Pharmacodynamics of the inhibition of GTP synthesis in vivo by mycophenolic acid

Pharmacodynamics of the inhibition of GTP synthesis in vivo by mycophenolic acid

Advan. Enzyme Regul. Vol. 34, pp. 107-117, 1994 Copyright ~ 1994 Elsevier ScienceLtd Printed in Great Britain. All rights reserved 0065-2571/94/$26.00...

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Advan. Enzyme Regul. Vol. 34, pp. 107-117, 1994 Copyright ~ 1994 Elsevier ScienceLtd Printed in Great Britain. All rights reserved 0065-2571/94/$26.00

Pergamon

0065-2571 (93)E0004-8 PHARMACODYNAMICS OF THE INHIBITION OF GTP SYNTHESIS IN VIVO BY MYCOPHENOLIC ACID TREVOR J. FRANKLIN and WILLIAM P. MORRIS Department of Cancer Research, Zeneca Pharmaceuticals, Alderley Park, Macclesfield SK10 4TG, U.K.

INTRODUCTION

Mycophenolic acid (MPA) (Fig. 1) is an antibiotic produced by several species of Pencillium and was probably first detected by Gosio in extracts of mouldy maize in 1896 (1, 2). Although the antimicrobial activity of MPA was described by Abraham in 1945 (3), it was not until 1952 that its structure was finally revealed (4). The antiproliferative activity of MPA against mammalian cells and against experimental tumors in rodents was reported by Carter et al. (5) and independently by Williams et al. (6). The site of action of the antibiotic was localized to inosine 5'-monophosphate dehydrogenase, E.C. 1.2.1.14 (IMPDH) (5, 7). MPA proved to be a potent and specific inhibitor of IMPDH and a detailed kinetic analysis of its interaction with IMPDH isolated from the protozoan Tritrichomonas foetus indicates that it is uncompetitive with IMP and non-competitive with NAD, and that its binding site may lie within the dinucleotide cleft of the enzyme (8). HO

!

O

oA.

/

FIG. 1. Mycophenolic acid.

Despite the promising activity of MPA against a wide range of experimental tumors in rodents (5, 6, 9), the compound was found to be inactive in human malignant diseases. The reason for this lack of activity is generally believed to be due to the rapid conjugation in vivo of the essential phenolic OH moiety to glucuronic acid. Indeed, the conversion to the biochemically inactive glucuronide is so rapid that it 107

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T. J. FRANKLINand W. P. MORRIS

frequently appeared in the plasma of patients dosed orally with MPA before the free acid itself (Dr D. S. Platt, personal communication). The clinical studies also showed that the plasma half life of unchanged MPA in intravenously dosed patients was very short, values of less than 30 min having been observed. Comparably short half lives have also been noted in the dog and rat (D. S. Platt, personal communication), (9). The discrepancy between the anti-tumor activity of MPA in rodents and its failure to affect human cancers may be due to the greater sensitivity of fast growing rodent tumors to the degree of inhibition of IMPDH achievable by a rapidly cleared drug, although direct evidence for this is lacking. The basis of the efficacy of MPA against human psoriasis is uncertain but it has been suggested that this may be due to the conversion of the glucuronide back to the parent compound by the high levels of 13-glucuronidase prevailing in psoriatic plaques (10). Although there is extensive pharmacokinetic data on MPA and its glucuronide in both animals and humans there appear to have been few attempts to determine the time course of the inhibition of IMPDH and GTP synthesis by MPA in vivo. Franklin and Cook (11) showed that there was maximal inhibition of the incorporation of [14C] hypoxanthine into nucleic acid guanine of Yoshida tumor cells taken from rats 1 hr after oral administration of the disodium salt of MPA. More recently we have examined the action of MPA on both the synthesis and levels of GTP and ATP in a proliferating (spleen) and non-proliferating (heart) tissue in the rat. We have also compared the efficacy of the free acid of MPA with that of its disodium salt and the recently introduced morpholinoethyl ester of MPA, otherwise known as Mofetil or RS-61443, which has striking immunosuppressive and graft-rejection suppressing properties in animals and humans (12) and which is said to lead to better bioavailability of the free acid by virtue of its greater solubility in the upper GI tract (13).

