L-Aspartate: a Review Jean L

(CANCER RESEARCH 48, 4441-4454, August 15, 1988]

Review Biochemistry and Clinical Activity of yV-(Phosphonacetyl)-L-aspartate: A Review Jean L. Grem,1Susan A. King, Peter J. O'Dwyer, and Brian Leyland-Jones

Investigational Drug Branch, Cancer Therapy Evaluation Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20892 [J. L. G., S. A. K., B. L-JJ, and Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [P. J. OJ

yV-(Phosphonacetyl)-L-aspartate (NSC 224131), a potent in K, = 2.7 x 10~8M), murine (spleen cells, KÂ¡= 2.6 x 1(T8 M), hibitor of de novo pyrimidine biosynthesis, was introduced into and human (spleen cells and HT-29 colon cancer cells, K\ = 1.1 x IO"8M; W138 fetal lung diploid fibroblast cells, K, = 0.85 x clinical trials in 1978 under the sponsorship of the Division of Cancer Treatment, National Cancer Institute. PALA2 demon 10~8M) origin (l, 3-5). Inhibition is competitive with respect strated a broad spectrum of activity against experimental tumor to carbamoyl phosphate and noncompetitive with respect to models and its biochemical and pharmacological effects were aspartate, suggesting that the natural substrates have an oblig well characterized. Phase I trials were followed by broad Phase atory binding order with carbamoyl phosphate binding to the II screening for antitumor activity. Unfortunately, PALA was enzyme prior to aspartate for catalysis to occur (3, 6). In inactive as a single agent. The emphasis then shifted to the addition to carbamoyl phosphate, ATP, Mg2+, Ca2+, and KC1 clinical application of PALA as a biochemical modulator of interfere with the inhibition of ACTase by PALA (7, 8). From other antimetabolites. This review summarizes the preclinical human cell lines and patient tissue samples, PALA has been and clinical studies of this investigational agent, focusing on found to bind approximately 1000 times more tightly than the difficulties encountered in translating the potent in vitro carbamoyl phosphate (4, 9). Structure-activity studies indicate effects of PALA to a successful pharmacological strategy in the that both carboxylate moieties are essential for biological activ clinic. ity (10). The biochemical effects of PALA have been examined in Mechanism of Action of PALA vitro and in vivo. Potent effects on de novo pyrimidine synthesis have been noted in cell-free systems at concentrations as low as The de novo synthesis of pyrimidines proceeds in the cytosol 0.1 MM(11,12). Swyryd et al. (\ 1) reported that PALA inhibited from simple components such as carbon dioxide, a source of the growth of SV40-transformed hamster kidney cells and nitrogen (glutamine), and a source of energy (ATP), resulting produced a dose-dependent inhibition of the incorporation of in the formation of carbamoyl phosphate (Fig. 1). The pyrimi radiolabeled precursors into UMP and CMP in a supernatant dine ring is formed in the next two steps. Three subsequent fraction of cell homogenates, with 5*95% inhibition at 10 nM. reactions result in the formation of UMP. A 10-fold higher concentration of PALA (100 MM)was neces PALA is a rationally synthesized compound, designed by sary to completely block the proliferation of a homogeneous Collins and Stark (1) to be a stable inhibitor of ACTase, the solution of cells, and the cytotoxicity could be reversed by 100 enzyme involved in the second step of de novo pyrimidine MMuridine. biosynthesis. PALA combines structural features of the two In vivo investigations have shown that effects of PALA on natural substrates of the enzyme (Fig. 2) and appears to put the UTP pools are also dose dependent (13). In BALB/c x DBA/ enzyme into a conformation closely resembling that associated 8 Fi (hereafter called CDSFi) mice bearing a spontaneously with the transition state (1). Uptake of PALA, a tetravalent metastasizing breast carcinoma, UTP pools in tumor tissue hydrophobic molecule, appears to be mediated by fluid phase were reduced to 60% of control 24 h after 100 mg/kg (300 mg/ endocytosis, with fusion of the endocytotic vesicle with the m2) PALA, a nontherapeutic dose. At 200 mg/kg, a dose that lysosome (2). Upon passage through the lysosomal membrane was marginally therapeutic, UTP pools were reduced to 45% into the cytosol, PALA may become highly charged, and thus of control. Even at the highest dose tested, 1000 mg/kg (3000 "trapped" intracellularly, permitting prolonged contact with mg/m2), UTP pools were not reduced below 25% of control. the target enzyme, ACTase. Specifically, administration of PALA in vivo or in vitro causes PALA binds tightly to ACTase of bacterial (Escherichia coli, substantial decreases in UTP, CTP, dCTP, dTTP, and dGTP pools (Table 1) (14-19). ATP, GTP, and dATP pools are either Received 4/7/88; accepted 5/20/88. unaffected or elevated. In a 5-h exposure of L1210 cells to 1 The costs of publication of this article were defrayed in part by the payment HIMPALA, DNA synthesis (as measured by incorporation of of page charges. This article must therefore be hereby marked advertisement in [3H]guanosine) was inhibited to 20% of control, but RNA accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' To whom requests for reprints should be addressed, at Investigational Drug synthesis was inhibited only to 80% of control (15). Simulta Branch, Cancer Therapy Evaluation Program, Division of Cancer Treatment, neous exposure to 10 MMdCyd and dThd restored DNA syn National Cancer Institute, Executive Plaza North, Room 731, Bethesda, MD 20892. thesis without affecting RNA synthesis (15). During a 48-h 2The abbreviations used are: PALA, A/-(phosphonacetyl)-L-aspartate; ACTase, exposure to PALA, 10 MMdCyd and dThd protected the cells aspartate carbamoyltransferase; dCyd, deoxycytidine; dThd, thymidine; from the growth-inhibitory effects of Â«100MMPALA. At higher CPS II, carbamoyl phosphate synthetase; PRPP, phosphoribosylpyrophosphate; MSKCC, Memorial Sloan-Kettering Cancer Center; CI, continuous infusion; PR, concentrations of PALA (1 mM), only uridine (50 MM)protected partial remission; FUra, 5-fluorouraciI; TS, thymidylate synthase; FdUMP, 5- the cells (15). Thus, inhibition of DNA synthesis may be the fluorodeoxyuridine monophosphate; FUTP, S-fluorouridine triphosphate; FUrd, 5-fluorouridine; FUDP, 5-fluorouridine diphosphate; FUMP, S-fluorouridine primary limitation on replication for cells grown in the presence monophosphate; MMPR, 6-methylmercaptopurine riboside; MTX, methotrex- of low concentrations of PALA, although inhibition of RNA ate; CR, complete remission; FdUrd, 5-fluorodeoxyuridine; NCCTG, North Central Cancer Treatment Group; ara-C, l-/3-D-arabinofuranosylcytosine; ara- synthesis may become an important factor during prolonged CMP. 1-ÃŸ-D-arabinofuranosylcytosine monophosphate; ara-CTP, 1-/3-D-arabino- exposure to high concentrations. furanosylcytosine triphosphate; ara-U, 9-/3-D-arabinofuranosyluracil; 5-aza-Cyd, Moyer and Handschumacher (20) used inhibition of pyrazo- 5-azacytidine; 5-aza-CMP, 5-azacytidine monophosphate; 5-aza-CTP, 5-azacytidine triphosphate; ID50, 50% inhibitory dose; DP, dipyridamole; NBMPR, nitro- furin-induced orotate and orotidine excretion by PALA as an benzyl-thioinosine; BCNU. 1.3-bis(2-chloroethyl)-l-nitrosourea. estimate of "total body" de novo pyrimidine biosynthesis. Pyr- 4441 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1988 American Association for Cancer Research. BIOCHEMISTRY AND CLINICAL ACTIVITY OF PALA

Bicarbonate * Glutamine tissue (20). Moyer et al. (15, 20) provided additional evidence Carbamoyl PhosphatÂ« cATP that the biochemical effects of PALA may be selective to tumor SynthÃ©tiseil x AOP â€¢P tissue. In mice bearing Lewis lung carcinoma, PALA produced Carbamoyl Phosphate + Aspanate a dose-dependent decrease in uridine and cytidine nucleotide

Asportate pools in tumor tissue but had no noticeable effect on nucleotide Transcarbamylase le. pools in the tissue samples of the liver and spleen (20). N-Carbamoyl Asparlate In summary, in vitro and in vivo data indicate that PALA

DihydroorolasÂ« potently inhibits ACTase and reduces UTP and CTP pools. I u Alterations of ribonucleotide pools can be detected at doses of Dihydroorotate PALA below that associated with cytotoxicity. Even at the

