Metabolism and Biochemistry of Mycophenolic Acid

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

Mycophenolic acid is a new, experimental oncolytic agent Labeled Mycophenolic Acid. Mycophenolic acid, labeled that interferes in the interconversion of inosine, xanthosine, uniformly or in the ring-methyl, ring-methoxy moieties, was and guanosine monophosphates. IMP dehydrogenase, which extracted after the incubation of uniformly labeled acetate or converts IMP -@XMP, and GMP synthetase, which converts methionine-5-' 4CH3 with a culture of Penicillium XMP -Ã·GMP, are inhibited by mycophenolic acid. The IMP stoloniferum. @ dehydrogenase from a human adenocarcinoma of the colon Measurement of Expired 4C02 . Rats or mice were dosed was more sensitive to mycophenolic acid than was the enzyme with labeled mycophenolic acid and were immediately placed from murine tumors. in respirometer chambers. Incoming air was drawn through the There is a correlation between the sensitivity of respirometer into a radiation detection chamber and an experimental tumors to mycophenolic acid and the relative infrared scanner for measurement of CO2 . The apparatus was activities of @3-glucuronidase and hypoxanthine-guanine comparable to that described by Tolbert et a!. ( 19). The @ phosphoribosyltransferase. Mycophenolic acid glucuronide collection of 4C02 was continuous over periods of 16 to 24 is unable to cross the cell membrane; therefore the hr. Urine and feces were collected from the respirometer intracellular concentration of the free acid depends on the rate chamber, and the total excreted radioactivity was measured. of hydrolysis of the glucuronide by @3-glucuronidase. A Distribution of Mycophenolic Acid in Tissues. Two mice mechanism of resistance to mycophenolic acid is the were sacrificed by exsanguination at each time period. Tissues circumvention of the block in the nucleotide interconversion. were excised, weighed, and dissolved in a minimum volume of @ GMP is resupplied by conversion of guanine to its nucleotide NCS. Samples of the dissolved tissues were removed for by hypoxanthine-guanine phosphoribosyltransferase. In measurement (8, 10). Urine samples were added directly to 15 tumors resistant to mycophenolic acid, the transferase activity ml of Diotol. The feces were dried, ground, and suspended in is high; in tumors sensitive to mycophenolic acid, the activity NCS. Blood samples were treated to remove the color by the is low. The relative activities of these two enzymes in tumors addition of 0.1 ml of whole blood to a scintillation vial in man may indicate the potential effectiveness of containing 2 ml of NCS. A saturated solution of benzoyl mycophenolic acid in humans. peroxide (0.4 ml) was added, and the vial was gently agitated Mycophenolic acid is initially secreted into the bile and until the color faded. Scintillation fluid was added to the vial, @ excreted in the urine and feces of animals. 4CO2 was not and the total radioactivity was measured. Bile samples were detected in the expired air of mice, rats, or marmosets given collected by cannulation of the bile duct in rats and mice. 1 4C-labeled mycophenolic acid. The only metabolite detected GMPSase. A 55% (NH@)2 504 precipitate of tissue in mice, rats, rabbits, and humans was mycophenolic acid homogenates was used as the source of the GMPSase enzyme glucuronide. (1). XMP-8.' 4C was used in the incubation mixture as described for the spectrophotometric assay (1). The reaction was stopped by immersion of the tubes in boiling water for 3 INTRODUCTION mm. The tubes were cooled, and the denatured protein was removed by centrifugation. A 25- to 50-j.zl sample of the supernatant was developed by ascending chromatography for Several preliminary reports have been published on the 18 hr with the use of 0.2 M ammonium formate at pH 5.0 on antitumor activity of mycophenolic acid and on the uptake Whatman DE-8l DEAE-cellulose paper. GMP moved 10.5 cm and distribution, the mode of action, and the possible in situ and XMP moved 3.4 cm from the origin. Since GMP could not activation by @-Gase'(5, 16, 18, 20). In a recent report, we be distinguished from xanthosine, a 2nd sample was compiled and updated the experimental oncolytic data (17). chromatographed for 4 hr with ethanol/i M ammomum This presentation brings together all of our results concerning acetate 75/30 (v/v), at pH 7.5, on Brinkman MN polygram the mode of action of mycophenolic acid as well as the 1st Cel-300 plates. The RF value for XMP was 0.1 1; for GMP, report on a 2nd mechanism of resistance to this drug. 0.10; and for xanthosine, 0.27. The amount of xanthosine was subtracted from the GMP level as determined by I The abbreviations used are: @-Gase, p-glucuronidase; GMPSase, GMP DEAE-cellulose chromatography. synthetase; XMP, xanthosine monophosphate; IMPDHase, IMP dehy drogenase; PRTase, hypoxanthine:guanine phosphoribosyltransferase; IMPDHase. The 45% ammonium sulfate precipitate was MAG, mycophenolic acid glucuromde. used as the source of the IMPDHase (7). ATP was added to the Received September 28, 1971;accepted May 9, 1972. incubation mixture (2) to inhibit the conversion of IMP -@

