metabolism and biochemistry of mycophenolic acid[cancer research 32, 1803â€”1809, september...

[CANCER RESEARCH 32, 1803â€”1809, September 1972

MATERIALS AND METHODS

Mycophenolic acid is a new, experimental oncolytic agentthat interferes in the interconversion of inosine, xanthosine,and guanosine monophosphates. IMP dehydrogenase, whichconverts IMP -@XMP, and GMP synthetase, which convertsXMP -Ã·GMP, are inhibited by mycophenolic acid. The IMPdehydrogenase from a human adenocarcinoma of the colonwas more sensitive to mycophenolic acid than was the enzymefrom murine tumors.

There is a correlation between the sensitivity ofexperimental tumors to mycophenolic acid and the relativeactivities of @3-glucuronidase and hypoxanthine-guaninephosphoribosyltransferase. Mycophenolic acid glucuronideis unable to cross the cell membrane; therefore theintracellular concentration of the free acid depends on the rateof hydrolysis of the glucuronide by @3-glucuronidase. Amechanism of resistance to mycophenolic acid is thecircumvention of the block in the nucleotide interconversion.GMP is resupplied by conversion of guanine to its nucleotideby hypoxanthine-guanine phosphoribosyltransferase. Intumors resistant to mycophenolic acid, the transferase activityis high; in tumors sensitive to mycophenolic acid, the activityis low. The relative activities of these two enzymes in tumorsin man may indicate the potential effectiveness ofmycophenolic acid in humans.

Mycophenolic acid is initially secreted into the bile andexcreted in the urine and feces of animals.@ 4CO2 was notdetected in the expired air of mice, rats, or marmosets given1 4C-labeled mycophenolic acid. The only metabolite detected

in mice, rats, rabbits, and humans was mycophenolic acidglucuronide.

INTRODUCTION

Several preliminary reports have been published on theantitumor activity of mycophenolic acid and on the uptake

and distribution, the mode of action, and the possible in situactivation by @-Gase'(5, 16, 18, 20). In a recent report, wecompiled and updated the experimental oncolytic data (17).This presentation brings together all of our results concerningthe mode of action of mycophenolic acid as well as the 1streport on a 2nd mechanism of resistance to this drug.

I The abbreviations used are: @-Gase, p-glucuronidase; GMPSase, GMP

synthetase; XMP, xanthosine monophosphate; IMPDHase, IMP dehydrogenase; PRTase, hypoxanthine:guanine phosphoribosyltransferase;MAG, mycophenolic acid glucuromde.

Received September 28, 1971;accepted May 9, 1972.

Labeled Mycophenolic Acid. Mycophenolic acid, labeleduniformly or in the ring-methyl, ring-methoxy moieties, wasextracted after the incubation of uniformly labeled acetate ormethionine-5-' 4CH3 with a culture of Penicilliumstoloniferum.

Measurement of Expired@ 4C02 . Rats or mice were dosedwith labeled mycophenolic acid and were immediately placedin respirometer chambers. Incoming air was drawn through therespirometer into a radiation detection chamber and aninfrared scanner for measurement of CO2 . The apparatus wascomparable to that described by Tolbert et a!. ( 19). Thecollection of@ 4C02 was continuous over periods of 16 to 24hr. Urine and feces were collected from the respirometerchamber, and the total excreted radioactivity was measured.

Distribution of Mycophenolic Acid in Tissues. Two micewere sacrificed by exsanguination at each time period. Tissueswere excised, weighed, and dissolved in a minimum volume ofNCS. Samples of the dissolved tissues were removed for@measurement (8, 10). Urine samples were added directly to 15ml of Diotol. The feces were dried, ground, and suspended inNCS. Blood samples were treated to remove the color by theaddition of 0.1 ml of whole blood to a scintillation vialcontaining 2 ml of NCS. A saturated solution of benzoylperoxide (0.4 ml) was added, and the vial was gently agitateduntil the color faded. Scintillation fluid was added to the vial,and the total radioactivity was measured. Bile samples werecollected by cannulation of the bile duct in rats and mice.