MATERIALS AND METHODS Materials. 8-[14C]Hypoxanthine (52 mCi/mmol) was obtained from Amersham International, UK, allopurinol from Sigma Chemical Co., Poole, Dorset, UK, tri-N-octylamine and Freon 11 from Aldrich Chemical Co., Gillingham, Dorset, UK. Mycophenolic acid, its sodium salt and the morpholinoethyl ester were all prepared at Zeneca Pharmaceuticals. In vivo labelling of acid-soluble purine nucleotides and their analysis by HPLC. Female albino Wistar rats (Alderley Park strain), 50-60 g body weight, were given ['4C]hypoxanthine, 10 p.C, by intraperitoneal injection at the same time as an intraperitoneal dose of allopurinol (to minimize the

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oxidation of [14C]hypoxanthine), 50 mg/kg of body weight 15 min before death. Spleens and hearts were dissected as quickly as possible after death and homogenized in ice-cold 10% trichloroacetic acid. The homogenate was held for 1 hr at 0°C before centrifugation to sediment the precipitated proteins and nucleic acids. The supernatant was neutralized with an equal vol of tri-N-octylamine (0.5 M) dissolved in Freon 11. Separation of GTP and ATP as single peaks was achieved on a Partisil T M 10 SAX column, 15 cm x 0.46 cm using isocratic elution with a mobile phase of 0.6 M ammonium phosphate buffer, pH 3.6 at a flow rate of 1.5-2.0 ml/min. The radioactivity of the effluent stream was monitored with a Berthold LB506-C detector and the UV with a Milton Roy SpectroMonitor 3100. The retention times of GTP and ATP were determined using pure standard compounds (Sigma Chemical Co.).

Administration of mycophenolic acid. The various forms of MPA were given orally at various intervals before the administration of [~4C]hypoxanthine and allopurinol, the insoluble free acid as an aqueous suspension and the disodium salt (MPAS) and morpholinoethyl ester (MPAME) as aqueous solutions. RESULTS Effect of disodium salt of mycophenolic acid on the synthesis and content of GTP in rat spleen and heart in vivo. MPAS was given orally to groups of 5 rats at various dose levels at 1 hr and 24 hr before the administration of allopurinol and [14C]hypoxanthine and the animals were killed 15 min later. HPLC analysis of the acid-soluble nucleotide extracts from spleens (Table 1A) indicated that even the lowest dose of MPAS (12.5 mg/kg) produced a marked effect on the incorporation of radiolabel into GTP 1.25 hr after dosing. The GTP content of the spleen was unaffected at 1.25 hr even at the highest dose of 100 mg/kg. In contrast, 24.5 hr after dosing with MPAS the effect of the lowest dose on the incorporation of radiolabel had disappeared, although synthesis was still depressed at 50 and 100 mg/kg. The GTP content of the spleen, however, was slightly reduced at 12.5 and 25 mg/kg and highly significantly reduced at the two highest doses of MPAS (by -33%, and -56%, respectively). The results from cardiac tissue were quite different. Firstly, the incorporation of radiolabel from [laC]hypoxanthine into GTP was negligible in both control and MPAS-dosed animals, probably due to minimal salvage of hypoxanthine and the low activity of IMPDH in heart muscle (14). Secondly, there was no effect of MPAS, even at the highest dose level (100 mg/kg) on the GTP content of the heart throughout the experiment (Table 1A).

