasma

levels showed a higher inter- and intra-monkey variation

than did the pharmacodynamics of suppression of lympho-

cyte proliferation (Figure 6).

0 2 10 240

10

20

50

40

30

24100 20

20

60

80

100

40

Time After Oral MMF Dose (h)

Mea

n P

CN

A E

xpre

ssio

n N

orm

aliz

ed to

Pre

trea

tmen

t Val

ues

(%)

SE

±

100 mg/kg PO MMF QD

50 mg/kg PO MMF QD

Time After Oral MMF Dose (h)

100 mg/kg PO MMF QD

50 mg/kg PO MMF QD

Mea

n M

PATr

ough

Pla

sma

Con

cent

ratio

n (m

g/L)

SE

±A

B

Figure 3: Pre-study on treated, but not transplanted animals

(n = 4). Complete pharmacokinetic (a) and pharmacodynamic

profiles (b) were performed on day 7 (MMF dose 100 mg/kg

orally once daily) and compared to day 28 (MMF dose 50 mg/kg

orally once daily) after starting the treatment. Mean mycophenolic

acid levels (a) were similar at all time-points except for higher

levels 2 h after dosing. In contrast, pharmacodynamic assays (b)

showed complete inhibition of lymphocyte proliferation at all time-

points only when 100 mg/kg MMF was administered. Fifty mg/kg

MMF given once daily, was not able to suppress lymphocyte

proliferation completely, suggesting an insufficient immuno-

suppressive effect of that dose.

Table 1: Demographic characteristics (all calculations: mean–SEM) for untreated and mycophenolate mofetil (MMF)-treated

cynomolgus monkeys after aortic transplantation to study

treatment of graft vascular disease

Untreated MMFa p-valuesb

Blood group B (n¼4),

AB (n¼ 2)

A (n¼ 6) – c

Cross-match neg. n¼6 n¼6 – c

MLR index (SI) 9.69– 5.73 7.3– 1.3 n.s.d

Preoperative weight (kg) 8.0– 0.27 6.81–0.03 0.03

Weight mismatch (kg) 1.0– 0.78 0.91–0.4 n.s.d

Transplant time (min) 112.8–3.8 128.6– 8.1 n.s.d

Graft ischemia time (min) 46.5– 3.7 61.6–12.1 n.s.d

Mean IVUS time (min) 41.1– 2.5 39.4–1.6 n.s.d

aMMF treatment from day 45–103.bMann–Whitney U-test.cSignificance not computed.dNot significant.

Klupp et al.

822 American Journal of Transplantation 2003; 3: 817–829

In a retrospective analysis after the end of the study, in vivo

PK/PD relationships were calculated. The coefficient of

correlation between MPA plasma levels and PCNA expres-

sion was much lower than in the in vitro study and than in

the phase I studies (r2¼ 0.58) and showed a high inter-

individual variation (range r2¼ 0.37–0.99). Calculated MPA

IC50s varied between 0.01 and 2.2 mg/L (mean 0.82 mg/L),

and the maximal effect of inhibition of PCNA expression

was 9.9 rel% (range 0.4–29.9 rel%).

Intravascular ultrasound studies and development of

graft vascular disease. In untreated controls, mid-graft

vessel volumes decreased from 192 –15 mm3 on day 21

to 160– 16 mm3 on day 105. After day 42, significant

intimal thickening was noted (p< 0.028; Figure 7), with

the most rapid increase between PODs 42 and 63

(44.6 –4.3 mm3). Lumen volumes decreased significantly

(p< 0.02) between POD 21 (167– 15 mm3) and POD

105 (105– 16 mm3), with a minimal lumen volume of

105

0

50

100

150

100

150

63 84 10563 84 10542 63

50

084

Rx1 Rx2 Rx3

Rx6Rx5Rx4

Time After Transplantation (Days)In

divi

dual

Tot

al M

MF

Dos

e (m

g/kg

/day

)

Figure 4: Individual dosage of myco-

phenolate mofetil in six cynomolgus

monkeys starting treatment on day

45 after aortic transplantation. Dose

was adjusted according to animal

tolerability, PK and PD, with the

intention of reaching an individual

maximal tolerated dose. A mean total

MMF dose of 99.2–4.2 mg/kg was

reached for 9 treatment weeks.