GMPSase. A 55% (NH@)2 504 precipitate of tissuehomogenates was used as the source of the GMPSase enzyme(1). XMP-8.' 4C was used in the incubation mixture asdescribed for the spectrophotometric assay (1). The reactionwas stopped by immersion of the tubes in boiling water for 3mm. The tubes were cooled, and the denatured protein wasremoved by centrifugation. A 25- to 50-j.zl sample of thesupernatant was developed by ascending chromatography for18 hr with the use of 0.2 M ammonium formate at pH 5.0 onWhatman DE-8l DEAE-cellulose paper. GMP moved 10.5 cmand XMP moved 3.4 cm from the origin. Since GMP could notbe distinguished from xanthosine, a 2nd sample waschromatographed for 4 hr with ethanol/i M ammomumacetate 75/30 (v/v), at pH 7.5, on Brinkman MN polygramCel-300 plates. The RF value for XMP was 0.1 1; for GMP,0.10; and for xanthosine, 0.27. The amount of xanthosine wassubtracted from the GMP level as determined byDEAE-cellulose chromatography.

IMPDHase. The 45% ammonium sulfate precipitate wasused as the source of the IMPDHase (7). ATP was added to theincubation mixture (2) to inhibit the conversion of IMP -@

SEPTEMBER 1972 1803

Metabolism and Biochemistry of Mycophenolic Acid

Martin J. Sweeney, David H. Hoffman, and Michail A. Esterman

The Lilly ResearchLaboratories, Eli Lilly and Company, Indianapolis, Indiana 46206

SUMMARY

on July 18, 2020. © 1972 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

% of totaldoseâ€•foundintheHr

afterSmallLargePer gofdoseStomachintestineintestineUrineFecesBloodLivertumorKidneys0.258.029.00.42.97.20.93.90.515.024.012.00.11.23.00.32.7123.019.07.22.50.40.72.10.20.7225.010.014.05.80.71.12.80.40.644.721.012.04.70.10.82.10.21.461.118.07.24.10.30.51.90.30.9160.30.70.355.023.00.30.00.00.0240.10.50.437.016.00.20.10.00.0

M. J. Sweeney, D. H. Hoffman, and M. A. Esterman

accounted for in the gastrointestinal tract after 15 mm,indicating a rapid uptake of mycophenolic acid-' 4C . In 24 hr,55% of the dose was excreted in the urine and < 23% wasexcreted in the feces. Tissues from these mice were dissolvedin NCS solution, and the total radioactivity was measured.Peak levels of radioactivity occurred in the tissues at 0.25 hr:blood, 2.9% of the dose; liver, 7.2%; kidney, 3.9%; and tumor,0.9% (Table 1).

When mycophenolic acid-' 4C was given i.m. intumor-bearing mice, only 5% of the dose remained at the siteof injection after 15 mm. The left leg, which received noinjection, showed a peak accumulation of 0.9% of theradioactivity at 15 mm. Urinary (38 to 50%) and fecal (3 1%)values were similar to those seen after administration of a doseof mycophenolic acid-' 4C p.o. The tissues from those micewere dissolved in NCS, and total radioactivity was measured.The peak levels were observed 15 mm after injection.However, the amounts were approximately twice as great asthose after a p.o. dose, with the exception of the lower valueof2.i% in the kidneys.

Excretion in Bile. Bile was collected from rats bycannulation for 4 hr after the administration of i.p. dose of 50mg of mycophenolic acid-' 4C per kg. Sixty-nine % of the dosewas excreted in the bile in the 1st hr, and a total of 78.4% wasexcreted in 3 hr. At 6 hr, an additional 14% of the dose wasfound in the urine.