T. J. FRANKLIN and W. P. MORRIS

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TABLE 1A. EFFECT OF ORALLY ADMINISTERED DISODIUM SALT OF MYCOPHENOLIC ACID ON GTP IN RAT SPLEEN AND HEART lN VIVO: DOSE AND TIME DEPENDENCE

Dose (mg/kg)

Time after administration of MPA (hr) 1.25 24.25 Sp. act. GTP:~ Total GTP Sp. act. GTP~: Total GTP (dis./min/~g) (~g) (dis./min/~g) (ttg)

Spleen 0 100 50 25 12.5

652.0 ___129.4 57.4 + 18.6t 43.2 + 9.4t 86.2 + 15.1t 200.6 + 49.2t

19.3 + 1.1 19.1 +_ 1.8 22.7 + 0.5 16.8 + 0.7 17.1 + 1.6

392.5 4- 21.4 168.8 4- 79.5t 273.6 4- 46.5¢ 351.5 4- 40.4 403.6 4- 100.5

35.4 _ 2.6 15.4 + 4.2* 23.5 4- 0.9* 29.4 4- 2.0 27.2 4- 1.1

Heart 0 I00 50 25 12.5

0 0 0 0 0

42.6 + 45.1 + 43.7 + 47.1 + 40.5 +

0 0 0 0 0

54.3 + 2.5 45.4 +_2.7 53.6 4- 3.3 46.2 + 4.3 50.7 + 6.2

2.8 4.0 3.1 2.5 3.5

The sodium salt of MPA was given orally in aqueous solution at the indicated doses and intervals before death. [14C] Hypoxanthine (10 tLC) and allopurinol (50 mg/kg) were given intraperitoneally 15 min before death. Values are means + S.E. of tissue extracts from 5 animals. *p < 0.01, tp < 0.05 different from undosed animals. $1ndicates specific activity of GTP.

Effect o f disodium salt o f mycophenolic acid on the A TP content o f rat spleen. I n a n u m b e r of tissue c u l t u r e lines M P A has b e e n f o u n d to i n d u c e a m a r k e d d e p r e s s i o n in G T P c o n t e n t whilst at the same time h a v i n g m i n i m a l effects o n the cellular c o n t e n t of A T P (15, 16, 17). W e were t h e r e f o r e surprised to find that t r e a t m e n t of rats with M P A S c o n s i s t e n t l y r e s u l t e d in a d o s e - d e p e n d e n t , significant d e p r e s s i o n in the r a d i o l a b e l l i n g of splenic A T P a n d also a r e d u c t i o n in the A T P c o n t e n t of s p l e e n a l t h o u g h these effects were r a t h e r less t h a n those o n splenic G T P ( T a b l e 1B). I n c o n t r a s t , t r e a t m e n t with M P A S had n o effect o n the A T P c o n t e n t of cardiac tissue. Since the c o n v e r s i o n of I M P to a d e n y l o s u c c i n i c acid is a n G T P - r e q u i r i n g r e a c t i o n , it is possible that the d e p r e s s i o n in splenic G T P b r o u g h t a b o u t by t r e a t m e n t with M P A S has a n adverse effect o n the synthesis of a d e n y l o s u c c i n a t e en route to A M P a n d A T P a l t h o u g h we have n o t yet sought e v i d e n c e for this. In a s e c o n d e x p e r i m e n t we carried out a m o r e d e t a i l e d a s s e s s m e n t over time of the effects of a single oral dose of M P A S (100 mg/kg) o n the r a d i o l a b e l l i n g of G T P a n d its c o n t e n t in rat spleen. C a r d i a c tissue was also i n c l u d e d in this e x p e r i m e n t b u t once again we were u n a b l e to detect any r a d i o l a b e l l i n g of cardiac G T P a n d its c o n t e n t was u n a f f e c t e d by M P A S over 24.5 hr (not shown). A n a l y s i s of splenic G T P c o n f i r m e d