SEPTEMBER 1972 1803

inosine and XMP â€”@xanthosine. The reaction was stopped by accounted for in the gastrointestinal tract after 15 mm, immersion of the tubes in boiling water for 3 mm. The tubes indicating a rapid uptake of mycophenolic acid-' 4C . In 24 hr, were cooled in an ice bath before they were centrifuged. A 55% of the dose was excreted in the urine and < 23% was sample of the supernatant was developed by ascending excreted in the feces. Tissues from these mice were dissolved chromatography for 2 hr with 0.2 M ammomum formate on in NCS solution, and the total radioactivity was measured. Brinkman DEAE-coated plastic plates. The RF for XMP was Peak levels of radioactivity occurred in the tissues at 0.25 hr: 0.34 and for IMP it was 0.64. All paper and plastic blood, 2.9% of the dose; liver, 7.2%; kidney, 3.9%; and tumor, chromatographic strips were put through a Vanguard Model 0.9% (Table 1). 880 chromatographic scanner with recorder, and the areas When mycophenolic acid-' 4C was given i.m. in under the curves were used to quantitate the purine tumor-bearing mice, only 5% of the dose remained at the site intermediates. of injection after 15 mm. The left leg, which received no PRTase. The activity of PRTase from tumors and ascites injection, showed a peak accumulation of 0.9% of the cells was measured with the use of homogenates according to radioactivity at 15 mm. Urinary (38 to 50%) and fecal (3 1%) the method of Seegmiller et al. (12). The reaction was stopped values were similar to those seen after administration of a dose by the addition of 0.2 ml EDTA. The GMP..8-' 4C formed of mycophenolic acid-' 4C p.o. The tissues from those mice from guanine-8-' 4C was measured after a 20-jil sample of the were dissolved in NCS, and total radioactivity was measured. incubation mixture was chromatographed by ascending The peak levels were observed 15 mm after injection. chromatography on cellulose MN 300 plates for I 6 hr with However, the amounts were approximately twice as great as ethanol/ammonium acetate, 75/30 (v/v). The spots were those after a p.o. dose, with the exception of the lower value identified by the distance moved from the origin as follows: of2.i% in the kidneys. guanine, 10.2 cm ; GMP, 1.7 cm ; uric acid, 5.5 cm ; and Excretion in Bile. Bile was collected from rats by guanosine, 10.7 cm. cannulation for 4 hr after the administration of i.p. dose of 50 13-Gase. Two % homogenates of tissues were assayed for mg of mycophenolic acid-' 4C per kg. Sixty-nine % of the dose @3-Gaseby the standard phenolphthalein method (13). Triton was excreted in the bile in the 1st hr, and a total of 78.4% was X_100 at 0.2% v/v was added to the homogenates to release excreted in 3 hr. At 6 hr, an additional 14% of the dose was bound enzyme. found in the urine. Bile samples were chromatographed on thin-layer plates, with amyl acetate/acetic acid/propanol/water (4/3/2/ 1). The RESULTS plates were scanned with UV light and radiological monitors. Only 1 radioactive spot with an RF of 0.33 was detected; it Absorption, Distribution, and Excretion. The lack of gave a negative phenol test with FeCl3 . The mycophenolic acid 1 4CO2 in the expired air of mice, rats, and marmosets standard had an RF of 0.70 and gave a positive phenol following the i.p. or p.o. administration of 60 mg of reaction. Since phenolic substances are detoxified and mycophenolic acid-' 4C per kg indicated that there was no excreted as glucuronides, a sample of the bile was incubated metabolic degradation of the compound. At least 90% of the with bacterial @3-Gaseandthen was chromatographed. The spot administered radioactivity was found in the urine and feces previously seen at RF 0.33 was absent, and a new spot within 24 hr. appeared at RF 0.70 that gave a positive phenol reaction with Uniformly labeled mycophenolic acid, 60 mg/kg, was given FeCl3. P.O. to C3H mice bearing the X5563 plasma cell myeloma. At The metabolite was extracted also from the urine of various times, 2 mice were sacrificed, and the total rabbits that received mycophenolic acid and was compared radioactivity in the gastrointestinal tract, urine, and feces was with chemically synthesized MAG. The X-ray powder measured. Only one-half of the administered radioactivity was defraction and nuclear magnetic resonance spectra of the