Bile samples were chromatographed on thin-layer plates,with amyl acetate/acetic acid/propanol/water (4/3/2/ 1). Theplates were scanned with UV light and radiological monitors.Only 1 radioactive spot with an RF of 0.33 was detected; itgave a negative phenol test with FeCl3 . The mycophenolic acidstandard had an RF of 0.70 and gave a positive phenolreaction. Since phenolic substances are detoxified andexcreted as glucuronides, a sample of the bile was incubatedwith bacterial @3-Gaseand then was chromatographed. The spotpreviously seen at RF 0.33 was absent, and a new spotappeared at RF 0.70 that gave a positive phenol reaction withFeCl3.

The metabolite was extracted also from the urine ofrabbits that received mycophenolic acid and was comparedwith chemically synthesized MAG. The X-ray powderdefraction and nuclear magnetic resonance spectra of the

inosine and XMP â€”@xanthosine. The reaction was stopped byimmersion of the tubes in boiling water for 3 mm. The tubeswere cooled in an ice bath before they were centrifuged. Asample of the supernatant was developed by ascendingchromatography for 2 hr with 0.2 M ammomum formate onBrinkman DEAE-coated plastic plates. The RF for XMP was0.34 and for IMP it was 0.64. All paper and plasticchromatographic strips were put through a Vanguard Model880 chromatographic scanner with recorder, and the areasunder the curves were used to quantitate the purineintermediates.

PRTase. The activity of PRTase from tumors and ascitescells was measured with the use of homogenates according tothe method of Seegmiller et al. (12). The reaction was stoppedby the addition of 0.2 ml EDTA. The GMP..8-' 4C formedfrom guanine-8-' 4C was measured after a 20-jil sample of theincubation mixture was chromatographed by ascendingchromatography on cellulose MN 300 plates for I 6 hr withethanol/ammonium acetate, 75/30 (v/v). The spots wereidentified by the distance moved from the origin as follows:guanine, 10.2 cm ; GMP, 1.7 cm ; uric acid, 5 .5 cm ; andguanosine, 10.7 cm.

13-Gase. Two % homogenates of tissues were assayed for@3-Gaseby the standard phenolphthalein method (13). TritonX_100 at 0.2% v/v was added to the homogenates to releasebound enzyme.

RESULTS

Absorption, Distribution, and Excretion. The lack of1 4CO2 in the expired air of mice, rats, and marmosets

following the i.p. or p.o. administration of 60 mg ofmycophenolic acid-' 4C per kg indicated that there was nometabolic degradation of the compound. At least 90% of theadministered radioactivity was found in the urine and feceswithin 24 hr.

Uniformly labeled mycophenolic acid, 60 mg/kg, was givenP.O. to C3H mice bearing the X5563 plasma cell myeloma. Atvarious times, 2 mice were sacrificed, and the totalradioactivity in the gastrointestinal tract, urine, and feces wasmeasured. Only one-half of the administered radioactivity was

Table 1A bsorption, distribution, and excretion of mycophenolic acid-'@ C and metabolites in

C3H mice bearing the X5563 plasma cell myeloma

a Total dose was 60 mg/kg, p.0, equivalent to 1.2 mg/mouse. Calculated for weight of the entire organ or volume (exceptfor the tumor).

1804 CANCER RESEARCH VOL. 32



Metabolism and Biochemistry ofMycophenolic Acid

= R Mycophenolic Acid Glucuronide (MAG)

Chart 1. Structures of mycophenolic acid and MAG.

EU,

E,â€”,.

MICE

EU,

Ea,

80

70

60

50

40

30

20

10@

MINUTES AFTER DOSING

Chart 2. Plasma concentrations after a p.o. dose of either free mycophenolic acid in mice (Xâ€”X) and in rats (oâ€”o) or MAG in mice (X- - -X)and in rats (0. - -o). Blood volumes were calculated at 7% of body weight; mice, 1.4 ml; rats, 14.0 ml. MA, mycophenolic acid.

synthetic and biosynthetic MAG were identical. Theglucuronyl moiety is associated with the phenolic hydroxygroup (Chart 1).