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111

TABLE lB. EFFECT OF ORALLY ADMINISTERED DISODIUM SALT OF MYCOPHENOLIC ACID ON ATP IN RAT SPLEEN AND HEART IN VIVO: DOSE AND TIME DEPENDENCE Time after administration of MPA (hr) 1.25 24.25 Total ATP Sp. act. ATP5 Total ATP (~g) (dis./min/ttg) (/~g)

Dose (mg/kg)

Sp. act. ATP5 (dis./min//~g)

Spleen 0 100 50 25 12.5

314.2 + 35.3 111.3 _+ 8.9t 122.2 + 9.4t 151.0 + 18.8t 263.3 + 21.0

155.3 170.8 197.7 141.1 145.4

+ + + + +

Heart 0 100 50 25 12.5

0 0 0 0 0

409.4 412.1 431.4 408. I 402.1

_+ 42.3 _+ 5.3 _+ 21.9 _+ 34.0 + 33.9

11.0 14.4 8.1 7.5 10.4

205.6 143.5 165.2 194.5 219.0

+ 17.2 + 16.2t + 4.9 _+ 18.5 _+ 57.0

0 0 0 0 0

294.4 126.5 188.8 225.6 212.6

+ 24.6 + 33.0* + 6.9t --_ 13.0¢ + 13.1"t

476.4 510.3 551.9 506.1 484.1

_+ 14.7 _+ 33.0 _+ 55.7 _+ 28.0 _+ 23.3

The sodium salt of MPA was given orally in aqueous solution at the indicated doses and intervals before death. [14C]Hypoxanthine (10/zC) and allopurinol (50 mg/kg) were given intraperitoneally 15 min before death. Values are means _+ S.E. of tissue extracts from 5 animals. *p < 0.01, tp < 0.05 different from undosed controls. $1ndicates specific radioactivity of ATP.

t h a t its r a d i o l a b e l l i n g b y [ 1 4 C ] h y p o x a n t h i n e w a s m a r k e d l y i n h i b i t e d w i t h i n 1.25 h r o f d o s i n g w i t h M P A S a n d r e m a i n e d s o f o r at l e a s t 6.25 h r ( T a b l e 2). A p a r t i a l r e c o v e r y o f r a d i o l a b e l l i n g o c c u r r e d b y 24.5 h r a f t e r d o s i n g . T h e

TABLE 2, TIME COURSE OF THE EFFECTS OF A SINGLE ORAL DOSE OF THE SODIUM SALT OF MYCOPHENOLIC ACID ON GTP AND ATP IN THE RAT SPLEEN IN VIVO

Time after dosing (hr)

GTP Sp. Act. (dis./min/~g)§

0 (undosed) 1.25 4.25 6.25 24.25

436.0 89.4 69.2 125.0 161.0

+ 100.0 +_ 9.65 + 17.45 + 32.95 + 0.9

ATP Total (/xg) 26.1 19.7 14.9 11.0 12.8

+ 3.4 _+ 1.2 _+ 0.8t + 1.2t + 0.95

Sp. Act. (dis./min/~g)§ 186.9 57.3 70.8 54.9 72.2

+ + + + +

19.8 2.4* 9.6* 5.0"t 23.4t

Total (~g) 336.2 284.4 211.6 153.7 212.4

-+ 41.4 + 14.7 + 14.35 + 10.65 + 9.25

The sodium salt of MPA was given orally in aqueous solution (100 mg/kg) at the indicated intervals before death. [t4C] Hypoxanthine (10/~C) and allopurinol (50 mg/kg) were given intraperitoneally 15 rain before death. Values are means + S.E. of spleen extracts from 5 animals. *p < 0.001, tp < 0.01, 5p < 0.(15 different from undosed animals. §Indicates specific radioactivity of resolved nucleotides.

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decline in the total G T P lagged behind the inhibition of radiolabelling. The data in Table 2 confirm the depressive effects of MPAS on both the radiolabelling and levels of splenic ATP.