105

+/–0

+15

–15

+/–0

+15

–1563 84 10563 84 10542 63 84

Rx1 Rx2 Rx3

Rx6Rx5Rx4

Time After Transplantation (Days)

Indi

vidu

al W

eigh

t Cha

nge

Afte

r S

tart

of T

reat

men

t (%

)

0–8

–6

–4

–2

21 42 63 84 105

0

2

4

6

8

10

A

B

controlMMFcontrol p = 0.02

Wei

ght C

hang

e A

fter

Tra

nspl

anta

tion

(mea

n %

± S

E)

Time After Transplantation (Days)

MMF treatment

Figure 5: Weight change normal-

ized to the preoperative weight as

an indicator of drug toxicity.

(a) From POD 63 on weight loss

was significantly different between

the two groups (p ¼ 0.02). (b)

Individual weight changes after start

of MMF treatment in combination

with gastrointestinal side-effects

resulted in dose adjustments

(compare to Figure 4).

Treatment of Graft Vascular Disease by MMF

American Journal of Transplantation 2003; 3: 817–829 823

87.8 –12 mm3 on day 105. However, this remodeling did

not have any clinical effects: mean intraluminal blood pres-

sure differences proximal and distal to the graft did not

change significantly between POD 21(1.5 –0.9 mmHg)

and POD 105 (5.3 –1.6 mmHg; p¼ 0.3).

Overall influence on graft vascular disease of delayed

MMF treatment starting 45 days after aortic transplant-

atio assessed by serial IVUS measurements. Before

starting MMF treatment on day 45, IVUS values were

not statistically significantly different between the control

42 63 84 105

Time After Transplantation (Days)

0

5

10

15

20

25

30

0

2

4

6

8

10

Mea

n A

bsol

ute

PC

NA

Exp

ress

ion

(%)

SE

±

Mea

n M

PATr

oug h

Pla

sma

Con

cent

r atio

n (m

g /L)

SE

±

PCNA expression

MPA plasma level

Figure 6: Pharmacokinetic and

pharmacodynamic monitoring of

animals treated with MMF after

aortic transplantation. Trough MPA

levels were measured twice weekly

and MPA levels of higher than 2 mg/L

were aimed for. Pharmacodynam-

ics was assessed by measuring

lymphocyte proliferation using flow

cytometry. After mitogen stimulation,

absolute intracellular PCNA expres-

sion was detected in cells, which

were in the S, G2 or M phase of the

cell cycle. Five per cent of absolute

PCNA expression or less was aimed

for.

Table 2: Means–SEM of hemogram and blood chemistry results from untreated and MMF treated cynomolgus monkeys with aortic

allografts on day 42 before start of treatment and at the end of the treatment on day 105 and the levels of significance for differences

p-value d

Control a p-value b MMF c p-value b (Control vs. MMF)

Hemogram & chemistry day 42 day 105 (42–105) day 42 day 105 (42–105) day 42 day 105