Plasma Concentrations. The initial studies withmycophenolic acid-' 4C did not distinguish between free andconjugated mycophenolic acid. In subsequent studies, the 2forms were measured in plasma (R. J. Bopp, R. E. Shirmer,and D. B. Myers. The Determination of Mycophenolic Acidand Its Glucuronide Metabolite in Biological Samples. J.Pharm. Sci., submitted for publication, 1972.). Both mice andrats were given p.o. doses of 45 mg of monosodiummycophenolic acid per kg, representing 0.9 mg/mouse and 9.0mg/rat. The peak concentration of mycophenolic acid andMAG in plasma occurred after 15 mm in mice. Both forms ofthe drug appeared in the same proportion. In rats, the freemycophenolic acid concentration peaked at 15 mm and theglucuronide peaked at 30 to 60 mm. The ratio ofmycophenolic acid to MAG was 3/1 at 15 mm. Although thetotal amount of drug given to the rats was 10-fold greater than

that given to mice, the total amount of free drug was 50-foldgreater in the rats (Chart 2).

Mode of Action. Cline et al. (3) reported that the inhibitionof virus growth by mycophenolic acid in cell cultures wasprevented when either guanine, guanosine, or GMP was addedto the medium. The hypoxanthine and xanthine metabolitesdid not prevent the inhibition by mycophenolic acid. Weperformed similar studies in cell cultures to determine whetherthe guanine metabolites would prevent mycophenolic acidfrom inhibiting the growth of cell cultures. L-cells were grownin purine-free Eagle's minimum essential medium. Theaddition of inosine, xanthosine, or guanosine to cultureswithout mycophenolic acid did not alter the growth rate ofthe L-cells. Alone, mycophenolic acid depressed the growth ofthe cells to 22% of that of the controls. When eitherxanthosine or inosine was added to the medium withmycophenolic acid, the rate of growth of the cultures was 24to 35% of that of the control culture. The addition ofguanosine at either 10 or 20 times the concentration of

SEPTEMBER 1972 1805

CH3 OR 0

HOOC

CH3

RHMycophenolic Acid (MA)

COOH

HHOHH

HO H

H OH

x

@ .@ .MAG

I@ p -1

15 30 60 90



Additionsâ€•Concentration (@tM)Final

cellcount (X 106 )bGrowth

rate(% ofcontrol)CNone1.06100Inosine44.01.0195Inosme88.01.07101Xanthosine44.00.9792Xanthosine88.00.9589Guanosine44.01.06100Guanosine88.01.06100Mycophenolic

acid4.40.2322Mycophenolicacid + inosine4.4 +44.00.2524Mycophenolicacid + inosine4.4 1-88.00.3432Mycophenolicacid + xanthosine4.4 +44.00.2725Mycophenolicacid + xanthosine4.4 +88.00.3735Mycophenolicacid + guanosine4.4 +44.00.8984Mycophenolicacid + guanosine4.4 + 88.00.9892

SourceTumor

responseto

mycophenolic acidâ€•G(XMPSaseIC,0b

10@ M)Walker

carcinosarcoma256(solid)3+8.4Meccalymphosarcoma3+7.6Adenocarcinoma7552+7.6C1498

leukemia(solid)2+8.8Ridgewayosteogenicsarcoma1+8.8Gardner

lymphosarcoma1+8.2Ehrlichascites tumorâ€”7.0

M. J. Sweeney, D. H. Hoffman, andM. A. Esterman

Table 2Effect ofpurine nucleosides on the activity of mycophenolic acid against L-cells

a Cells were grown in purine-free MEM for 24 hr prior to the addition of mycophenolic acidand/or the nucleosides.Mediawere replaced each 24 hr.

b Initial cell count was 0.13 X 106 cells; final cell count was taken 4 days after additions

were made.C Cell count at 4 days with no additions was arbitrarily set as the 100% growth rate. Results

with 10 X (44.0 @M)nucleosides are averages of 3 tests; 20 x (88.0 @M)nucleosides areaverages of 2 tests.