Table 1 @ A bsorption, distribution, and excretion of mycophenolic acid-' C and metabolites in C3H mice bearing the X5563 plasma cell myeloma

theHr % of totaldoseâ€•foundin

ofdoseStomachintestineintestineUrineFecesBloodLivertumorKidneys0.258.029.00.42.97.20.93.90.515.024.012.00.11.23.00.32.7123.019.07.22.50.40.72.10.20.7225.010.014.05.80.71.12.80.40.644.721.012.04.70.10.82.10.21.461.118.07.24.10.30.51.90.30.9160.30.70.355.023.00.30.00.00.0240.10.50.437.016.00.20.10.00.0afterSmallLargePer g

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

1804 CANCER RESEARCH VOL. 32

CH3 OR 0

HOOC

CH3 RHMycophenolic Acid (MA)

COOH H H = R Mycophenolic Acid Glucuronide (MAG) OHH HO H H OH Chart 1. Structures of mycophenolic acid and MAG.

80 MICE

E 60 E U, U, 50 E E a, 40 ,â€”,. 30

20 x @ 10@ .@ . MAG @ I p -1 15 30 60 90 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. The that given to mice, the total amount of free drug was 50-fold glucuronyl moiety is associated with the phenolic hydroxy greater in the rats (Chart 2). group (Chart 1). Mode of Action. Cline et al. (3) reported that the inhibition Plasma Concentrations. The initial studies with of virus growth by mycophenolic acid in cell cultures was mycophenolic acid-' 4C did not distinguish between free and prevented when either guanine, guanosine, or GMP was added conjugated mycophenolic acid. In subsequent studies, the 2 to the medium. The hypoxanthine and xanthine metabolites forms were measured in plasma (R. J. Bopp, R. E. Shirmer, did not prevent the inhibition by mycophenolic acid. We and D. B. Myers. The Determination of Mycophenolic Acid performed similar studies in cell cultures to determine whether and Its Glucuronide Metabolite in Biological Samples. J. the guanine metabolites would prevent mycophenolic acid Pharm. Sci., submitted for publication, 1972.). Both mice and from inhibiting the growth of cell cultures. L-cells were grown rats were given p.o. doses of 45 mg of monosodium in purine-free Eagle's minimum essential medium. The mycophenolic acid per kg, representing 0.9 mg/mouse and 9.0 addition of inosine, xanthosine, or guanosine to cultures mg/rat. The peak concentration of mycophenolic acid and without mycophenolic acid did not alter the growth rate of MAG in plasma occurred after 15 mm in mice. Both forms of the L-cells. Alone, mycophenolic acid depressed the growth of the drug appeared in the same proportion. In rats, the free the cells to 22% of that of the controls. When either mycophenolic acid concentration peaked at 15 mm and the xanthosine or inosine was added to the medium with glucuronide peaked at 30 to 60 mm. The ratio of mycophenolic acid, the rate of growth of the cultures was 24 mycophenolic acid to MAG was 3/1 at 15 mm. Although the to 35% of that of the control culture. The addition of total amount of drug given to the rats was 10-fold greater than guanosine at either 10 or 20 times the concentration of