Dihydroorotate I â€žAO highest doses, de novo synthesis is not completely inhibited. Of Dehydrogenase 1 * NAO special interest is the observation in mice that PALA may Uracil Orotate selectively diminish UTP and CTP in tumor tissue while spar Orolali ing normal tissues. Such observations suggest that lower doses Phosphoriboiyl transfÃ©ras* of PALA might be useful in modulating the biochemical profile of cancer cells. Orotidylate

Ribos* t P Preclinical Antitumor Activity â€¢co, â€¢Urd Phos phorylise -*â€¢Uridine Monophosphate Undine Uracil The spectrum of antitumor activity displayed by PALA is I ATP unusual (Table 2) (19, 21-30). PALA is not active against the

| V ADP RibonucleotidÂ« relatively sensitive murine leukemia models but is active against ReductasÂ« Uridine Diphosphate dUDP a wide variety of murine solid tumor lines including three lines considered resistant to most drugs: Lewis lung, Pan 02, and B16 melanoma (21, 23, 24, 26). PALA is active against B16 i melanoma and Lewis lung on every schedule tested when ad Uridine Triphosphate dUMP ministered i.p. (for 1 day; daily for 9 days; days 1, 5, and 9) and 1,- GluUm.nÂ« . ATP â€¢M,O f CTP Synthetase there was no apparent schedule dependency. PALA displays * Gluumale * AOP * P. * 2 M modest activity against the s.c. implanted MX-1 mammary and Cytidine Triphosphate- CDP- -dCDP- dCMP LX-1 lung xenografts (27) but is inactive against other human solid tumor models (21, 28-30). Fig. 1. Pathways of de novo pyrimidine biosynthesis. Urd, uridine. Mechanisms of Resistance. There are several mechanisms which account for resistance to PALA. In mammalian cells, the first three enzymes of the de novo pyrimidine pathway, CPS II,

CH2 ACTase, and dihydroorotase, are associated in a multifunc = 03P-0-C â€”NH, tional protein complex (31). CPS II is considered the rate- CH controlling enzyme in vivo and is subject to allosteric regulation, with UTP serving as a feedback inhibitor (32-34). The specific Carbamoyl Phosphate Aspartate -coo- activity of ACTase is approximately 100-fold higher than that of CPS II (35). ACTase = 03P-CH2 CH2 Early during the preclinical investigation of PALA, several I c. CH-coo- laboratories reported that stable resistant mutant cell lines had been developed which displayed increased ACTase activity (11). Kempe et al. (36) noted that the increased ACTase activity in NH2 CH2 PALA SV40-transformed cells with acquired resistance to PALA oc CH curred in parallel with the activities of CPS II and dihydroor otase. Stable resistant cells were found to contain a large H amount of a poly so muÃpolyadenylate-containing RNA with the N Carbamoyl-Aspartate capacity to direct the synthesis of the multifunctional protein in vitro (37). Therefore, the increase in the activity of the Fig. 2. Structures of PALA, carbamoyl phosphate, aspartate, and yv-carbamoyl ACTase appeared to result from an increase in the rate of aspanate. synthesis. It was later proved that the gene coding for the multifunctional enzyme complex was amplified (38). The de azofurin inhibits orotidylate decarboxylase (Fig. 1), leading to gree of amplification roughly correlated with the degree of accumulation of orotate and orotidine. PALA inhibited urinary overproduction of mRNA and protein in the mutants. excretion of orotate and orotidine in a dose-dependent manner Some tumors with intrinsic resistance to PALA have also in mice. However, even at maximally tolerated doses, 500 mg/ been shown to have higher ACTase activity compared to sen kg ( 1500 mg/m2), residual de novo synthesis was occurring as sitive cells. Jayaram et al. (39) reported that the mean specific evidenced by incomplete inhibition of orotate and orotidine activity of ACTase in nine PALA-sensitive tumors was 241 Â± excretion (approximately 80%). The effect diminished to <40% 126 (SD) nmol/mg/h, compared to 526 Â±99 nmol/mg/h in inhibition by 72 h. The effect of 500 mg/kg PALA on pyrazo- four PALA-resistant tumors. There was overlap, however, be furin-induced accumulation of orotate and orotidine in spleen tween the sensitive and resistant tumors. ACTase from sensitive and Lewis lung tissue at 3 h following the drug administration and resistant tumors did not appear to differ qualitatively, included complete inhibition in Lewis lung tissue and 50% inasmuch as the K\ and time course of recovery from enzyme inhibition of that observed with pyrazofurin alone in spleen inhibition were similar. The intratumoral content of PALA in 4442 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1988 American Association for Cancer Research. BIOCHEMISTRY AND CLINICAL ACTIVITY OF PALA

Table 1 Effect of PALA on nucleotide pools Nucleotidc levels (% of control) systemIn Tumor dGTP13 dTTP dATP vitroexperimentsChinese 22)*L1210hamster ovary cells (14)Â°,500 MMPALA (h 2115 41 78 5)Humanleukemia (15), 1 mM PALA (h <389 20 172 24)Humanpromyelocytic leukemia HL-60 (16), 500 MMPALA (h 19 144 45 72)In ovarian carcinoma 2008 (17), 50 MMPALA (h

(mouse)Lewisvivo experiments 24)Lewislung (15), 250 mg/kg PALA (h 24)LI lung (18), 400 mg/kg PALA (h 24)P388/0210 leukemia (15), 250 mg/kg PALA (h 24)P388/acivicin-resistantleukemia (19), 200 mg/kg PALA (h 24)Human (19), 200 mg/kg PALA (h ovarian carcinoma 2008 (17) 200 mg/kg PALA (h 24)DTP910132129893947CTP94792739783779ATP88106100225214107180105GTP132112951481619228678dCTP " Reference. * Hour of measurement.

Table 2 In vivo antitumor activity' of PALA

Active activity >67%)Murine(ILS/GI (ILS/GI34-67%)i.p. 0-33%)i.p.(ILS/GI leukemiai.p. P388/ara-Ci.p. P388s.c. L1210i.p. P388/5-FUrai.p. L5178Yi.p. P338/AT-125Murine P815s.c.

solidtumorss.c. Lewislungi.v. B16melanomai.p. osteosarcomai.v.Ridgway Lewislungi.p. colon26s.c. B16melanomas.c. Pan02s.c. plasmacytomas.c.MOPC 315 glioma26s.c. colon26s.c. colon38i.p. Ehrlichascitess.c. CDSFimammary's.c. 13762mammarys.c. ovarianHumanM5076