Table 3 mycophenolic acid than was the enzyme from the Meccalymphosarcoma (Table 5). The amide and glucuronide ofmycophenolic acid also showed comparable inhibition ofIMPDHase from the Mecca lymphosarcoma, the Ca-755mammary carcinoma, and the Ridgeway osteogenic sarcoma(Table 6).

LandschUtz Ascites Cells. Franklin and Cook (8) also foundthat IMPDHase from the LandschUtz ascites tumor wasinhibited by mycophenolic acid. The K, for mycophenolic acidwas 3.03 X 10-s M, and showed mixed inhibition whenevaluated against the enzyme from these LandschÃ¼tzascitescells. For a comparison of the relative importance of theinhibition of IMPDHase and GMPSase by mycophenolic acid, asample of the LandschUtz ascites cells was obtained from theImperial Chemicals Industries, Ltd. (Macclesfield , Cheshire,England) by our laboratories. The GMPSase was purified fromthe LandschUtz ascites cells. The K1 for mycophenolic acid was8 X l08 M as determined from a Lineweaver-Burk plot ofactivity versus concentration of substrate (Chart 3). Theinhibition of GMPSase was also of the mixed type as was thatof IMPDHase. IMPDHase and GMPSase were equally sensitiveto mycophenolic acid, and the relative importance of the twois not known.

Activation by @3-Gase.The glucuronide of mycophenolicacid represents about 50% of the circulating form of the drugin mouse plasma and 30 to 50% of the drug form in the plasmain rats. In general, the glucuronide of any drug is a detoxifiedform and is considered metabolically inactive (15). Theinability ofglucuronides freely to penetrate the cell membranereduces the biological activity of the parent compound. In cellcultures, the MAG is inactive at 25 times the activeconcentration of mycophenolic acid. However, if MAG ispreincubated with j3-Gase, cell growth is inhibited (Table 7).These data, along with the data from Tables 4 and 6, whichshow that MAG was active against the cell-free preparations of

Inhibition by mycophenolic acid of GMPSasefrom various tumors

a@ @sto 100% inhibition of growth; 2+, 50 to 74%; 1+, 30 to49%; â€”,0 to 29%. Data were obtained from previous studies (17).

b@ , concentration of mycophenolic acid giving 50% inhibition ofenzyme activity.

mycophenolic acid in the medium resulted in growth rates of84 and 92% of that of the control culture (Table 2).

The interconversion of the purine monophosphates wasconsidered a possible site of inhibition. Therefore, bothIMPHDase and GMPSase were partially purified from severaltumors. The mycophenolic acid inhibited GMPSase fromtumors that were either sensitive or resistant to it (Table 3).

The activities of several derivitives of mycophenolic acid,including MAG, were compared with those of mycophenolicacid. The carboxamide and MAG, which showed no in vitroeffect on cell growth, inhibited the GMPSase to the samedegree as did mycophenolic acid (Table 4).

IMPDHase was purified from tumors that were eithersensitive or resistant to mycophenolic acid. Regardless of thesource of the IMPDHase, mycophenolic acid showed similarinhibition of the enzymes. However, IMPDHase from a humanadenocarcinoma of the colon and from the LandschUtz ascitescells was more sensitive to the inhibitory action of




IC50a forIMPDHase

Compound Source ofenzyme (Xl04M)Mycophenolic

acid Mecca lymphosarcoma 9Mycophenolic acid Adenocarcinoma 755 6Mycophenolic acid Ridgeway osteogenic 5

sarcoma

Mycophenolicacid Meccalymphosarcoma 4â€”glucuronide

Mycophenolic acidâ€”amide Adenocarcinoma 755 4(CONH2)

Mycophenolic acidâ€”amide Ridgeway osteogenic 5(CONH2)sarcomaa

IC, @,, concentration of mycophenolic acid that causes a 50%inhibition of enzyme activity.