SEPTEMBER 1972 1805

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

cell rate control)CNone1.06100Inosine44.01.0195Inosme88.01.07101Xanthosine44.00.9792Xanthosine88.00.9589Guanosine44.01.06100Guanosine88.01.06100MycophenolicAdditionsâ€•Concentration (@tM)Final count (X 106 )bGrowth(% of

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

a Cells were grown in purine-free MEM for 24 hr prior to the addition of mycophenolic acid and/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)nucleosidesare averages of 3 tests; 20 x (88.0 @M)nucleosidesare averages of 2 tests. Table 3 mycophenolic acid than was the enzyme from the Mecca Inhibition by mycophenolic acid of GMPSasefrom varioustumors lymphosarcoma (Table 5). The amide and glucuronide of mycophenolic acid also showed comparable inhibition of response to IC,0b IMPDHase from the Mecca lymphosarcoma, the Ca-755 @ M)Walker SourceTumor mycophenolic acidâ€•G(XMPSase10 mammary carcinoma, and the Ridgeway osteogenic sarcoma (Table 6). (solid)3+8.4Meccacarcinosarcoma256 lymphosarcoma3+7.6Adenocarcinoma LandschUtz Ascites Cells. Franklin and Cook (8) also found 7552+7.6C1498 that IMPDHase from the LandschUtz ascites tumor was (solid)2+8.8Ridgewayleukemia inhibited by mycophenolic acid. The K, for mycophenolic acid sarcoma1+8.8Gardnerosteogenic was 3.03 X 10-s M, and showed mixed inhibition when lymphosarcoma1+8.2Ehrlich ascites tumorâ€”7.0 evaluated against the enzyme from these LandschÃ¼tzascites cells. For a comparison of the relative importance of the @ a @sto 100% inhibition of growth; 2+, 50 to 74%; 1+, 30 to inhibition of IMPDHase and GMPSase by mycophenolic acid, a 49%; â€”,0to 29%. Data were obtained from previous studies (17). sample of the LandschUtz ascites cells was obtained from the @ b , concentration of mycophenolic acid giving 50% inhibition of Imperial Chemicals Industries, Ltd. (Macclesfield , Cheshire, enzyme activity. England) by our laboratories. The GMPSase was purified from the LandschUtz ascites cells. The K1 for mycophenolic acid was mycophenolic acid in the medium resulted in growth rates of 8 X l08 M as determined from a Lineweaver-Burk plot of 84 and 92% of that of the control culture (Table 2). activity versus concentration of substrate (Chart 3). The The interconversion of the purine monophosphates was inhibition of GMPSase was also of the mixed type as was that considered a possible site of inhibition. Therefore, both of IMPDHase. IMPDHase and GMPSase were equally sensitive IMPHDase and GMPSase were partially purified from several to mycophenolic acid, and the relative importance of the two tumors. The mycophenolic acid inhibited GMPSase from is not known. tumors that were either sensitive or resistant to it (Table 3). The activities of several derivitives of mycophenolic acid, Activation by @3-Gase.The glucuronide of mycophenolic including MAG, were compared with those of mycophenolic acid represents about 50% of the circulating form of the drug acid. The carboxamide and MAG, which showed no in vitro in mouse plasma and 30 to 50% of the drug form in the plasma effect on cell growth, inhibited the GMPSase to the same in rats. In general, the glucuronide of any drug is a detoxified degree as did mycophenolic acid (Table 4). form and is considered metabolically inactive (15). The IMPDHase was purified from tumors that were either inability ofglucuronides freely to penetrate the cell membrane sensitive or resistant to mycophenolic acid. Regardless of the reduces the biological activity of the parent compound. In cell source of the IMPDHase, mycophenolic acid showed similar cultures, the MAG is inactive at 25 times the active inhibition of the enzymes. However, IMPDHase from a human concentration of mycophenolic acid. However, if MAG is adenocarcinoma of the colon and from the LandschUtz ascites preincubated with j3-Gase, cell growth is inhibited (Table 7). cells was more sensitive to the inhibitory action of These data, along with the data from Tables 4 and 6, which show that MAG was active against the cell-free preparations of