tumorsÂ° MX-1mammarys.c. CX-1colons.c. LX-1 lungInactive CX-2colons.c. lymphomas.c.diffuse histiocytic lymphomai.e.lymphoblastic U 251glioblastomai.e. TE 671medulloblastomas.r.c. MX-1mammarys.r.c. LX-1lungs.r.c. colonRef.21-2421,22,2419,2421,23,242121,23,25,26242121272121212821,2829293030282828fromCX-1 PALA administered i.p.; level of activity derivedModerateincreased Jife span (ILS) or growth inhibition (GI), whichever was higher. sensitive and resistant tumors in vivo was similar as well, thesized uracil nucleotides via the de novo pathway at a rate suggesting that differences in drug uptake did not account for comparable to the increased CPS II activity. It is also possible resistance (39). The murine leukemia lines of a former National that in the presence of PALA, carbamyl phosphate, the product Cancer Institute tumor panel (L5178Y, P388, and LI210) of CPS II activity, accumulates to a sufficiently high concentra resistant in vitro to PALA (50% inhibitory concentration, 58 tion that the inhibitory effect of PALA on ACTase is effectively Â¿IM,average)tended to have a higher ACTase activity (57 nmol/ abolished (42). This hypothesis may explain the ability of mg protein/min, average) and a faster doubling time (15.3 h, resistant cells to overcome inhibition of flux through the de average) compared to three sensitive solid tumor cell lines novo pathway despite partial inhibition of ACTase (43). (Lewis lung, B16 melanoma, and glioma 26: 50% inhibitory Sensitivity also appears to be related to the duration of concentration, 3.1 MM;ACTase activity, 25.7 nmol/mg protein/ depletion of nucleotide pools. PALA at both 25 and 250 mg/ min; doubling time, 28.7 h) (24). Leyva et al. (40) also reported kg produced large changes in the CTP and UTP pools of Lewis that the ACTase activity of resistant human and murine lymph- lung within 3 h (UTP, 48 and 39% of control; CTP, 45 and oid cell lines (NC37, CCRF-CEM, L1210) was approximately 54% of control, respectively) with effects persisting for at least 3-fold higher (73.2 nmol/mg protein/h) than that of two sen 48 h (UTP, 17 and 4% of control; CTP, 9% of control, respec sitive melanoma cell lines (B16 and IPC 48; 22.8 nmol/mg tively) (15). The UTP and CTP pools in the more resistant protein/h). However, no correlation was found in these studies L1210 were also depressed to 14 and 28% of control 3 h between drug sensitivity and doubling times. following 250 mg/kg PALA, but in contrast to Lewis lung, Increased activity of CPS II has also been implicated in recovery was evident by 24 h. Similar results were obtained by resistance. Kensler et al. (18) found that variants of Lewis lung examination of the flux through the de novo pyrimidine pathway carcinoma with acquired resistance to PALA had a 2-3-fold by measuring the incorporation of radiolabeled precursor from increase in CPS II activity compared to the sensitive lines. A 13CO2into the uracil nucleotide pool of s.c. implanted Lewis PALA-resistant human breast cancer cell line has been de lung and L1210 tumor (43). Flux was inhibited by 75-80% 1 h scribed which has a 5-fold increase in CPS II activity compared after PALA (400 mg/kg) in both tumors, but a lag of 4 h was to the parental strain (41). This MCF-7/PALA-R subline syn needed before a reduction in the total uracil nucleotide pool 4443 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1988 American Association for Cancer Research. BIOCHEMISTRY AND CLINICAL ACTIVITY OF PALA was observed. The activity of the de novo pathway remained studies conducted in pregnant Swiss albino mice (54, 55). In depressed for up to 48 h after PALA in Lewis lung but recovered dogs and monkeys, toxicity was manifested as anorexia, diar to 70% of control by 12 h in LI210. ACTase activity in situ rhea, emesis, sedation, convulsions, flaking skin, and alopecia was also determined. Both tumors had only 4% of control (21). Laboratory analysis revealed leukocytosis in dogs and ACTase activity l h after PALA, a rapid increase in activity leukopenia in monkeys. Lymphocytopenia was noted in both between 1 and 4 h, and a slower recovery between 4 and 72 h species, as well as increased creatine phosphokinase, blood urea (Lewis lung, 38% of maximal activity; L1210, 45% of maximal nitrogen, and creatinine. A single i.v. dose of 480 mg/kg (9600 activity). Thus, there was little difference in the restitution of mg/m2) produced lethality in the dog, as did 60 mg/kg/day for ACTase activity in these tumors. Nevertheless, the LI210 cells 5 days (1200 mg/m2/day for 5 days). The lethal dose on the appeared to overcome the inhibition of flux through the de novo daily for 5 days schedule in the monkey was 400 mg/kg/day pathway. (4800 mg/m2/day) (21). Mammalian cells also possess a salvage pathway for pyrimidine synthesis. Pyrimidine bases such as uracil can be converted Preclinical and Clinical Pharmacology to the nucleotide level by the sequential action of uridine phosphorylase (in the presence of a ribose 1-phosphate donor) Several methods have been used for the assay of PALA in and uridine kinase (Fig. 1). Preformed uridine can be directly biological fluids. These include competitive protein binding converted to UMP by the action of uridine kinase. Alternatively, (56), ACTase inhibition (57-59), high pressure liquid chroma- orotate phosphoribosyltransferase can convert uracil to UMP tography (60), and a variety of methods involving gas chroma- in the presence of PRPP. Uridine kinase is sensitive to feedback tography (61-64). The sensitivities of these assays are generally inhibition by UTP and CTP (44). Since exposure to PALA around 0.5 /Â¡AI,althoughone method has obtained a lower limit causes a reduction in UTP and CTP pools, PALA could stim of 2 nM (59). ulate utilization of cytidine and uridine by uridine kinase. Pharmacological studies with PALA have been performed in Indeed, Kensler et al. (18) demonstrated that homogenates of the mouse, rat, dog, and monkey, as well as in humans during resistant and sensitive Lewis lung tumors prepared 24 h after the Phase I trials. [I4C]PALA administered i.v. to mice was i.p. administration of PALA (400 mg/kg) to tumor-bearing found to have a rapid initial half-life (/..) of 46 min with mice had an increased capacity to phosphorylate uridine and approximately 80% of the drug excreted unchanged in the urine cytidine compared to control homogenates. The activity of by 24 h (65). The values for the rat, dog, and monkey were uridine/cytidine kinase after PALA treatment was also greater similar with initial f./,values of 59, 59, and 65 min and urinary in the resistant lines than in the sensitive line. Increased pyrim- excretion of 65,85, and 70%, respectively (65). A f./,ÃŸofaround idine nucleoside-phosphorylating activity has also been noted 1-1.25 days was also detected in the mouse (66). Fecal excretion in naturally resistant lines as compared to sensitive lines (39). accounted for <5% of drug elimination in all four species tested Further evidence that the salvage pathway is important in (65). PALA demonstrated limited distribution in mouse tissues overcoming the cytotoxicity of PALA is derived from in vitro with the exception of kidney and bone (65). PALA accumulated and //; vivo experiments in which uridine and cytidine can in the bone of mice to high concentrations (3000 x KÂ¡for reverse the PALA effect (17, 45-47). In tissue culture medium ACTase) approaching levels of 400 ÃŸM24h after a single dose supplemented with nondialyzed calf serum, the physiological of 400 mg/kg i.p. (67). The t* of PALA in bone was 23 days, concentrations of pyrimidines which are present may be suffi possibly a consequence of chelation of PALA with divalent cient to meet the requirements for normal growth (48). Karle cations. [I4C]PALA did not concentrate in tumor tissue of mice et al. (48) demonstrated that when de novo pyrimidine synthesis bearing LI210 or Lewis lung tumors (65). There was also no was inhibited by 95% in LI210 cells by PALA, the uracil evidence of PALA metabolism as measured by Chromatographie nucleotide pool continued to expand and the cells continued to methods (65). take up [14C]uridine. Thus, neoplastic cells have the capacity to The clinical pharmacokinetics of PALA is summarized in decrease their dependency on the de novo pathway and utilize Table 3 (56, 60, 68-70). Following i.v. administration, plasma the salvage pathway. levels decayed in a bi- or triexponential manner with a terminal In summary, resistance to PALA has been attributed to tv, between 4.8 and 12.7 h. The volume of distribution (VD) was increased activity of ACTase or CPS II, the ability to overcome found to be slightly larger than extracellular fluid volume, inhibition of the de novo pathway, and repletion of UTP and suggesting limited access of PALA to tissue compartments (56). CTP pools via the salvage pathway. Weber et al. (49-52) have Measurable concentrations of PALA were attained in the cere- demonstrated that neoplastic cells have an increased enzymatic brospinal fluid in humans (68, 71) with PALA levels 12-40% capacity to salvage purines and pyrimidines. The mechanism(s) of concurrent plasma concentration 8 h following administra underlying clinical resistance to PALA, however, have not been tion (71). In brain biopsies, PALA was found to concentrate clearly defined. into intracerebral tumor greater than muscle (temporalis), and the concentration was especially low in edematous brain tissue. PALA was also measurable in tears at concentrations as high Preclinical Toxicology as 0.6 HIM;the average plasma/tear concentration ratio was 3.7 The toxicological evaluation of PALA was conducted in mice, (70). Total body clearance was linearly related to creatinine dogs, and monkeys. In C57BL x DBA/2 mice, acute toxicity clearance, suggesting that clearance of the drug is primarily by testing revealed that the single i.p. injection 10% lethal dose glomerular filtration (68). No metabolites of PALA were de was 1079 mg/kg (3237 mg/m2), 50% lethal dose 1587 mg/kg tected in any study. (4761 mg/m2), and 90% lethal dose 2333 mg/kg (6999 mg/m2) (21). Hematological evaluation of mice given 180 to 290 mg/ Clinical Pharmacodynamics kg/day for 9 days i.p. revealed reticulocytopenia and mild lymphopenia but granulocytes and platelets were unaffected PALA has been shown to cause a reduction in plasma uridine (53). Histopathological evaluation showed gastrointestinal le levels in mice and humans. Karle et al. reported that adminis sions (53). PALA was found to have embryolethal effects in tration of PALA (200 mg/kg i.p. daily for 1 or 4 days) to mice