Comparison of the pharmacodynamic action of mycophenolic free acid with its disodium salt and morpholinoethyl ester on GTP metabolism in rat spleen. Lee et al. (13) compared the pharmacokinetics of mycophenolic free acid (MPA) with those of its morpholinoethyl ester ( M P A M E ) in cynomolgous monkeys and found that the latter compound was more reproducibly absorbed after oral administration and gave higher areas under the curves in plots of plasma concentrations of total mycophenolic acid, i.e., free acid + mycophenolic acid glucuronide, vs. time over 24 hr. This was attributed to the greater aqueous solubility of M P A M E compared with MPA, and improved absorption from the upper GI tract. However, the data did not provide a direct comparison of the abilities of M P A and

% Reduction in specific radioaetivify o f GTP

"1 8O

.z -I--

MPA MPAS

* /I

70

60

50 4O 30 -~ 20 10 0 10

y I 30

I I 50 70 D o s e (mg/kg)

I 90

I 110

FIG. 2. Dose-responses of mycophenolicfree acid (MPA), its disodium salt (MPAS) and its morpholinoethyl ester (MPAME) against the radiolabelling of GTP in rat spleen. All compounds were dosed orally, MPA as an aqueous suspension, MPAS and MPAME in aqueous solutions, 4 hr before the intraperitoneal injection of [14C]hypoxanthine (10 p,C) and allopurinol (50 mg/kg). The animals were killed 15 min later. Plotted values are derived from the means of data from 5 animals in each group. **p < 0.01, *p < 0.05 different from undosed animals, calculated from specific activity data.

INHIBITION OF GTP SYNTHESIS IN V1VO

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MPAME to deliver higher plasma levels of MPA itself rather than the biochemically inactive glucuronide. MPAME itself was not detected in the plasma, probably because of rapid conversion to the free acid by esterase activity in vivo (13). We have compared the action of MPAME on GTP metabolism in rat spleen in vivo with that of MPA and the freely water-soluble MPAS. Figure 2 shows the dose-response plots of all three compounds against the radiolabelling of GTP when administered orally 4.25 hr before death. Although there was a trend towards the inhibition of the radiolabelling of GTP by the free acid in this experiment, it did not achieve statistical significance even at the highest dose of 100 mg/kg. MPAME was clearly more effective than the free acid but it appeared to be rather less effective than MPAS, although a more detailed analysis would be necessary to confirm this. Treatment with MPAS and MPAME produced comparable, statistically significant depressions in the GTP content of spleen whereas the free acid was without effect (Fig. 3).

% Reduction in total GTP

40

30

2o

10

0 12.5

25

50

100

Dose (mg/kg) FIG. 3. Effects of mycophenolic free acid (MPA), its disodium salt (MPAS) and its morpholinoethyl ester (MPAME) on GTP levels in rat spleen in vivo. The data were obtained from the same experiment referred to in Fig. 2. ***p < 0.001, **p < 0.01, *p < 0.05 different from undosed animals, calculated from GTP contents.

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T. J. FRANKLINand W. P. MORRIS