@@i.oolL2:o

AntitumorRR'IC,,,â€•activity1'OHCOOH7.6

x10@+++O-glucuronicacidCOOH7.4 X10@'NTCCONHC2H5COOH7.2X

l0@+++OCH3COOH7.4x10â€•-SHCOOHNI-OHCONH29.OX

i0â€•-

AntitumorRIC,,,activity-CH2COOHl.2X

10'NT-CH2CH=CCH3COOHNI--(CH2)2CHCH3(CH2)2COOHNI-Mycophenolic

acid7.6 X iO@+++

Tumor systemMolarconcentration

of mycophenolic acid%inhibition

ofIMPDHaseMecca

lymphosarcoma2.0 X [email protected] 10@

l1.0x 10-a15_Ox 10-a22

4279

100Adenocarcinoma

7552.0 X [email protected] [email protected] [email protected] 10-a20

3162

95Ridgeway

osteogenic sarcoma3.8 X 10â€•7.6x l0@

15.0x i0-@4078

100Mecca

lymphosarcoma (mouse) 9.0 x 10@LandschUtz ascites cells (mouse) 2.5 X l08Adenocarcinoma of the colon (man) 1.7 X l0850â€•

50â€•50â€•


Table 4

Inhibition of GMPSaseby derivativesof mycophenolic acidTable 6

Effect of mycophenolic acid and derivativeson JMPDHase

a IC, ,, , concentration of mycophenolic acid giving 50% inhibition ofenzyme activity.

b Data were obtained from previous studies (17). +++, 100%inhibition of the growth of the Mecca lymphosarcoma in mice; â€”,noactivity.

C The abbreviations used are: NT, not tested due to insufficient

quantity; NI, no inhibition.

Table 5Inhibition ofJMPDHase by mycophenolic acid

a These values are reported at the concentration that gave 50%inhibition.

SEPTEMBER 1972 1807

._:!__. io-4

[XMP]

Chart 3. The Lineweaver-Burk plot of GMPSase from the LandschÃ¼tzascites cells. Substrate concentration: o, XMPat 10@ M;X, XMPwithmyocophenolic acid at 2 X l0' M. The K1 calculated from these data

was 2 x 108 M for mycophenolic acid.

IMPDHase and GMPSase, confirm the hypothesis that theMAG does not penetrate the cell membrane. The sensitivity orresistance of certain tumors to mycophenolic acid may bedependent on their ability to hydrolyze the glucuronide via13-Gase. A survey of j3-Gase activities in a broad spectrum oftumors indicated a correlation between drug sensitivity andhigh enzyme activity. The resistant tumors showed lowenzyme activity. The only ascites tumor that responded tomycophenolic acid was the Walker 256 ascites, which had 4times the j3-Gase activity of those cells that did not respond(Table 8).

Guanine Salvage Pathway. The ability of GMP to reduce theinhibitory effect of mycophenolic acid on cell cultures andviruses in vitro indicated that no metabolic block occursbetween GMP and DNA-RNA synthesis. Therefore, the cellularsynthesis of GMP in some tumors could be sufficient tocircumvent the block of the conversion of XMP â€”@GMP bymycophenolic acid. Guanosine or guanine can be obtained viathe diet or by the salvaging of the metabolites from nucleic



% inhibition of growth(after additionto medium)onDay

DayDayAdditionsto mediaâ€•1 23None0

00j3-Gase,1 1.25 units/ml0 212013-Gase,2.25 units/ml0 04MAG,11.25@g/ml0

00MAG,2.25 @sg/ml0 00Mycophenolic

acid, 1.5 @zg/ml25 5856Mycophenolicacid + jl-Gase, 2.25 units/ml25 5860MAG,

11.25 @ig,+@-Gase,11.25 units/mi70 8893k'MAG,2.25 big,+ @-Gase,2.25 units/mi14 2 1 O@