Table 3 Clinical pharmacokinelics of PALA serum Assay and Total body excretion(%)85 concentration methodEnzymeEnzymeEnzymemodelBiexponentialBiexponentialBiexponentialTriexponentialn1974-910-11Doseschedule0.8-5 (min)5610041M(h)5.34.8282.7tv,~r(h)clearance1.60ml/kg/min85.2ml/min12.7 OiM)89-1705329-3680137-400900Ref.68566960,70 g/m2,60-min at 24h80 i.v.0.6-2 g/m2,15-min at 16h(n for5 i.v. daily =2)Majority days4 gasChromatograph)Highand CI1-4g/m2, 24-h by30 hÂ¿80 performanceliquidchromatographyKinetic g/m2,60-mini.v. 1.43ml/kg/minya(ml/kg)309333290Urinaryat 10h(B 2daystv,adaily for = 2)Peak

produced a 56% decrease in plasma uridine levels, which re toxicities were dermatitis, diarrhea, and mucositis, the signs of mained depressed for at least 96 h (72). Serum uridine levels of which generally appeared 5-10 days after PALA administra five patients receiving a 15-min infusion of 1-2 g/m2/day for 5 tion. Other toxicities included mild nausea and vomiting and days were measured (73). Predose uridine levels were 2.6-6.4 very minimal myelosuppression. Paresthesias and facial tin ÃÃM(therange for plasma uridine was 1.9-8.0 pM in normal gling occurred in some patients on the bolus schedules and were volunteers). Within the first 24 h after PALA, the uridine levels related to the rapid administration of the drug (77, 78). Three decreased 7-65%. Maximum decreases in all patients were 37- patients also experienced acute neurotoxicity in the form of 85%, but uridine levels only rarely fell below 1.9 /IM. The seizures after receiving multiple PALA doses (77, 82). No renal significance of the decrease in plasma uridine is not clear and or hepatic toxicity were observed on any study with the excep might result from either export of uridine from the liver or tion of minor elevations of aspartate aminotransferase and uric accelerated uptake due to increased activity of the salvage acid (79, 82). pathway. No correlation has been made between the decrease Phase II. Partial and minor responses were observed in Phase in plasma uridine and antitumor effect. I studies in patients with melanoma, chondrosarcoma, and The inhibition of leukocyte ACTase activity was measured in colon cancer (78, 80, 82). These data stimulated a broad Phase patients participating in a Phase I trial of PALA given as an II evaluation of PALA. The results of these trials are summa i.v. bolus daily for 5 days (74). ACTase activity was inhibited rized in Table 5 (83-102). A few cancer patients were noted to by 80% in one patient within 24 h of administration of 1500 have clearing of concurrent psoriasis during their chemother mg/m2 PALA, and ACTase activity had recovered to only 50% apy, but very few patients had a antitumor response (103). of baseline 11 days after cessation of therapy. Moore et al. (4) Toxicity on these Phase II trials was consistent with that measured PALA concentration and inhibition of ACTase in predicted from the Phase I experience, involving mainly the tumor biopsies taken from patients participating in a Phase I cutaneous and gastrointestinal systems. Neurotoxicity (sei trial. The degree of inhibition of ACTase activity (17-87%) zures, headaches, lethargy, confusion) was observed sporad appeared to correlate with PALA dose (1-6 mg/m2). UTP levels ically. Of nine patients who developed delayed-onset seizures, were decreased relative to purine nucleotides in 9 of 10 tumor seven had focal brain lesions (104). Myelosuppression was specimens by 16-72% but were decreased by >50% in only 3 infrequently observed but proved to be a major toxicity in the patients. one study in which 50% of patients with lymphoma experienced Investigators at MSKCC measured the dose response of leukopenia or thrombocytopenia (96). While a proportion of PALA on pyrazofurin-induced orotic aciduria and orotidinuria patients on the Phase II trials were more heavily pretreated as an estimate of inhibition of "total body pyrimidine synthesis" than is currently desirable, it is clear that PALA had minimal (75). A low dose of PALA, 250 mg/m2, was as effective as activity as a single agent. higher doses (2 g/m2) in decreasing the amount of urinary orotate/orotidine. This PALA dose was subsequently adopted PALA in Combination: Biochemical Rationale and Clinical Re as the lowest modulatory dose of PALA for use with the highest sults tolerable dose of the effector agent. The effect of low dose PALA and FUra. The most extensively studied combination PALA in terms of modulating the UTP and CTP pools in involving PALA is in conjunction with FUra. FUra exerts its tumor and normal tissues was not measured. A limitation of cytotoxic effect by at least two mechanisms: incorporation of this methodology is that it can lead to an underestimation of FUra to FUTP into RNA; and inhibition of TS (Fig. 3) (105- the rate of de novo biosynthesis and an overestimation of the 107). Incorporation into DNA may also contribute to cytotox- degree of enzyme inhibition since only some of the intermedi icity (108-110). Pretreatment with PALA may enhance the ates of the de novo pathway are measured (43,76). Furthermore, cytotoxicity of FUra by several potential mechanisms. These there is no accounting for the capacity of cells to utilize salvage include: (a) depletion of UTP pools by PALA favoring incor pathways to replete nucleotide pools. poration of FUTP into RNA; (b) depletion of UTP and CTP In summary, there is evidence to suggest that PALA can pools by PALA resulting in increased uridine/cytidine kinase inhibit ACTase activity in tumor and normal tissues in humans activity (decreased feedback inhibition) and favoring increased and is capable of inhibiting the de novo pathway of pyrimidine formation of FUMP; (c) decreased orotate and increased PRPP synthesis. Definitive documentation in humans of a selective by PALA favoring the synthesis of FUMP by orotate phos- depletion of ribonucleotide pools in tumor tissue is lacking. phoribosyltransferase; (d) depletion of UDP and CDP by PALA resulting in decreased dUMP and less competition with Clinical Trials with PALA Alone FdUMP for TS binding; and (e) decreased dCTP pools causing Phase I. Phase I clinical trials provided information on tol further inhibition of DNA synthesis beyond that resulting from erable doses of PALA on a number of schedules utilizing both the FdUMP-mediated blockade of TS and subsequent dTTP bolus and CI administration (Table 4) (77-82). Dose-limiting pool depletion. 4445 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1988 American Association for Cancer Research. BIOCHEMISTRY AND CLINICAL ACTIVITY OF PALA

Table 4 Phase I trials with PALA Dose Recommended dose Dose/course Dose-limiting Ref.Bolus Schedule n (g/m2/day) (g/m2/day) (g/m2) toxicity

(q2w)Â° 58 0.8-15 6.0 Skin rash 77 Mucositis8.7

24hCI(q3w)0.5-10.5Bolus 27 (q3w) 8.7 Skin rash 78 Mucositis Diarrhea1.5(qw)

0.1-2.0Bolusdaily for 5 days (q2-3w) 34 7.5 Skin rash 79 Stomatitis Diarrhea1.25(q3-4w) daily for 5 days (q6w) 25 0.1-1.25 801.3(q3w) 6.25 Diarrhea

5-day0.8-1.755-day CI (q3w) 10 6.5 Skin rash 78 Mucositis Diarrhea1.8

0.15-2.0BolusCI (q3w) 29 9.0 Diarrhea 81 Mucositis rash3.75-4.5 Skin weekly for 3 weeks 30 0.9-6.75 11.25-13.5 Skin rash 82 Diarrhea1.0

.0' Bolus daily for 5- 10 days (q2w) 17 1 for 5 days 5.0 Skin rash 77 Mucositis+ q2w, every 2 weeks (other schedules are similarly denned).

Table 5 Summary of Phase II single agentPALAResponse studies of UTP pool was increased 3-fold due to PALA-mediated UTP (%) depletion. PALA (1-3.3 IHM) enhanced FUra incorporation ofNo. Nn. of patients RRÂ° into RNA in vitro in the B16 melanoma line, two human Ref.BladderDisease studies evaluated CR PR (MR + PR) melanoma lines (IGR3 and M5), and the human MCF-7 breast 1 17 0 0 0 83 carcinoma (112, 113). Exposure of the MCF-7 cells to 33 /uM Breast 2 59 0 5 8 84,85 PALA for 19 h resulted in a 2.9-fold increase in PRPP levels Cervix 1 33 0 0 0 86 relative to control, an increase in the [3H]FUra/32P ratio by 3.4- Colorectal 3 70 0 1 1 87, 88, 89 Head and neck 3 62 0 3 5 90,91 fold, and a decrease in the clonogenic survival from 96% of Lung Non-small cell 3 101 03 3 92,93,94 control observed with 10 pM FUra alone to 37% of control for Small cell 2 20 0 0 0 94,95 the combination (113). PALA has also been shown to increase Lymphoma 1 31 0 0 0 96 [14C]FUMP incorporation into RNA when used in combination Melanoma 2 52 0 6 12 97,98 Ovarian 1 32 0 0 0 99 with FUrd in AS-30D hepatoma ascites cells (114). Further Renal cell 2 58 0 2 3 83,100 studies conducted in Sarcoma 180 cells revealed that PALA Sarcoma102Â° 2 47 0 1 2 101, and FUra had synergistic biochemical effects only after pretreat- RR, response rate. Studies were conducted on weekly, daily for 2 days, daily ing the cells for >12 h with PALA (115). As compared to FUra for 5 days, and everyschedules.Urd 2- to 3-week alone, the combination of PALA pretreatment and FUra pre vented the FUra-mediated increase in dUMP levels, increased UMP UDP UTP the levels of free FdUMP (2.5-fold), and enhanced the forma