DISCUSSION MPA is active against a broad range of experimental tumors in rodents when given orally once daily (5, 6, 9). The limited information available on the pharmacokinetics of MPA in rats and mice indicates that when it was given orally (45 mg/kg) as the sodium salt the compound was rapidly absorbed in both species achieving peak plasma concentrations of the free acid within 15-30 min although markedly higher plasma concentrations were reached in rats (9). The free acid was rapidly cleared, especially in the rat, plasma levels falling to less than 20% of the peak values within 1 hr of dosing (9). This pharmacokinetic profile of rapid absorption followed by rapid clearance seems scarcely congruent with the effective antitumor regime of once daily oral dosing. However, the pharmacodynamic results presented in this report indicate that the biochemical action of MPAS in a proliferative tissue such as spleen is sustained for considerably longer than might be predicted from the pharmacokinetic data, suggesting that mycophenolic acid is retained in sensitive cells up to 24 hr after oral administration depending on the size of the dose given. Kinetic analysis of the inhibition of mammalian IMPDH by MPA gave K i values between 30-40 nM (11) and it is unlikely that this degree of affinity could account for sustained retention of the inhibitor within cells. It has been suggested that MPA may form a covalent complex with IMPDH although no direct evidence for this was provided (18). If such a complex were formed relatively slowly from an initial reversible enzyme-inhibitor complex it is possible that it could be missed in short-term kinetic studies. This possibility may repay further investigation. Alternatively, free MPA may be retained in some extra-vascular tissue or cellular compartment in exchange with the enzyme in the spleen after it ceases to be detectable in plasma. Earlier studies had shown that the reduction of GTP levels caused by MPA in proliferating glioma cells in culture led to a fall in the cyclic AMP response to I$-adrenergic stimulation, probably because of diminished coupling between the agonist-receptor complex and adenylyl cyclase via the G-protein-GTP transducer (15). It was possible, therefore, that MPA might depress neuro-endocrine responses in vivo which involve G-protein coupling. However, administration of MPA at 100 mg/kg to rats, a dose level causing marked inhibition of GTP synthesis in spleen, had no effect on the following activities which are considered to be G-protein-mediated: normal heart rate and blood pressure, the pressor response to the ¢x~-agonist methoxamine, the gastric acid secretion response to the H 2 agonist dimaprit and normal gastrointestinal motility (P. W. Marshall, personal communication). Our results which indicate an absence of any effect of GTP in rat cardiac tissue may point to a possible explanation for the lack of pharmacological activity in non-proliferating tissues. We

INHIBITION OF GTP SYNTHESIS IN VIVO

l 15

suggest that inhibition of IMPDH causes a depletion of GTP in proliferating cells where there is a continual requirement for guanine nucleotides for the biosynthesis of nucleic acids. The absence of pharmacological activity of MPA on neuroendocrine functions in non-proliferating tissues would be consistent with their low levels of IMPDH and minimal net consumption of guanine nucleotides. The morpholinoethylester of mycophenolic acid has been introduced into clinical practice for the treatment of refractory kidney transplant rejection (19) and refractory rheumatoid arthritis (20) largely on the basis of a perceived improvement in bioavailability due to enhanced water solubility in the upper GI tract compared with the free acid. Our results provide evidence for an advantage of MPAME over free MPA insofar as the inhibition of GTP synthesis and the resulting depression of GTP levels in a proliferating lymphoid tissue, the spleen, in rats are concerned. However, in a three-way comparison between free MPA, MPAME and the freely water-soluble MPAS, all three compounds being given orally, we found that the morpholinoethyl ester was possibly rather less effective than MPAS against GTP metabolism, at least over the dose range and time period investigated. It would be interesting to compare the pharmacokinetics of plasma free MPA and also its glucuronide, following the oral administration of MPAME and MPAS. Weber has made a compelling case for the importance of IMPDH as a chemotherapeutic target in proliferating malignant cells (21). Although there are several nucleoside analogs which are metabolized to competitive inhibitors of IMPDH within cells (22, 23), MPA and a few semi-synthetic close analogs are the only direct inhibitors of this enzyme. In principle, MPA has considerable advantages over the nucleoside prodrugs because of its intrinsic potency and specificity and lack of undesirable effects such as the inhibition of DNA repair and the induction of chromosomal breaks (12). The challenge remains to devise a successor to MPA which retains its potency and specificity whilst at the same time possessing sufficient metabolic stability in vivo to afford effective anti-cancer therapy in human patients. SUMMARY

Mycophenolic acid is effective against a wide range of experimental tumors in rodents when given orally, despite rapid metabolism to the inactive glucuronide derivative and rapid clearance from plasma. In the light of this, the pharmacodynamic action of mycophenolic acid on the radiolabelling of GTP and ATP by [~4C]hypoxanthine in spleen and heart has been investigated in vivo in the rat as a preliminary to studies in tumor tissue. The data indicate that inhibition of GTP, and more surprisingly, ATP synthesis in spleen was sustained for at least 24.25 hr after single