Hemoglobin (mg/dL) 13.2–0.3 12.8– 0.3 0.5 12.6– 0.3 11.1– 0.8 0.1 0.3 0.1

Hematocrit (%) 41 –0.8 40.5– 1.1 0.4 39.9– 0.9 35.5– 0.8 0.3 0.1 0.1

RBC (103/nL) e 6.5 – 0.1 6.3 – 0.2 0.2 6.1– 0.2 5.2 –0.4 0.05 0.07 0.03

WBC (/nL) e 9.2 – 1.3 8.2 – 0.6 0.7 11.2– 0.9 7.5 –1.4 0.1 0.3 0.7

Platelet count (/nL) 356– 33 331– 23 0.5 399– 30 306–33 0.1 0.3 0.6

Total protein (mg/dL) 7.1 – 0.1 6.9 – 0.1 0.2 6.8– 0.2 6.3 –0.2 0.1 0.2 0.01

Albumin (mg/dL) 3.9 – 0.1 3.9 – 0.2 0.9 3.9– 0.1 3.8 –0.2 0.3 0.5 0.3

Amylase (IU/L) 303– 31 278– 33 0.2 340– 50 261–20 0.4 0.8 1.0

Lipase (IU/L) 10.8– 1.6 15– 2.9 0.2 9.4– 1.9 6.7 –1.5 0.05 0.6 0.02

Creatinine (mg/dL) 1.2 – 0.1 1.1 – 0.05 0.3 0.9– 0.05 0.8 –0.1 0.3 0.08 0.09

BUN (mg/dL) e 13.5–1.8 11.5– 0.5 0.7 10.2– 0.8 9 –1.6 0.8 0.2 0.5

Total bilirubin (mg/dL) 0.2– 0.05 0.18– 0.02 0.8 0.2– 0.05 0.13– 0.02 0.02 0.8 0.09

Conj. bilirubin (mg/dL) 0.05–0.02 0.03– 0.01 0.5 0.04– 0.001 0.03– 0.01 0.01 1.0 0.3

AST (IU/L) e 40 –3 35– 2 0.3 34– 3 24– 1 0.04 0.3 0.01

ALT (IU/L) e 19 –5 33– 16 0.6 24– 3 18– 3 0.3 0.3 0.8

AP (IU/L) e 164– 17 141– 14 0.5 229– 19 192–15 0.3 0.04 0.06

Cholesterol (mg/dL) 98 –6 92– 8 0.8 94– 4 75– 8 0.05 0.7 0.2

Triglycerides (mg/dL) 49 –6 74– 12 0.06 55– 5 50– 8 0.5 0.4 0.1

Glucose (mg/dL) 42.1–5.3 55.8– 7.1 0.13 45.3– 4.3 53.3– 4.3 0.4 0.5 0.7

LDH (IU/L) e 508– 96 413–71 0.5 460– 83 273–30 0.1 0.7 0.2

CPK (IU/L) 356– 80 233–71 0.2 488– 205 285–147 0.18 0.6 0.8

aControl animals did not receive any immunosuppressive treatment from day 0 to 105.bWilcoxon signed-rank test for two dependent samples.cMMF treatment from days 45 to 103.dMann–Whitney U-test for independent samples.

RBC – Red blood cell count, WBC – White blood cell count, BUN – urea, AST – aspartate amino transferase, ALT – alanine amino

transferase, AP – alkaline phosphatase, LDH – lactate dehydrogenase, CPK – Creatinine phosphokinase.

Klupp et al.

824 American Journal of Transplantation 2003; 3: 817–829

and the treatment groups: mean vessel volumes for animals

in the treatment group were 166– 15 mm3 vs. 183– 17 mm3

in the control group; intimal volumes 25–3 mm3 vs.

25–2 mm3; and lumen volumes 141– 13 mm3 vs.

158– 17 mm3, respectively. The intraluminal blood pres-

sures differences were not significantly different (control:

3.2– 2 mmHg, MMF 5.5– 1.9 mmHg; p¼ 0.3).

In contrast to the control group (p¼ 0.028), VV in the

treatment group did not change significantly between

the beginning of the study (154 – 18 mm3) and day 105

(138– 13 mm3). However, due to high inter-individual

differences within the animals of the MMF group, no

statistically significant differences in mean VV were

found between treatment and control group on day 105

(p¼ 0.3).

Lumen volumes (LV) did not differ significantly among

animals in the control and MMF treatment groups. On

POD 105, mean LV was 104 –16 mm3 in the control and

90 –6 mm3 in the treatment group. Cross-sections with

minimal lumen areas (LA) were 7.8 –0.5 mm2 and

8.7– 1.2 mm2 and with maximal LA 9.7– 0.7 and

11.5– 1.8 mm2 in MMF and control groups, respectively.