1806 CANCER RESEARCH VOL. 32

Table 4 Table 6 Inhibition of GMPSasebyderivativesof mycophenolic acid Effect of mycophenolic acid and derivativeson JMPDHase

IC50a for IMPDHase l04M)MycophenolicCompound Source ofenzyme (X

acid Mecca lymphosarcoma 9 Mycophenolic acid Adenocarcinoma 755 6 Mycophenolic acid Ridgeway osteogenic 5 sarcoma AntitumorRR'IC,,,â€•activity1'OHCOOH7.6 Mycophenolicacid Meccalymphosarcoma 4 â€”glucuronide 10@+++O-glucuronic x Mycophenolic acidâ€”amide Adenocarcinoma 755 4 10@'NTCCONHC2H5COOH7.2XacidCOOH7.4 X (CONH2) l0@+++OCH3COOH7.4x Mycophenolic acidâ€”amide Ridgeway osteogenic 5 10â€•-SHCOOHNI-OHCONH29.OX (CONH2)sarcomaa

i0â€•- IC, @,, concentration of mycophenolic acid that causes a 50% inhibition of enzyme activity.

AntitumorRIC, ,,activity-CH2COOHl.2X 10'NT-CH2CH=CCH3COOHNI--(CH2)2CHCH3(CH2)2COOHNI-Mycophenolic

acid7.6 X iO@+++

a IC, ,, , concentration of mycophenolic acid giving 50% inhibition of enzyme activity. @@i.oolL2:o b Data were obtained from previous studies (17). +++, 100% inhibition of the growth of the Mecca lymphosarcoma in mice; â€”,no ._:!__. io-4 activity. [XMP] C The abbreviations used are: NT, not tested due to insufficient quantity; NI, no inhibition. Chart 3. The Lineweaver-Burk plot of GMPSase from the LandschÃ¼tz @ ascites cells. Substrate concentration: o, XMPat 10 M;X, XMPwith Table 5 myocophenolic acid at 2 X l0' M. The K1 calculated from these data Inhibition ofJMPDHase by mycophenolic acid was 2 x 108 M for mycophenolic acid.

concentration inhibition IMPDHase and GMPSase, confirm the hypothesis that the IMPDHaseMeccaTumor systemMolar of mycophenolic acid% of MAG does not penetrate the cell membrane. The sensitivity or resistance of certain tumors to mycophenolic acid may be lymphosarcoma2.0 X i0@ 7.6x 10@ 42 dependent on their ability to hydrolyze the glucuronide via l1.0x 10-a 79 13-Gase. A survey of j3-Gase activities in a broad spectrum of 100Adenocarcinoma 15_Ox 10-a22 tumors indicated a correlation between drug sensitivity and high enzyme activity. The resistant tumors showed low 7552.0 X l0@ enzyme activity. The only ascites tumor that responded to 3.8x l0@ 31 7.6X l0@ 62 mycophenolic acid was the Walker 256 ascites, which had 4 95Ridgeway 19.Ox 10-a20 times the j3-Gase activity of those cells that did not respond (Table 8). osteogenic sarcoma3.8 X 10â€• Guanine Salvage Pathway. The ability of GMP to reduce the 7.6x l0@ 78 100Mecca 15.0x i0-@40 inhibitory effect of mycophenolic acid on cell cultures and viruses in vitro indicated that no metabolic block occurs lymphosarcoma (mouse) 9.0 x 10@ between GMP and DNA-RNA synthesis. Therefore, the cellular LandschUtz ascites cells (mouse) 2.5 X l08 50â€• synthesis of GMP in some tumors could be sufficient to Adenocarcinoma of the colon (man) 1.7 X l0850â€• 50â€• circumvent the block of the conversion of XMP â€”@GMPby a These values are reported at the concentration that gave 50% mycophenolic acid. Guanosine or guanine can be obtained via inhibition. the diet or by the salvaging of the metabolites from nucleic