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o r a l d o s e s o f t h e d i s o d i u m salt o f m y c o p h e n o l i c acid, i n d i c a t i n g that t h e i n h i b i t o r is r e t a i n e d in sensitive cells for c o n s i d e r a b l y l o n g e r t h a n m i g h t be e x p e c t e d f r o m the p h a r m a c o k i n e t i c profile in the p l a s m a in this species. G T P a n d A T P levels b e c a m e d e p r e s s e d in rat s p l e e n s u b s e q u e n t to t h e inhibition of nucleotide radiolabelling. The persistence of mycophenolic acid in p r o l i f e r a t i n g cells m a y a c c o u n t for the e f f e c t i v e n e s s of o n c e daily dosing against rapidly growing experimental tumors. In c o n t r a s t with s p l e e n , t h e r e was n o i n c o r p o r a t i o n of r a d i o l a b e l f r o m [ l n C ] h y p o x a n t h i n e into e i t h e r G T P o r A T P in rat h e a r t a n d m y c o p h e n o l i c acid h a d no effect on the c a r d i a c c o n t e n t o f e i t h e r n u c l e o t i d e . T h e lack o f effect o f m y c o p h e n o l i c acid o n c a r d i a c G T P levels is c o n s i s t e n t with the a b s e n c e o f a n y p h a r m a c o l o g i c a l a c t i o n on c a r d i a c f u n c t i o n s a s s o c i a t e d with receptor-G-protein-GTP interactions. T h e ability o f t h e m o r p h o l i n o e t h y l e s t e r o f m y c o p h e n o l i c acid (a clinically effective i m m u n o s u p p r e s s i v e a g e n t ) to inhibit G T P synthesis a n d d e p r e s s G T P levels in rat s p l e e n in v i v o was c o m p a r e d with t h a t o f m y c o p h e n o l i c free acid a n d its d i s o d i u m salt. T h e e s t e r d e r i v a t i v e was c l e a r l y m o r e effective t h a n the p o o r l y w a t e r - s o l u b l e free acid b u t s h o w e d c o m p a r a b l e activity with the f r e e l y s o l u b l e d i s o d i u m salt.

REFERENCES 1. B. GOSIO, Richerche batteriologiche e chimiche sulle alterazioni del mais, Rivista d'lgiene e Sanita Pubblica, Ann. 7, #21,825-849 (1896). 2. B. GOSIO, Richerche batteriologiche e chimiche sulle alterazioni del mais. Rivista d'Igiene e Sanita Pubblica, Ann.4 #22, 869-888 (1896). 3. E. P. ABRAHAM, The effect of mycophenolic acid on the growth of St. aureus in heart broth, Biochem. J. 39, 398-408 (1945). 4. J. H. BIRKINSHAW, H. RAISTRICK and D. J. ROSS, Studies in the biochemistry of micro-organisms 86. The molecular constitution of mycophenolic acid, Biochem. J. 50, 630-634 (1952). 5. S.B. CARTER, T. J. FRANKLIN, D. F. JONES, B. J. LEONARD, S. D. MILLS, R. W. TURNER and W. B. TURNER, Mycophenolic acid: an anti-cancer compound with unusual properties, Nature 223,848-850 (1969). 6. R.H. WILLIAMS, D. H. LIVELY, D. C. DELONG, J. C. CLINE, M. SWEENEY, G. A. POORE and S. H. LARSEN, Mycophenolic acid: antiviral and antitumor properties, J. Antibiotics 21,463-464 (1968). 7. T. J. FRANKLIN and J. M. COOK, The inhibition of nucleic acid synthesis by mycophenolic acid, Biochem. J. ll3, 515-524 (1969). 8. L. HEDSTROM and C. C. WANG, Mycophenolic acid and thiazole adenine dinucleotide inhibition of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: implications on enzyme mechanism, Biochemistry 29, 849-854 (1993). 9. M. J. SWEENEY, D. H. HOFFMAN and M. A. ESTERMAN, Metabolism and biochemistry of mycophenolic acid, Cancer Res. 32, 1803-1809 (1972). 10. W.W. EPINETYE, C. M. PARKER, E. L. JONES and M. C. GREIST, Mycophenolic acid for psoriasis, J. Am. Acad. Dermatol. 17,962-971 (1987). II. T. J. FRANKLIN and J. M. COOK, Inhibition of guanine nucleotide synthesis in Yoshida ascites cells, Biochem. Pharmacol. 20, 1335-1338 (1971). 12. A. C. ALLISON and E. M. EGUI, Mycophenolate mofetil, a rationally designed immunosuppressive drug, Clin. Transplantation 7, 96-112 (1993).