Urd"NAKinase UMP UDP tion of the FdUMP-TS ternary complex. .^Urd Kmase Kinase In vivo, the effects of FUrd (8 /imol/kg), PALA (200 ^mol/ FUrd FUMP FUDP FUTP Phosphorlase / ^" kg), and the combination have been studied in rats bearing the / Orotate Phosphoribotyl- Ribonucleotide i.p. implanted hepatoma ascites cell line AS-30D (116). Thirty ReducÃaseFUradThd/ transferasÂ«+ PRPP days after tumor transplantation, rats treated with FUrd or PALA were 20 and 50% tumor free, respectively, while 60% of \ dThd dTMP the rats receiving PALA 1 h prior to FUrd were tumor free. Phosprtorylase \KinaseFdUrd Kinase The addition of galactosamine (500 ^mol/kg) to PALA fol FdUMPFdUDPLdUMP lowed 1 h later by FUrd further improved the tumor-free survival to 80%. When the incorporation of radiolabeled FUrd 1 Thymidylate into the RNA of either host liver or Morris hepatoma 7777 SynthasedTMP (implanted in the thigh) was measured, pretreatment with PALA and galactosamine resulted in enhanced incorporation Fig.FUra.A 3. Metabolism of with apparent selectivity for the hepatoma (248% of FUrd alone control) as opposed to host liver (72% of FURd alone control). number of these biochemical effects have been noted in In CDSFi mice bearing syngeneic spontaneously metastasizing preclinical models of the combination. Human MDA mammary breast carcinoma, pretreatment with a nontherapeutic dose of carcinoma cells demonstrated synergistic growth-inhibitory ef PALA (100 mg/kg) followed 24 h later by FUra (100 mg/kg) fects at all levels of the combination tested (FUra, 2.5 x 10~7- resulted in a decrease in tumor weight at day 20 from 13% 1.5X 10~5M;PALA,6x 10-'-3.7x 10~3M)(1 11). The addition (FUra alone) to 4% of control (13). Exposure to 100 mg/kg of PALA was found to cause increased incorporation of FUra PALA followed by [3H]FUra (50 mg/kg, 80 /iCi/kg) resulted in into rRNA, tRNA, and mRNA. While the total FUTP pool a 2.3-fold increase in [3H]FUra incorporated into tumor RNA, was not enhanced, the proportion of FUTP in the total FUTP with minimal effect on bone marrow or intestinal tissue. In- 446.046 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1988 American Association for Cancer Research. BIOCHEMISTRY AND CLINICAL ACTIVITY OF PALA creasing the dose of PALA to 200 mg/kg increased the FUra- was less than 10 min at all dose levels of FUra/PALA, indicat RNA in the intestine without improving the therapeutic index ing that PALA did not alter the plasma clearance of FUra. (13). In the majority of Phase I studies of PALA/FUra, diarrhea Miller et al. (117) explored the effect of PALA on the and mucositis were dose limiting (Table 6). Ataxia and myelo- pharmacodynamics of FUra in normal beagles. PALA, 50 mg/ suppression were dose limiting on the more recent Phase I trial kg (1000 mg/m2), was given i.v. 18-24 h prior to FUra, 10 mg/ by Ardalan and Singh (125) of weekly PALA, 250 mg/m2, kg (87 iiCi/kg; 200 mg/m2), and various tissues were sampled followed by a 24-h CI of FUra at doses exceeding 2600 mg/ l h later. ACTase activity was reduced to 37, 7, and 21% of m2/week. The Phase II evaluation of the combination has been control in intestinal mucosa, liver, and bone marrow, although conducted primarily in patients with colorectal cancer (Table the UTP + UDP pools were reduced only in the bone marrow 7) (126-130). Single arm studies have produced results no better (to 39% of control). Following PALA pretreatment, a higher than that expected with FUra alone. Two prospectively random amount of FUTP + FUDP (124 and 169%) and FUra-RNA ized trials have directly compared the combination to FUra (200%) was found in intestinal mucosa and liver, respectively. alone (129, 130). Both of these trials utilized the daily for 5 No significant changes of the FUra ribonucleotide pools and days schedule. Neither revealed a significant difference in re FUra-RNA were noted in other tissues. sponse by arm. Promising results observed in the Phase I trial The combination of PALA and FUra has been tested exten by Ardalan using a sequential low dose PALA/high dose FUra sively in the clinic (Table 6) (75, 118-125). Most trials did not CI schedule have prompted confirmatory single arm studies in incorporate biochemical end points. O'Connell et al. (122) did colon, gastric, and pancreatic cancer. measure the in vitro incorporation of initiated FUra into human PALA plus Methotrexate or MMPR as Modulator of FUra. leukocyte RNA after PALA treatment by obtaining blood sam An additional strategy to increase the incorporation of FUra ples from four patients treated with a daily for 5 days schedule. into RNA is to expand the intracellular levels of PRPP with These investigators could not demonstrate decreased [3H]FUra MTX or MMPR. Increased availability of PRPP is postulated incorporation into normal human leukocyte RNA. Leukocytes to increase the amount of FUMP formed by orotate phospho- from peripheral blood may not be an appropriate model, how ribosyltransferase. Increased FUTP formation coupled with the ever, due to the low rate of RNA synthesis in these nonpro- UTP pool depletion caused by PALA would favor incorporation liferating cells. Ardalan et al. (118) measured the level of of FUTP into RNA. Major et al. (Il 3) found that pretreatment ACTase activity in peripheral leukocytes in four patients re of MCF-7 breast carcinoma cells with PALA (0.01 mg/ml) for ceiving a 5-day CI of PALA/FUra. At a steady state concentra 19 h with either MTX (1 ^M) or MMPR (1 ^M) during the last tion of 20 fiM PALA (achieved with a dose of 940 mg/m2/day 6 h followed by a 3-h exposure to 10 ^M FUra resulted in a 7- for 5 days), the leukocyte ACTase activity decreased to 90% of and 9-fold increase, respectively, in the ratio of [3H]FUra/32P baseline over the last 4 days of the treatment. Nucleotide into RNA. In comparison, use of a single modulator resulted triphosphate pools were not measured. Pharmacological studies in a 1.7-3.4-fold increase. Furthermore, the loss of clonogenic by Erlichman (119) found that the mean elimination tv,of FUra survival was greatest with a three drug combination compared

Table 6 Combination trials of PALA and FUra: Phase I Institution/ sourceUSC" schedulePALA, and Toxicity18 dose(mg/m2)PALA, 940 mg/m2/dx5 CI DLT: mucositis/diarrhea 940 dx5 breast)1PR (colon, Ardalan et al. FUra, 180-325 mg/m2/dx5 CI N&V30Other: rash, FUra, 250dxSPALA, (118)NCI-MB Simultaneousq3-4wPALA, i.v.

& CPB 700-1500 mg/m2/d dl-5 i.v. DLT: mucositis/diarrhea 1000 dx5 PR(melanoma)3 Erlichman et al. FUra,q3wPALA, 100-250 mg/m2/d d2-5 i.v. Other: conjunctivitis, myelosuppression, FUra, 200dx4PALA, (119)SFCIMeshad rash16 CNS,

500-1 175 mg/m2/d dl-5 CI DLT: mucositis 750 dx5 PR (lung, colon, et al. FUra,q4wPALA, 185-430 mg/m2/d d2-6 i.v. Other: diarrhea, alopecia, rash, FUra, 275dxSNot histiocytoma)1 (120)SFCIWeiss myelosuppression34

850 mg/m2/d dl-5 CI DLT: mucositis recommendedPALA, PR(colon)2 et al. FUra,CIPALA, 300-630 mg/m2/d d2-6 Other: N&V, diarrhea, rash, phlebitis, (121)Mayo myelosuppression21

625 mg/m2/dx5 i.v. DLT: Stomatitis 625 dxS PR (colon, unknown O'Connellal.(122)M et FUra,q3-5wPALA, 100-350 mg/m2/dx5 i.v. Other: diarrhea, rash, FUra,q4wPALA, 250-300 dxS primary)3 myelosuppression27

DA 800-400 mg/m2/dx5 i.v. DLT: diarrhea/stomatitis/rashRecommended 400 dxS PR (colon) Bedikian et al. FUra, 400-200 mg/m2/dx5 i.v. q4w orn FUra, 200 dxSComment2 (123, 124)Dose PALA, 2000-1000 mg/m2/wx4 i.v. 24 DLT: diarrhea/stomatitis/rash PALA, 1500/w 4 PR (colon) FUra, 480-240 mg/m2/wx4 i.v. (PALA Other: N&V, myelosuppression, anorexia, FUra, 350/w 3 h before FUra) conjunctivitis