Overall, the progression of intimal volume did increase

significantly between start of treatment on day 45 and

the end of the study on day 105 in both the control

(p< 0.028) and MMF groups (p< 0.043). In a general

linear model, evaluating the overall difference in

development of intimal volumes between days 42 and

105, there were no significant differences among animals

in the control and MMF treatment groups (p¼ 0.3;

Figure 7). The progression of graft vascular disease

decreased in MMF-treated animals, but the differences

between MMF and control groups were not statistically

significant on day 84 (43– 7 vs. 52 – 4 mm3, respectively,

p¼ 0.32) or on day 105 (47 –7 vs. 55 – 2 mm3, respec-

tively, p¼ 0.37).

Overall influence of MMF treatment on graft vascular

disease assessed by histology. Grafts from untreated

animals showed histological signs of graft vascular dis-

ease that were indistinguishable from human allograft

vasculopathy. The GVD consisted of concentric fibrointimal

proliferation of smooth muscle cells and myofibroblasts,

containing scattered macrophages and lymphocytes in a

collagenous matrix of intima. Smooth muscle cell a-actin

staining confirmed that the intima in allografts was com-

prised predominantly of smooth muscle cells. The internal

elastic membrane was partly fractured, and partly pre-

served with reduplication. The medial layer was obscured

by fibrous tissue that extended into the surrounding

adventitial soft tissue. Perivascular inflammation was min-

imal in one graft, focal in one, band-like in one and multifocal

in three. No calcifications or cholesterol plaques were

noticed. Semi-quantitative assessment of GVD was mild in

one animal, moderate in two animals and severe in three.

In contrast, in the MMF-treated animals GVD was mild in

two animals, moderate in three and severe in one graft.

This histology did not differ significantly from results in

the untreated controls (p¼ 0.3). Also, the damage to the

internal elastic membrane and the perivascular inflam-

mation were not statistically different.

The histomorphometric analysis correlated highly with the

IVUS morphometry (r2¼ 0.88, p< 0.001). However,

despite the pressurized preservation with formalin, the

grafts shrank, and intimal areas measured by histomorpho-

metry were by 31.6% smaller than areas measured by

IVUS.

Mean intimal areas measured by histomorphometry failed

to be statistically different between the control

(4.2– 0.4 mm2) and MMF treatment groups (2.9– 0.5 mm2;

p¼ 0.078). Differences in mean intimal thickening, how-

ever, were close to being significant between the groups

(control: 504– 44 mm, MMF 410– 47 mm; p¼ 0.065).

The medial areas were significantly lower in the MMF-

treated animals (1.5 –0.1 mm2) than in the untreated

animals (2.3 – 0.3 mm2 (p¼ 0.016). However, there was

no difference in lumen areas between the groups

(control: 4.2– 0.5 mm2; MMF: 3.9 – 0.4 mm2; p¼ 0.87).

Individual development of graft vascular disease after

delayed MMF treatment starting on day 45 after aortic

transplantation. In contrast to the grafts from control

animals, the extent of development of graft vascular dis-

ease showed a high variation among the MMF-treated

monkeys. Whereas the IVUS measurements in all the

untreated animals showed a homogenous distribution in

intimal volume between 47 and 67 mm3 in the aortic grafts

on day 105, the animals in the treatment group could be

divided into two subpopulations: four out of six animals

showed significantly less intimal thickening, than each of

the control animals (p< 0.02; Figure 8). In the other two

Mea

n IV

US

–In

timal

Vol

ume

SE

(m

m)

±3

Time After Transplantation (Days)

0

10

20

30

40

50

60

70

0 21 42 63 84 105

controlMMF

MMF treatment

p = 0.3

Figure 7: Mean intimal volume in the mid-segment of aortic

allografts from untreated and MMF treated cynomolgus

monkeys assessed by IVUS studies at 3-week intervals.