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13. W . A . LEE, L. GU, A. R. M1KSZTAL, N. CHU, K. LEUNG and P. H. NELSON, Bioavailability improvement of mycophenolic acid through amino ester derivatization, Pharmaceutical Res. 7, 161-166 (1990). 14. G. WEBER, H. NAKAMURA, Y. NATSUMEDA, T. SZEKERES and M. NAGAI, Regulation of GTP synthesis, Advan. Enzyme Regul..32, 57-69 (1992). 15. T. J. FRANKLIN and P. A. TWOSE, Reduction in IB-adrenergic response of cultured glioma cells following depletion of intracellular GTP, Eur. J. Biochem. 77, 113-117 (1977). 16. M . B . COHEN, J. MAYBAUM and W. SADEE, Guanine nucleotide depletion and toxicity in mouse T lymphoma (S-49) cells, J. Biol. Chem. 256, 8713-8717 (1981). 17. K. KIGUCHI, F. R. COLLART, C. HENNING-CHUBB and E. HUBERMAN, Induction of cell differentiation in melanoma cells by inhibitors of IMP dehydrogenase: altered patterns of IMP dehydrogenase expression and activity, Cell Growth and Differentiation 1,259-270 (1990). 18. D . J . HUPE, B. A. AZZOLINA and N. D. BEHRENS, IMP dehydrogenase from the intracellular parasitic protozoan Eimeria tenella and its inhibition by mycophenolic acid, J. Biol. Chem. 261, 8363-8369 (1986). 19. H. W. SOLLINGER, F. O. BELZER, M. H. DEIERHOI, A. DIETHELM, T. A. GONWA, R. S. KAUFFMAN, G. B. KLINTMALM, S. V. McDIARMID, J. ROBERTS, J. T. ROSENTHAL and S. B. TOMLANOVICH, RS-61443 (mycophenolate mofetil). A multicenter study for refractory kidney transplant rejection, Ann. Surg. 216, 513-519 (1992). 20. R. GOLDBLUM, Therapy of rheumatoid arthritis with mycophenolate mofetil, Clin. and Exptl. Rheumatology 11 (Suppl. 8), Sl17-Sl19 (1993). 21. G. WEBER, Biochemical strategy of cancer cells and the design of chemotherapy, Cancer Res. 43, 3466-3492 (1983). 22. Y. YAMADA, Y. NATSUMEDA and G. WEBER, Action of the active metabolites of tiazofurin and ribavirin on purified IMP dehydrogenase, Biochemistry 27, 2193-2196 (1988). 23. H. N. JAYARAM, K. GHAREHBAGHI, N. J. JAYARAM, J. REISER, K. KROHN and K. D. PAULL, Cytotoxicity of a new IMP dehydrogenase inhibitor, benzamide riboside, to human myelogenous leukemia K562 cells, Biochem. Biophys. Res. Commun. 186, 1600-1606 (1992).