USC PALA, 250 mg/m2/w i.v. dl 21 DLT: Diarrhea/ataxia/myelosuppression PALA, 250/w 2 CR, 8 PR (colon/ Ardalan et al. FUra, 750-3400 mg/m2/w 24-h CI versus Other: N&V, rash FUra, 2600/w pancreas) (125) FUra, 1000-3400 mg/m2/w 24-h CI 20 See above FUra, 2600/w 2 PR

MSKCC PALA, 250-2000 mg/m2/wx3 i.v. 68 DLT (3 h): diarrhea/mucositis PALA, 250/w 1 PR Casper et al. FUra, 200-1200 mg/m2/wx3 i.v. (FUra DLT (24 h): myelosuppression FUra, 750/w (24 h) (rhabdomyosarcoma) (75) 3 or 24 h after PALA) Other: N&V, rash Â°USC, University of Southern California; NCI-MB & CPB, National Cancer Institute Medicine Branch and Clinical Pharmacology Branch; MDA, M.D. Anderson Hospital and Tumor Institute; d, day; w, week; dx5, daily for 5 days; q3-4w, every 3-4 weeks; dl-5, days 1-5; wx4, weekly for 4 weeks; DLT, dose-limiting toxicity; N&V, nausea and vomiting; CNS, central nervous system; SFCI, Sidney Farber Cancer Institute. 4447 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1988 American Association for Cancer Research. BIOCHEMISTRY AND CLINICAL ACTIVITY OF PALA

Table 7 Phase II trials of PALA and FUra FUra/ Institution/SourceSFCIÂ° schedulePALA, and Ã©valuable21 FdUrd16 850 mg/mVd d 1-5 CI colon PR2 Weiss et al.(126)City FUra.q4wPALA, 300 mg/m2/d d2-6 i.v. 8 pancreas 0 PR 4breast10 420 2PR02 other10

of Hope 940 mg/m2/dx5 CI colon PR06 Presant27)ECOGMuggia et al. ( 1 FUra.q3-4wPALA, 250-400 mg/m2/dx5 CI 3sarcoma23 00

1500 mg/m2 i.v. dl colorectal PR Â«-fa/.(128)Dose FUra, 400-800 mg/m2 i.v. dl -8 cycle q2w or PALA, 1000 mg/m2 i.v. dl, 8 I4colorectalPrior 0Response2 3 PR FUra, 300-600 mg/m2 i.v. d2, 9Patients

MDA Randomize (Arm C replaced A) Mann et al. A. PALA, 800 mg/m2/dx5 i.v. q2w versus 19 breast Not specified 1 PR (P< 0.01 between A and B) B. PALA, 400 mg/m2/dx5 i.v. 35 breast Not specified 10 PR (P > 0.01 between B and C) FUra, 300 mg/m2/dx5 i.v. q3w versus (responders had prior FUra) C. FUra. 400 mg/m2/dx5 i.v. q3w 11 breast Not specified 2 PR (as above)

NCCTG Randomize Buroker et al. Arm A (q5w) 69 colorectal (130) FUra, 500 mg/m2/dx5 i.v. 37 measurable 11 PRorCR Arm B (qSw) FUra, 300 mg/m2/dx5 i.v. 65 colorectal PALA, 625 mg/m2/dx5 i.v. 34 measurable 4 PR or CR Arm C (q4w) dThd, 45 g i.v. dl 67 colorectal FUra, 300 mg/m2 i.v. dl 34 measurable 6 PR or CR Arm D (qSw) FUra, 500 mg/m2/dx5 i.v. 66 colorectal Levamisole, 50 mg p.o. q8h dl4-16, d21-23 40 measurable 9 PR or CR Arm E(qlOw) FUra, 300 mg/m2/dx5 i.v. dl-5, d36-40 68 colorectal MeCCNU, 30 mg/m2 p.o. d2-6 38 13 PRorCR Oncovin, l mg/m2 i.v. dl, 36 No arm significantly better than Streptozotocin, 500 mg/m2/w i.v. FUra alone

Fox Chase PALA, 250 mg/m2/w i.v. dl Pancreas Ongoing trial O'Dwyer FUra, 2600 mg/m2/w CI d2 Colorectal

SWOG/Ardalan PALA, 250 mg/m2/w i.v. dl Gastric Proposed or ongoing FUra, 2600 mg/m2/w CI d2 Pancreas * SFCI. Sidney Farber Cancer Institute; ECOG, Eastern Cooperative Oncology Group; MDA, M.D. Anderson Hospital and Tumor Institute; SWOG, Southwest Oncology Group; d, day; w, week; dl-5, days 1-5; q4w, every 4 weeks; q3-4w, every 3-4 weeks; MeCCNU, rranj-l-(2-chloroethyl)-3-(4-methylcyclohexyl)-l- nitrosourea. to a two drug regimen. Martin et al. (131) have demonstrated PRPP content in serial biopsies from a skin metastasis in improved antitumor activity with combinations that have mul creased 25-fold from 39 to 961 pmol/mg (133). A Phase II trial tiple modulatory effects upon FUra in tumor-bearing mice. On is presently being conducted for patients with adenocarcinoma a weekly for 3 weeks i.p. schedule, the best results were obtained of unknown primary. in mice bearing advanced mammary carcinoma with the com PALA plus dThd as Modulators of FUra. dThd, like PALA, bination of PALA (100 mg/kg) followed in 17 h by methotrex- has been shown to enhance incorporation of FUra into RNA ate (300 mg/kg) followed in 2.5 h by FUra (150 mg/kg) with in preclinical models (134, 135). The mechanism of this effect leucovorin and uridine rescue initiated 2 h after FUra. Data has been suggested to involve the effect of dThd on the phar- pooled from multiple experiments confirmed that the tumor macokinetic behavior of FUra (136-138). This subject has weight on day 28 was <5% of control, with a PR (>50% recently been reviewed (139, 140). reduction in tumor volume) rate of 27-32%. These results were Clinical studies of the three drug combination PALA/dThd/ significantly better than any of the individual combinations FUra have been performed (141-144). Again, studies were not and, of importance, the toxicity to the host was not enhanced conducted with strict biochemical end points. Serial tumor by PALA. biopsies obtained from two patients receiving the three drugs Two clinical trials have been conducted at MSKCC and the simultaneously revealed that (a) ACTase was inhibited by 75% Dana Farber Cancer Institute with the three drug combination but returned to baseline within 48 h, (b) pyrimidine base and of PALA/FUra/MMPR, but the results are not yet published. nucleoside pools decreased after PALA administration but in An additional Phase I trial using the 250 mg/m2 dose of PALA creased after FUra/dThd, (c) UMP and UDP pools decreased with 75 mg/m2 MMPR in combination with escalating doses after PALA and increased after FUra/dThd, and (d) UTP levels of FUra administered as a 24-h CI is planned in an effort to remained constant (145). Pharmacokinetic studies showed that build upon the encouraging preliminary results seen with this the tv, of both FUra and FdUrd were significantly prolonged to schedule of PALA/FUra. Two schedules of PALA/MTX/FUra 4-4.5 h on both schedules. Basseches et al. (146) at the Mayo were tested by investigators at MSKCC (132, 133). Bone mar Clinic reported that in four patients with normal renal function row aspirates from five patients showed a 1.8-fold increase in tested with PALA/dThd/FUra, the average tv, of FUra was 3.0 PRPP content (0.7-2.4 pmol/mg) 24 h post-MTX/PALA. The h. 4448 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1988 American Association for Cancer Research. BIOCHEMISTRY AND CLINICAL ACTIVITY OF PALA