Treatment of Graft Vascular Disease by MMF

American Journal of Transplantation 2003; 3: 817–829 825

animals (Rx5, Rx6), intimal proliferation progressed

unhindered and did not respond to the MMF treatment.

These resultswereconfirmedbyqualitativeandquantitative

histological analysis (Figure 9).

Correlations between the mixed leukocyte reaction, the

MMF dose, pharmacokinetic and pharmacodynamic

values and graft vascular disease (Table 3). The stimula-

tion index of MLR correlated positively with intimal

volume measured by IVUS on day 105 and intimal area

measured by histomorphometry (r2¼ 0.88, p¼ 0.02 and

r2¼ 0.86, p¼ 0.03, respectively). Mean total daily MMF

dose correlated highly with the magnitude of GVD on

day 105 (r2¼ 0.89, p< 0.015; Figure 10). In the two

animals that did not respond to MMF treatment, the

daily MMF dose had to be reduced substantially for a

longer period of time, due to toxicity (animals Rx5, Rx6;

Figures 4 and 8). Although, the absolute or relative per

cent expression of lymphocyte proliferation (PCNA) in

blood collected at the end of dosing intervals did not

correlate with the extent of GVD, the number of days

when inhibition of proliferation by MMF did not attain

95% of Emax correlated inversely with the magnitude of

intimal volumes on day 105 (r2¼ 0.82, p< 0.046).

Discussion

Conventional immunosuppressive agents fail to be com-

pletely effective in therapy and prevention of GVD (29,30).

Currently, only sirolimus (SRL) (3,21,29,31), and myco-

phenolate mofetil (MMF) appear to merit attention.

MMF has – besides its immunosuppressive effects on

lymphocytes – an inhibitory effect on smooth muscle cell

proliferation (4,9). In different balloon catheter injury

models in rodents, 40 mg/kg MPA (9) and 30 mg/kg

MMF (10) proved to be effective in preventing intimal

thickening. Our group first showed (32) that MPA is able

to reduce the incidence and severity of GVD in a Brown

Norway-to-Lewis transplant model. In addition, MMF

reduced the obliteration of renal arteries at a dose of

15 mg/kg/day (33) after rat kidney transplantation. Models

of isolated vessel transplantations have an advantage, in

Time After Transplantation (days)

1058463422100

20

40

60

80 controlIn

divi

dual

IVU

S –

Intim

al V

olum

e (m

m3 )

105846342210

MMF

MMF treatment

Rx6Rx5Rx4Rx3Rx2Rx1

Figure 8: Progression of intimal

volume (IV) in individual monkeys

in the control and in the MMF

treatment group as assessed by

IVUS. In four out of six animals in

the treatment group, the rate of

progression of intimal volume was

lower than in all animals of the

control group (p¼ 0.02). In animals

Rx5 and Rx6 MMF dose reduction

was necessarily due to weight loss

and gastrointestinal side-effects

(compare Figures 4 and 5b).

C1 C2 C3 C4 C5 C6 mean mean Rx1 Rx2 Rx3 Rx4 Rx5 Rx6

p = .87

p = .078

p = .016

0

2

4

6

8

10

12

14

16

18

Control MMF

His

tolo

gy–

Ves

sel A

reas

(m

m)2

Medial AreaIntimal AreaLumen Area

Figure 9: Histomorphometry from

the aortic allografts in untreated

and MMF treated animals on day

105 after transplantation, indivi-

dually (C1-C6, control; Rx1-Rx6,

MMF treatment) and summarized

as means + SE. Intimal area failed to

be significantly lower in the MMF

treated animals compared to the

untreated controls (2.9–0.5 mm2 and

4.2– 0.4 mm2, respectively; p¼ 0.078).

The medial area was significantly lower

in the MMF-treated animals than in the

control group (1.5 – 0.1 mm2 and

2.3–0.3 mm2, respectively; p¼ 0.016).