SEPTEMBER 1972 1807

Table 7 showed activity against cell-free enzymes but were inactive Effect ofmycophenolic acid and MAG on the against the intact tumors (17). The diversity and responses of growth of L-cells in culture various tumors and ascites tumors to mycophenolic acid may be based on their 13-Gase and PRTase activities. In Table 8, % inhibition of growth (after addition tumors are listed in order of decreasing sensitivity to onDayto medium) mycophenolic acid and are compared with the @3-Gaseand PRTase activities. No single enzyme activity can fully explain DayAdditions Day the mycophenolic acid sensitivity. Some exceptions occur in 3None0 to mediaâ€•1 2 each enzyme correlation. However, if both enzymes are 0j3-Gase, 0 considered, a definite correlation between enzyme activities 2013-Gase,1 1.25 units/ml0 21 and tumor sensitivity emerges. Therefore, control of the 4MAG,11.25@g/ml02.25 units/ml0 0 transport of mycophenolic acid and the alternate route of GMP synthesis may account for the diverse responses to this 0MAG, 0 0Mycophenolic2.25 @sg/ml0 0 Table 8 56Mycophenolicacid, 1.5 @zg/ml25 58 g3-GaseandPRTaseversussensitivity of tumors to mycophenolic acid 60MAG, acid + jl-Gase, 2.25 units/ml25 58 inhibition or 93k'MAG,11.25 @ig,+@-Gase,11.25units/mi70 88 prolongation 2.25 big,+ @-Gase,2.25units/mi14 2 1 O@ mycophenolic acid of life a The media were preincubated with the additions for 6 hr before being added to the cell cultures. This permitted the hydrolysis of MAG MAWalker Tumora-Gase (unitsâ€•)PRTase(unitsb)% by in the medium with gI-Gase. 256carcinosarcoma19160+++@Mecca b After 24 hr, 6.4 j@gMAG per ml were hydrolyzed to give 4.25 @tg mycophenolic acid per ml of medium. lymphosarcoma42168++-i Ca-l55mammary30273-â€˜--FC1498 @ C After 24 hr, 0.6 MAG per ml was hydrolyzed to give 0.4 @g mycophenolic acid per ml of medium. (solid)22459+Gardnerleukemia lymphosarcoma25372+X5563 myeloma204441-C3H acid turnover. The only means for converting them to GMP is mammary18332+Ridgeway by the salvage pathway that is controlled by PRTase. Tumors sarcoma19101+Ca-ll5mammary32566â€”B-82osteogenic with low PRTase activities, unable to synthesize sufficient (solid)44480â€”S-180(solid)20223â€”Walkerleukemia amounts of GMP, should be more susceptible to mycophenolic acid than are tumors with high PRTase activities. The PRTase activity was assayed in all of the tumors and ascites cells that ascites9687+++Ll2l0ascites25587â€”L1210-Illascites28513L1210-Vascites24523â€”L1210TGresistant'@250â€”Ehrlich256 were sensitive or resistant to mycophenolic acid. There was a partial correlation between high PRTase activity and resistance to mycophenolic acid. Those tumors that were sensitive to mycophenolic acid had low PRTase activities. However, not all TGresistant150â€”Ehrlich tumors with low PRTase activities were sensitive (Table 8). ascites12336â€”S-l80ascites9471â€”Mecca

lymphosarcoma42168+++Gardner DISCUSSION lymphosarcoma25372+Ca-l55mammary30273++C3H