Tumora-Gase (unitsâ€•)PRTase(unitsb)%

inhibition orprolongation

mycophenolicacid of life

byMAWalker

256carcinosarcoma19160+++@Meccalymphosarcoma42168++-i

Ca-l55mammary30273-â€˜--FC1498leukemia(solid)22459+Gardner

lymphosarcoma25372+X5563myeloma204441-C3Hmammary18332+Ridgeway

osteogenicsarcoma19101+Ca-ll5mammary32566â€”B-82

leukemia(solid)44480â€”S-180(solid)20223â€”Walker

256ascites9687+++Ll2l0ascites25587â€”L1210-Illascites28513L1210-Vascites24523â€”L1210TGresistant'@250â€”Ehrlich

TGresistant150â€”Ehrlichascites12336â€”S-l80ascites9471â€”Mecca

lymphosarcoma42168+++Gardnerlymphosarcoma25372+Ca-l55mammary30273++C3H

mammary18336+Ca-ll5mammary32566â€”Mecca

lymphosarcoma42168+++B-82leukemia(solid)44480â€”Mecca

lymphosarcoma42168+++Ridgewayosteogenic sarcoma19101+

M. J. Sweeney, D. H. Hoffman, and M. A. Esterman

Table 7Effect ofmycophenolic acid and MAG on the

growth of L-cells in culture

showed activity against cell-free enzymes but were inactiveagainst the intact tumors (17). The diversity and responses ofvarious tumors and ascites tumors to mycophenolic acid maybe based on their 13-Gase and PRTase activities. In Table 8,tumors are listed in order of decreasing sensitivity tomycophenolic acid and are compared with the @3-GaseandPRTase activities. No single enzyme activity can fully explainthe mycophenolic acid sensitivity. Some exceptions occur ineach enzyme correlation. However, if both enzymes areconsidered, a definite correlation between enzyme activitiesand tumor sensitivity emerges. Therefore, control of thetransport of mycophenolic acid and the alternate route ofGMP synthesis may account for the diverse responses to this

Table 8g3-Gaseand PRTaseversussensitivity of tumors to mycophenolic acid

a The media were preincubated with the additions for 6 hr beforebeing added to the cell cultures. This permitted the hydrolysis of MAGin the medium with gI-Gase.

b After 24 hr, 6.4 j@gMAG per ml were hydrolyzed to give 4.25 @tgmycophenolic acid per ml of medium.

C After 24 hr, 0.6@ MAG per ml was hydrolyzed to give 0.4 @g

mycophenolic acid per ml of medium.

acid turnover. The only means for converting them to GMP isby the salvage pathway that is controlled by PRTase. Tumorswith low PRTase activities, unable to synthesize sufficientamounts of GMP, should be more susceptible to mycophenolicacid than are tumors with high PRTase activities. The PRTaseactivity was assayed in all of the tumors and ascites cells thatwere sensitive or resistant to mycophenolic acid. There was apartial correlation between high PRTase activity and resistanceto mycophenolic acid. Those tumors that were sensitive tomycophenolic acid had low PRTase activities. However, not alltumors with low PRTase activities were sensitive (Table 8).

DISCUSSION

Mycophenolic acid is a metabolically stable compound.There was no detectable â€˜4C02 in the expired air of mice,rats, and marmosets that received either uniformly labeled ormethyl-labeled mycophenolic acid. The only metabolicalteration of the drug detected was its conjugation withglucuronic acid, the normal mechanism for the detoxificationof phenolic compounds. In urine, 90 to 100% of the excretedmycophenolic acid is recovered as MAG. This extensivedetoxification probably accounts for the low toxicity inanimals (17).