Neurotoxicity was dose limiting on three studies which used Only limited clinical investigation of the combination has relatively low doses of PALA but high doses of dThd (141- been conducted (147, 148). Investigators at MSKCC treated 143). Gastrointestinal toxicity was also common. When PALA seven leukemia patients with PALA/dThd/ara-C (147). Only was increased on one trial to 4 g/m2 in combination with FUra/ minor and inconsistent changes were noted following flash dThd, leukopenia became dose limiting (144). Only a limited incorporation of [3H]ara-C into nucleotides and blast DNA. No number of Phase II studies of this combination have been PR were observed. In a study at M. D. Anderson Hospital and conducted. A 27% response rate (1 CR, 9 PR) was noted in Tumor Institute, four of seven leukemia patients Ã©valuablefor colorectal patients by O'Connell et al. (144). Since a number of response experienced marrow aplasia following therapy (148). the responses occurred in patients with anaplastic Grade IV Cellular pharmacology studies indicated that dTTP levels in colorectal cancer, a separate Phase II study was initiated by the leukemic blasts were increased 1.5-4.7-fold at 24 h over base NCCTG for these patients. This regimen is also being studied line, while levels of CTP and UTP were not affected. An by the NCCTG in patients with gastric carcinoma and lym- increase in ara-CTP accumulation in vitro in leukemia blasts phoma. Results from these trials have not yet been published. obtained prior to and 23 h following treatment was detected in PALA in Combination with ara-C. ara-C is activated to ara- four of seven samples, but a correlation with antileukemic CMP by dCyd kinase (Fig. 4). The sequential action of nucleo- response could not be made. tide kinase converts ara-CMP to the triphosphate level, ara- PALA in Combination with 5-aza-Cyd. 5-aza-Cyd is converted CTP interferes with DNA synthesis by at least two mechanisms: to 5-aza-CMP by undine kinase, and upon subsequent anabol- incorporation of the fraudulent nucleotide into DNA; and in ism to 5-aza-CTP, is incorporated into RNA. 5-aza-Cyd inhibits hibition of DNA polymerase. PALA-mediated depletion of RNA, protein, and DNA synthesis. Theoretically, PALA could dCTP could have several consequences. Since dCyd kinase is enhance the formation of 5-aza-CMP by decreasing the levels under feedback regulation by dCTP, a reduced dCTP pool of UTP and CTP which are feedback inhibitors of uridine/ would lead to increased activity of the enzyme and increased cytidine kinase. The reduction in UTP and CTP would favor conversion of ara-C to the nucleotide level. Incorporation of the incorporation of 5-aza-CTP into RNA by RNA polymerase. ara-CTP into DNA might be favored if the level of the compet L1210 and P388 cells exposed to 300 MMPALA for 3 h followed ing substrate, dCTP, were reduced. by l h exposure to 75 MM[l4C]-5-aza-Cyd increased by 1.5-fold Grant et al. (16) found that preincubation of HL-60 cells the incorporation of 5-aza-Cyd into the acid-soluble fraction of with PALA, 100 MM,for 12 h followed by 1 /tM [3H]ara-C for the cells, and the 5-aza-CTP level was twice that measured with 45 min resulted in a 211% increase in the total intracellular exposure to 5-aza-Cyd alone (149). Pretreatment with PALA accumulation of ara-C. In addition, this combination was as reduced the UTP and CTP pools in these cells by only 45-50%. sociated with an 88% inhibition of DNA synthesis as measured Furthermore, a 3-fold increase in the amount of 5-aza-Cyd by incorporation of [3H]dThd, a 2.25-fold increase in the for incorporated into RNA was observed. Pretreatment with 40 MM mation of ara-CTP, and a 2.5-fold increase in the ara-CTP PALA for 3 h followed by 1 MM5-aza-Cyd (IDSOaza-Cyd alone, retained after 4 h. Martin et al. (135) evaluated the combination 5 MM)inhibited the cell growth of both leukemic lines by 90%, of PALA, dThd, and ara-C in CDSFi mice bearing advanced and clonogenic survival was reduced by 80%. In vivo studies mammary tumors. Three weekly doses of PALA (200 mg/kg have not been performed, and no clinical trials have been i.p.) followed 24 h later by high dose ara-C (1000 mg/kg) conducted. resulted in an average tumor weight 41% of control compared Enhancement of PALA Cytotoxicity by Nucleoside Transport to 64% with ara-C alone. The best results were observed when Inhibitors. The ability of physiological amounts of uridine and dThd (a total of 4000 mg/kg given in divided doses) was cytidine to protect cells from the cytotoxicity of PALA has combined with PALA/ara-C; the tumor weight was 15% of been alluded to previously. DP and NBMPR, reversible inhib control, significantly better than that observed with either itors of facilitated nucleoside transport in mammalian cells, PALA/ara-C or dThd/ara-C. With low dose ara-C (200 mg/ have been combined with antimetabolites such as PALA in an kg), tumor weight reduction on the evaluation day was 81% of effort to prevent nucleoside salvage. Chan et al. (46) showed control for ara-C alone, while sequential PALA followed by that DP inhibited the incorporation of extracellular [3H]uridine dThd and ara-C resulted in a tumor weight 34% of control, into the nucleotide fraction of T-242 melanoma and T-219 compared to 77% with PALA/ara-C and 46% with dThd/ara- colon carcinoma cells in a dose-dependent manner. While uri C. Mortality studies performed on mice bearing Lewis lung dine uptake was inhibited 50% at concentrations <<>.1 MM, carcinoma (refractory to ara-C at its maximum tolerated dose) increasing the concentration of DP by 2 logs did not completely showed that all animals receiving PALA (200 mg/kg) and dThd inhibit uridine uptake. Clonogenic assays conducted in the (total dose, 4000 mg/kg) followed 3 h later by ara-C (20 mg/ presence of PALA Â±1 MMDP for 10-14 days showed that DP kg) (i.p. daily for 10 days beginning 8 days after transplantation) reduced the viability of T-242 melanoma cells from 90% to were alive on day 49, while no animals receiving any single 16% (5 MM PALA) and from 73% to 8% (100 MM PALA). drug survived, and only 20% of the mice treated with a two Exposure to 5 MM PALA + 1 MM DP did not significantly drug combination survived. reduce the viability of colony-forming unit-granulocyte-macro- phage cells. At the higher concentration of PALA, however, dCyd dCMP â€¢dCDP dCTP . DP reduced the viability of the normal bone marrow cells to <5%. These data suggested selectivity for the tumor cell line at DNA dCyd dCMP dCDP low concentrations of PALA. Chan extended these observations Kinase Kinase Kinase in the ovarian carcinoma 2008. The IDso of PALA was in excess Ara-C â€¢Ara-CMP Ara-CDP Ara-CTP of 500 MMin this resistant line for cell growth and viability assays, but concomitant exposure to 1 MMDP reduced the ID5o dCyd CMP to 70 and 5 UM, respectively. A 24-h exposure to PALA/DP Deaminase Deaminase reduced the UTP and CTP pools to <6% and <26%, respec Ara-U Ara-UMP tively. Pharmacological concentrations of uridine reversed the Fig. 4. Metabolism of ara-C. effect of PALA/DP on ribonucleotide pools and toxicity. In 4449 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1988 American Association for Cancer Research. BIOCHEMISTRY AND CLINICAL ACTIVITY OF PALA tissue culture medium the concentration of DP used represents of this regimen will await the results of these trials. free drug. In contrast, DP is greater than 99% protein bound PALA/Alanosine and Other Purine Biosynthesis Inhibitors. in vivo, and the free and pharmacologically active concentration Alanosine, an antibiotic resembling L-aspartic acid, inhibits two of DP is thus approximately 2 logs lower than the total plasma enzymes of the de novo pathway of purine synthesis (154). The concentration. combination of L-alanosine and PALA were synergistic in clon In vivo, the effect of PALA (200 mg/kg) and DP (100 mg/ ogenic assays against human renal carcinoma and melanoma kg) on the ribonucleotide pools of the ovarian 2008 xenograft cell lines (155). Therapeutic synergism of the combination was was examined (17). Twenty-four h following PALA alone, the demonstrated in mice bearing P388/ara-C-resistant leukemia DTP and CTP pools were 47 and 79%, respectively, of control. (156). The Phase I trial of the combination PALA/alanosine DP administered alone did not affect the UTP and CTP con was conducted at the Mayo Clinic (155). Stomatitis was dose centration. The combination of PALA plus DP, however, re limiting on both the daily for 5 days and every 3 week schedule. duced the UTP and CTP pools to below detection. Finally, 100 No myelosuppression was observed. The Mayo Clinic and the mg/kg DP given s.c., which produces a peak plasma level of NCCTG tested this combination in colorectal cancer and mel 11.5 MMtotal drug, shifted the 50% lethal dose of PALA in anoma, but no significant activity was seen (157, 158). Thera non-tumor-bearing mice from 1000 mg/kg to 400-500 mg/kg. peutic synergy in an in vivo system has also been noted for the In regard to NBMPR, Erlichman et al. (150) reported that a combination of PALA/tiazofurin (159). No clinical trials have 72-h exposure of B16 melanoma cells to 100 MMPALA alone been conducted. or in combination with 10 MMNBMPR reduced the clonogenic PALA in Combination