Mycophenolic acid is a metabolically stable compound. There was no detectable â€˜4C02 in the expired air of mice, mammary18336+Ca-ll5mammary32566â€”Mecca rats, and marmosets that received either uniformly labeled or methyl-labeled mycophenolic acid. The only metabolic lymphosarcoma42168+++B-82 alteration of the drug detected was its conjugation with (solid)44480â€”Meccaleukemia glucuronic acid, the normal mechanism for the detoxification lymphosarcoma42168+++Ridgeway of phenolic compounds. In urine, 90 to 100% of the excreted osteogenic sarcoma19101+ mycophenolic acid is recovered as MAG. This extensive detoxification probably accounts for the low toxicity in a Unit, activity of enzyme releasing 1 @tgofphenolphthalein per mg animals (17). protein in 1 hr at 37Â°,pH4.5. The biological activity of mycophenolic acid results from its b Unit, activity of enzyme forming 1 nmole of GMP per mg protein in lhrat3lÂ°,pH7.6. @ interference in the interconversion of inosine, xanthosi.ne, and C 75 to 100% inhibition of tumor growth or prolongation of life guanosine nucleotides. The 2 enzymes controlling these in animals with ascites tumors or leukemia; ++, 50 to 74%;+, 30 to conversions, IMPDHase and GMPSase, are inhibited by 49%; â€”,less than 30% and considered as no response. Data were mycophenolic acid. Both of these enzymes were equally obtained from previouspublication (17). inhibited whether they were purified from tumors that were d TG, thioguanine resistant. Both the Ll2lO TG- and Ehrlich TG-resistant ascites tumors were obtained from Dr. LePage, M. D. sensitive to mycophenolic acid or from those that were Anderson Hospital. These ascites tumors have no detectable PRTase resistant to it. Several derivatives of mycophenolic acid also activity.