Klupp et al.

826 American Journal of Transplantation 2003; 3: 817–829

that no initial immunosuppressive therapy is needed to

secure graft function (34,35) and GVD can be investigated

independently. In an ACI-to-Lewis model of orthotopic

aortic transplantation, intimal proliferation was reduced

by 76.1% with the daily treatment of 40–30 mg/kg MMF

(8). MMF was less, but still significantly effective, when

treatment was delayed by 2 weeks or 4 weeks.

These results in rodents encouraged us to investigate the

effect of MMF after allogenic aortic transplantation in

non-human primates. As previously published, this pro-

cedure has many advantages over the rodent studies

(19,21,36,37). Additionally, this present study investigated

the effect of MMF on advanced GVD: in the first 6 weeks

after orthotopic aortic transplantation cynomolgus

monkeys did not receive any immunosuppressive treat-

ment at all. During this time, intimal proliferation developed

unhindered and drug efficacy in treating advanced

lesions could be studied. Our protocol was designed in

such a way that each animal was treated with an indi-

vidualized, maximal tolerated dose of MMF. We intended,

thereby, to evaluate the efficacy of ideal MMF treatment

for GVD.

To determine the maximal tolerated dose for a 2-month

period of treatment in cynomolgus monkeys, various pre-

studies were performed. Previous toxicological studies in

non-human primates (Roche data on file) showed that

MMF at a dose of 150 mg/kg/day leads to gastrointestinal

toxicity, necessitating MMF withdrawal within 4 weeks. In

our phase I pharmacokinetic and pharmacodynamic pre-

studies, we showed that, with 100 mg/kg/day MMF QD

dosing, all animals suffered from severe weight loss and

gastrointestinal toxicity (Figure 2). After 14 and 21 days of

MMF treatment, in all animals the dose had to be reduced

stepwise to 50 mg/kg/day. However, the pharmacokinetics

and pharmacodynamics in this experiment showed that the

lower dosage was not able to provide sufficient MPA

plasma levels for inhibition of lymphocyte proliferation, as

measured by intracellular PCNA expression.

In rodent studies (27,28,38), we showed earlier that PK

and PD parameters correlate highly with the immunosup-

pressive efficacy of MMF. We were even able to prove

that sustained suppression of lymphocyte proliferation

throughout the dosing interval was necessary to prevent

graft rejection and to show the beneficial effect of BID vs.

QD dosing (27).

With our BID therapeutic regimen in cynomolgus monkeys,

we were able to dose the animals starting on day 45 after

transplantation for 2 months with a mean total daily MMF

dose of 99.2– 4.2 mg/kg. The individual susceptibility to

MMF toxicity showed a high variation, resulting in daily

MMF doses between 30 and 120 mg/kg (Figure 4). How-

ever, none of the animals was withdrawn from the study

due to uncontrollable toxicity.

The primary endpoint of our present study was the intimal

thickening on day 105 after transplantation. Three days

before beginning treatment in the MMF group, the intimal

volume was identical in both groups. By day 105, IV in the

mid-graft in the control group increased to 55.2 –2.8 mm3,

and in the MMF group to 47.8 – 7.2 mm3 and there was no

overall difference between the groups (p ¼0.3). The dif-

ferences in histomorphometry between the two groups

just failed to be significant (p¼ 0.07), but the postmortem

shrinkage of the grafts made histomorphometry less

reliable than IVUS measurements.

However, and most important, there was a clear inter-

animal variability for progression of GVD in the MMF

0

20

40

60

80

0 80 90 100 110 120

Mean Total Daily MMF Dose (mg/kg/day)Between Day 45–103 After Aortic Transplantation

IVU

S –

Intim

al V

olum

e D

ay 1

05 (

mm

3 )

r2 = 0.89p < 0.015

Rx6Rx5Rx4Rx3Rx2Rx1

Figure 10: Total daily MMF dose (mg/kg/day) correlates

significantly with the intimal volume on day 105 assessed

using IVUS morphometry (r2 = 0.89; p< 0.015).