The biological activity of mycophenolic acid results from itsinterference in the interconversion of inosine, xanthosi.ne, andguanosine nucleotides. The 2 enzymes controlling theseconversions, IMPDHase and GMPSase, are inhibited bymycophenolic acid. Both of these enzymes were equallyinhibited whether they were purified from tumors that weresensitive to mycophenolic acid or from those that wereresistant to it. Several derivatives of mycophenolic acid also

a Unit, activity of enzyme releasing 1 @tgof phenolphthalein per mgprotein in 1 hr at 37Â°,pH 4.5.

b Unit, activity of enzyme forming 1 nmole of GMP per mg proteinin lhrat3lÂ°,pH7.6.

C@ 75 to 100% inhibition of tumor growth or prolongation of life

in animals with ascites tumors or leukemia; ++, 50 to 74%;+, 30 to49%; â€”, less than 30% and considered as no response. Data wereobtained from previouspublication (17).

d TG, thioguanine resistant. Both the Ll2lO TG- and EhrlichTG-resistant ascites tumors were obtained from Dr. LePage, M. D.Anderson Hospital. These ascites tumors have no detectable PRTaseactivity.





drug in tumors of similar morphology. The Mecca and Gardnerlymphosarcomas differ in their response to mycophenolic acidand in j3-Gase and PRTase activities. The Ca-755 and Ca-i 15mammary carcinomas show identical j3-Gase activities.However, the controlling factor may be the high level ofPRTase activity of the Ca-i 15 carcinoma as compared with thelow activity in the Ca-755. The Mecca lymphosarcoma andB-82 leukemia (solid) showed different responses that seem tocorrelate only with the PRTase activity, since both tumorshave similar (3-Gase activities. Finally, both the Meccalymphosarcoma and the Ridgeway osteogenic sarcoma havelow PRTase activities, and their differences in response seemto rely on the transport of the mycophenolic acid as mediatedby j3-Gase activities (Table 8).

The lack of response to mycophenolic acid by ascites tumorcells may be due to high PRTase activities. Also, the inabilityof the cells to hydrolyze MAG must have some control,especially in the thioguanine-resistant ascites cells, since thosecells have no PRTase . j3-Gase activity in some ascites tumorcells is comparable to other solid tumors. However, it is alsopossible that the @3-Gasein these cells is not associated with orcannot be transported to the cell membrane. Similarpossibilities are considered also in the continuous membranesystems in which enzymes may be carried to the cell surfaceby lysosomes or vesicles (9, 11, 14).

The measurement of the j3-Gase and PRTase activities inbiopsy specimens of human tumors may indicate the potentialtherapeutic effectiveness of mycophenolic acid in man.Tumors displaying high 13-Gase/lowPRTase activities would bemore likely candidates for treatment than would tumors withlow j3-Gase/high PRTase activities.

The detoxification of mycophenolic acid by glucuronidation may account for the relatively low toxicity. This could bean additional asset for differential therapy. Therapy that madeuse of compounds in the form of glucuronides, or that werecapable of forming glucuronides, has been proposed before (4,6, 21). The high activities of @3-Gasein many tumors shouldprovide a high concentration of the active drug to thesetissues, in preference to the lower (3-Gase-containing normaltissues. An additional advantage of drugs that are rapidlydetoxified is their use in regional perfusion. The active drugcan be administered directly to the tumor for a maximumconcentration effect. Any drug that bypasses or perfuses thetumor and enters into the general circulation will be detoxifiedby the liver.

ACKNOWLEDGMENTS

We are appreciative of the assistance of Mr. Robert Johnson in thestudies on tissue culture. The preparation of the labeled mycophenolicacid by Dr. W. Max Stark and Mr. Steven Larsen is also greatlyappreciated. Special thanks are extended to Dr. Roger Schirmer for theassays of plasma levels of mycophenolic acid and to Dr. Robert Hosleyfor his advice in preparing the manuscript.

REFERENCES

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SEPTEMBER 1972 1809



1972;32:1803-1809. Cancer Res Martin J. Sweeney, David H. Hoffman and Michail A. Esterman Metabolism and Biochemistry of Mycophenolic Acid

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