1808 CANCER RESEARCH VOL. 32

drug in tumors of similar morphology. The Mecca and Gardner Phosphate and L-Glutamine. Arch. Biochem. Biophys., 79: lymphosarcomas differ in their response to mycophenolic acid 91â€”110,1959. and in j3-Gase and PRTase activities. The Ca-755 and Ca-i 15 2. Atkinson, M. R., Morton, R. K., and Murray, A. W. Inhibition of mammary carcinomas show identical j3-Gase activities. Inosine 5'-Phosphate Dehydrogenase from Ehrlich Ascites-Tumor However, the controlling factor may be the high level of Cells by 6-Thioinosine 5'-Phosphate. Biochem. J., 89: 167â€”172, 1963. PRTase activity of the Ca-i 15 carcinoma as compared with the 3. Cline, J. C., Nelson, J. D., Gerzon, K., Williams, R. H., and low activity in the Ca-755. The Mecca lymphosarcoma and DeLong, D. C. In Vitro Antiviral Activity of Mycophenolic Acid B-82 leukemia (solid) showed different responses that seem to and Its Reversal by Guanine-Type Compounds. AppI. Microbiol., correlate only with the PRTase activity, since both tumors 18: 14â€”20,1969. have similar (3-Gase activities. Finally, both the Mecca 4. DeDuve, C. Lysosomes and Chemotherapy. In: R. J. C. Harris lymphosarcoma and the Ridgeway osteogenic sarcoma have (ed.), Biological Approaches to Cancer Chemotherapy, pp. low PRTase activities, and their differences in response seem 101â€”112.New York: Academic Press, Inc., 1966. to rely on the transport of the mycophenolic acid as mediated S. Esterman, M. A., and Sweeney, M. J. Mycophenolic Acid: Studies by j3-Gase activities (Table 8). of the Possible Mode of Action. Proc. Am. Assoc. Cancer Res., 11: 24, 1970. The lack of response to mycophenolic acid by ascites tumor 6. Fishman, W. H. @3-Glucuronidase. In: Chemistry of Drug cells may be due to high PRTase activities. Also, the inability Metabolism, pp. 124â€”149. Springfield, Ill: Charles C Thomas, of the cells to hydrolyze MAG must have some control, Publisher, 1961. especially in the thioguanine-resistant ascites cells, since those 7. Franklin, T. J., and Cook, J. M. The Inhibition of Nucleic Acid cells have no PRTase . j3-Gase activity in some ascites tumor Synthesis by Mycophenolic Acid. Biochem. J., 113: 515 â€”524, cells is comparable to other solid tumors. However, it is also 1969. possible that the @3-Gaseinthese cells is not associated with or 8. Herberg, R. J. Determination of Carbon-14 and Tritium in Blood cannot be transported to the cell membrane. Similar and Other Whole Tissues. Liquid Scintillation Counting of Tissues. possibilities are considered also in the continuous membrane Anal. Chem., 32: 42â€”46,1960. systems in which enzymes may be carried to the cell surface 9. Ide, H., and Fishman, W. H. Dual Localization of @-Glucuronidase and Acid Phosphatase in Lysosomes and in Microsomes. II. by lysosomes or vesicles (9, 11, 14). Membrane-Associated Enzymes. Histochemie, 20: 300â€”321,1969. The measurement of the j3-Gase and PRTase activities in 10. Kobayashi, Y., and Maudsley, D. V. Practical Aspects of Liquid biopsy specimens of human tumors may indicate the potential Scintillation Counting. Methods Biochem. Analysis, 1 7: 55 â€”133, therapeutic effectiveness of mycophenolic acid in man. 1969. Tumors displaying high 13-Gase/lowPRTase activities would be 11. Koenig, H., and Jibril, A. Acidic Glycolipids and the Role of Ionic more likely candidates for treatment than would tumors with Bonds in the Structure-Linked Latency of Lysosomal Hydrolyases. low j3-Gase/high PRTase activities. Biochim. Biophys. Acta, 65: 543â€”545,1969. The detoxification of mycophenolic acid by glucuronida 12. Seegmiller, J. E., Rosenbloom, F. M., and Kelley, W. N. Enzyme tion may account for the relatively low toxicity. This could be Defect Associated with a Sex-Linked Human Neurological Disorder an additional asset for differential therapy. Therapy that made and Excessive Purine Synthesis. Science, 155: 1682â€”1684,1967. 13. Sigma Bulletin No. 105, December 1958. use of compounds in the form of glucuronides, or that were 14. Smith, R. E. Phosphohydrolases in Cell Organelles: Electron capable of forming glucuronides, has been proposed before (4, Microscopy. Ann. N. Y. Acad. Sci., 166: 525â€”564, 1969. 6, 21). The high activities of @3-Gaseinmany tumors should 15. Smith, R. L., and Williams, R. T. Implications of the Conjugation provide a high concentration of the active drug to these of Drugs and Other Exogenous Compounds. In: G. J. Dutton (ed.), tissues, in preference to the lower (3-Gase-containing normal Glucuronic Acid, p. 476. New York: Academic Press, Inc., 1966. tissues. An additional advantage of drugs that are rapidly 16. Sweeney, M. J., Cline, J. C., and Williams, R. H. Antitumor and detoxified is their use in regional perfusion. The active drug Antiviral Activities of Mycophenolic Acid. Proc. Am. Assoc. can be administered directly to the tumor for a maximum Cancer Res., 10: 90, 1969. concentration effect. Any drug that bypasses or perfuses the 17. Sweeney, M. J., Gerzon, K., Harris, P. N., Holmes, R., Poore, G. A., tumor and enters into the general circulation will be detoxified and Williams, R. H. Experimental Antitumor Activity and by the liver. Precinical Toxicology of Mycophenolic Acid. Cancer Res., 32: 1795â€”1802,1972. 18. Sweeney, M. J., Hoffman, D. H., and Poore, G. A. Possible in Situ ACKNOWLEDGMENTS Activation of Mycophenolic Acid by @-Glucuronidase. Cancer Res., We are appreciative of the assistance of Mr. Robert Johnson in the 31:477â€”478,1971. studies on tissue culture. The preparation of the labeled mycophenolic 19. Tolbert, B. M., Kirk, M., and Baker, E. M. Continuous C' @O2and acid by Dr. W. 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SEPTEMBER 1972 1809

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

Cancer Res 1972;32:1803-1809.

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