Table 3: Correlation between mixed leukocyte reaction (MLR),

ischemia time, pharmacokinetics, pharmacodynamics and graft

vascular disease measured by intravascular ultrasound (IVUS) and

histomorphometry on day 105 after aortic transplantation in the

mycophenolate mofetil (MMF) treatment group

IVUS (IV) Histology (IA)

r2 p r2 p

MLR 0.88 0.02 0.86 0.03

Ischemia time 0.18 0.5 0.34 0.3

Mean daily MMF dose 0.89 0.015 0.85 0.03

Mean MPA trough levels 0.07 0.9 0.02 0.9

Days with MPA trough 0.15 0.7 0.21 0.6

<2.0 mg/L

Mean PCNA expression 0.16 0.7 0.21 0.7

Days with PCNA expression 0.14 0.7 0.10 0.8

>5.0 abs%

Days with PCNA expression 0.82 0.046 0.80 0.058

<95% Emax

IV –intimal volume, IA – intimal area, MPA – mycophenolic acid,

PCNA – proliferating cell nuclear antigen.

Treatment of Graft Vascular Disease by MMF

American Journal of Transplantation 2003; 3: 817–829 827

treatment group: four out of six MMF-treated animals

showed an intimal volume that was lower than in each

of the control animals. In the other two animals, progres-

sion of GVD was unhindered (Figure 8). In the treatment

group, the stimulation index (SI) of the MLR correlated

with GVD, and both ‘non-responding’ animals (Rx5, Rx6)

had SI’s above 10.

Mean total daily MMF dose correlated highly with IV meas-

ured by IVUS on day 105 and with IA as measured by

histomorphometry (Figure 10; Table 3). In both ‘non-

responding’ animals, the dose of MMF had to be reduced

substantially for more than 50% of the treatment days due

to MMF toxicity.

Neither MMF pharmacokinetics nor trough pharmaco-

dynamic markers correlated with IV or IA on day 105

(Table 3). However, taking the drug sensitivity of each

animal into account, the number of days when 95% of

inhibition of maximal lymphocyte proliferation was not

reached correlated significantly with the extension of

GVD. This finding, as well as the results from our rodent

studies (27), supports the hypothesis that an insufficient

immunosuppressive effect, measured by pharmaco-

dynamics in the peripheral blood, predicts acute or chronic

rejection of the graft.

In summary, there was no overall statistically significant

difference in progression of GVD between the untreated

control and the MMF treatment groups. However, there

was a high correlation between the total daily MMF dose

and intimal hyperplasia. Depending on the individual

tolerability of MMF in cynomolgus monkeys, MMF was

effective for treatment of advanced GVD after aortic trans-

plantation. This efficacy was achieved when intimal prolif-

eration had already proceeded unhindered for more

than 6 weeks after transplantation due to lack of any

immunosuppression. Since we have shown in rodent

studies that MPA and SRL more effectively inhibit intimal

thickening than either drug alone (9), combination

therapy of MMF and SRL may be even more effective

for controlling GVD in non-human primates or patients.

With this study in non-human primates, the foundation

has been laid for randomized, double-blind clinical,

studies investigating the value of MMF for prevention or

treatment of GVD.

Acknowledgments

We gratefully acknowledge the technical assistance of Randi Shorthouse

and the editorial support of Kate Hinke. We also thank Dr Elsie Eugui for

her excellent advice. This study was funded in part by Roche Laboratories,

Inc. (Nutley, NJ). It has also been supported by the Ralph and Marian Falk

Trust and by the Hedco Foundation. Dr Jochen Klupp received grants (KL

1214/1–1/1–2) from the ‘Deutsche Forschungsgemeinschaft’; Dr Teun van

Gelder, from the Dutch Kidney Foundation, and Dr Camille Dambrin, from

the French Federation of Cardiology.

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