with Inhibitors of de Novo Pyrimidine survival from 100% to 1% when the cells were grown in medium Synthesis. Several investigators have described synergy with the supplemented with nondialyzed fetal calf serum. Exposure to combination of PALA and acivicin. Acivicin, an antibiotic 100 MMPALA in medium supplemented with dialyzed fetal resembling L-glutamic acid, inhibits L-glutamine-dependent calf serum, in which the concentration of preformed nucleosides amidotransferases such as CPS II and CTP synthetase (160). is negligible, reduced the viability to 2% of control. Coadmin- Acivicin (0.2 MMfor 20 h) and PALA (30 MMfor 20 h) were istration of 5 or 30 MMuridine with 100 MMPALA partially synergistic in vitro against LI210 as determined by isobologram protected the cells (viability, 40 and 80% of control, respec method (161). The combination inhibited pyrazofurin-induced tively). These observations indicated that physiological amounts total orotidine and orotate in LI210 cells by 63% and resulted of pyrimidine nucleosides present in the nondialyzed serum in a more than additive depletion of dCTP pools. Mice bearing were sufficient to circumvent the PALA-mediated inhibition of s.c. implanted PALA-resistant Lewis lung (LL/PALA-C) were the de novo pyrimidine pathway. In vivo in C57BL mice bearing treated with acivicin, 10 mg/kg (30 mg/m2), plus PALA, 20 B16 melanoma, the 50% lethal dose of PALA was reduced mg/kg (60 mg/m2), every other day during days 1-9. The from 250 mg/kg/day for 4 days to 160 mg/kg/day for 4 days combination produced more than 80% inhibition of tumor for the combination (200 mg/kg of NBMPR-P). The combi growth at day 21, and 3 of 10 mice were long term survivors nation of PALA at 150 mg/kg/day for 4 days with NBMPR-P (162). In contrast, animals treated with either single agent alone at 100 mg/kg/day for 4 days produced a comparable growth had minimal inhibition of tumor growth and none were long delay observed with PALA at a higher, equitoxic dose (300 mg/ term survivors. In addition, Ardalan et al. (19) showed that kg/day for 4 days). The conclusion was that salvage of physio P388 leukemia cells with acquired resistance to acivicin were logical levels of uridine may be an important resistance mech collaterally sensitive to PALA. Furthermore, PALA given at anism to PALA, and, although the combination of PALA and 200 mg/kg (600 mg/m2) to mice bearing P388/acivicin cells NBMPR was synergistic in vitro, this did not lead to an im produced a marked depletion of UTP and CTP in the tumor, provement in therapeutic index in vivo. compared to a minimal effect on P388-sensitive cells. The A Phase I trial of PALA plus DP has been conducted by combination of PALA and acivicin has also been shown to be investigators at the University of California at San Diego (151, synergistic against a ( I)HI , spontaneous metastasizing breast 152). A fixed dose of p.o. DP (50 mg/m2 every 6 h) was tumor and P388/ara-C in vivo (159, 163). administered with escalating doses of PALA (500-4500 mg/ The combination of acivicin, PALA, and FUra has been m2 every 3 weeks). Diarrhea, mucositis, rash, and abdominal reported to have therapeutic synergism in vivo (164). This pain were dose-limiting toxicities at doses >3900 mg/m2. synergy could not be attributed to enhancement of FUra incor Plasma uridine levels were measured in ten patients on this poration into RNA compared to that seen with PALA/FUra. study. DP alone caused a reduction from 3.49 Â±1.28 MMto The combination of PALA/acivicin/ara-C was not synergistic 2.29 Â±0.70 MMat 9 h after the first p.o. dose and 2.46 Â±0.61 in vivo or in vitro (163, 165). UM after 1 week of DP. Seven h after PALA administration, No clinical trials of the combinations of PALA/acivicin or the uridine level had been further reduced to 0.87 Â±0.23 MM. PALA/acivicin/FUra have been performed. Acivicin has shown Uridine levels remained depressed for 6 or 11 days in two minimal activity in Phase II screening, with the exception of patients treated at 4200 mg/m2 PALA. The clinical significance modest activity against high grade gliomas (166). A possible of the decline in uridine levels in the presence of DP is not clinical application of the combination would be in patients clear and the effects of PALA alone on uridine levels were not with primary brain tumors. measured in this study [Levy et al. (153) noted that 1 mg Other Combination Trials with PALA. One Phase I trial of NBMPR-P increased the plasma /â€¢/,andconcentration of uri- the combination PALA/BCNU was conducted by investigators dine]. Two PRs were observed in 38 patients Ã©valuablefor at the Mayo Clinic (167). A fixed dose of PALA (5000 mg/m2) response (one each in non-small lung cancer and soft tissue was combined with BCNU (40-240 mg/m2) every 6 weeks. The sarcoma) (151, 152). A Phase II trial of PALA and p.o. DP has dose-limiting toxicity of this study was myelosuppression. A been initiated at MSKCC in soft tissue sarcoma, and a trial at Phase II trial of the combination in colorectal cancer using 200 Purdue is planned in non-small cell lung cancer. Pharmacolog mg/m2 BCNU was conducted by these same investigators, but ical monitoring of total and free DP levels will be done at both activity was insufficient to warrant further investigation. Inves the peak and trough following p.o. administration. Since PALA tigators at Mayo also added dibromodulcitol or dianhydroga- is considered to be a weak "effector" agent, further evaluation lactitol to the two drug combination and conducted a trial in 4450 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1988 American Association for Cancer Research. BIOCHEMISTRY AND CLINICAL ACTIVITY OF PALA brain tumors. Results of this study have not been published. a low PALA dose may selectively enhance the effect of FUra Because of the lack of myelotoxicity with PALA, an empirical in human cancer cells with sparing of normal tissues. Improve trial of PALA (3000 mg/m2 on days 1 and 2), vindesine (3 mg/ ment in the anticancer activity and the therapeutic index of the m2 on days 1 and 8), and cisplatin (30 mg/m2 on days 1-5) was PALA-containing regimens over that observed with the maxi evaluated in melanoma patients by Voigt et al. (168) for the mally tolerated dose of the effector agent alone remains to be European Organization for Research and Treatment of Cancer. demonstrated. Of 21 patients, 9 objective responses were noted including 5 complete remissions. Median duration of complete response References was 5 months. No further trials of this combination were conducted. 1. Collins, k. I). and Stark, G. R. Aspartate transcarbamylase. Interaction with the transition state analogue /V-(phosphonacetyl)-L-aspartate. J. Biol. Chem., 246:6599-6605, 1971. 2. White, J. C., and Mines, L. H. Role of endocytosis and lysosomal pH In Discussion uptake of jV-phosphonacetyl)-L-aspartate and its inhibition of pyrimidine synthesis. Cancer Res., 44: 507-513, 1984. Extensive clinical evaluation revealed that despite its broad 3. Hoogenraad, N. J. Reaction mechanism of aspartate transcarbamylase from spectrum of antitumor activity against murine solid tumors, mouse spleen. Arch. Biochem. Biophys., 161: 76-82, 1974. 4. Moore, E. C., Friedman, J., Valdivieso, M., Plunkett, W., Marti, J. R., et PALA is inactive as a single agent in human malignancies. al. Aspartate carbamoyltransferase activity, drug concentrations, and pyrim Pharmacological and biochemical monitoring in the preclinical idine nucleotides in tissue from patients treated with yV-(phosphonacetyl)- and clinical setting indicate that PALA can inhibit ACTase L-aspartate. Biochem. Pharmacol., 31: 3317-3321, 1982. 5. Tsuboi, K. K., Edmunds, H. N., and Kwong, L. K. Selective inhibition of activity in tumor and normal tissue. Preclinical data emerged pyrimidine biosynthesis and effect on proliferative growth of colonie cancer which demonstrated synergism between PALA and agents such cells. Cancer Res., 37: 3080-3087, 1977. 6. Roberts, M. F., Opella, S. J., Schaffer, M. H., Phillips, H. M., and Stark, as FUra and ara-C. Therefore, interest shifted to combination G. R. Evidence from "C NMR for protonation of carbamyl-P and iV- trials. The initial clinical trials of PALA/FUra were disappoint (phosphonacetyl)-L-aspartate in the active site of aspartate transcarbamyl ing. Although several studies indicated that the high doses of ase. J. Biol. Chem., 251: 5976-5985, 1976. 7. Moore, E. C. JV-(Phosphonacetyl)-L-aspartate inhibition of the enzyme PALA used appeared to be modulating the toxicity of FUra, complex of pyrimidine biosynthesis. Biochem. Pharmacol., 31: 3313-3316, this did not translate into higher response rates. However, these 1982. 8. White, J. C., and HiÃ±es,L. H. Binding of radiolabeled yV-(phosphonacetyl)- trials used high doses of PALA which mandated a lower dose i -aspan ate to aspartate transcarbamylase from Ehrlich ascites tumor cells. of FUra, the effector agent. In addition, the dose ratios, se Biochem. Pharmacol., 33: 3645-3648, 1984. quence, and interval between drugs may not have been optimal. 9. BÃ¢illon,J., (ink-hard. M., Malaise, E. P., and HervÃ©,G.Kinetic parameters of aspartate transcarbamylase in human normal and tumoral cell lines. Preclinical studies indicated that noncytotoxic concentra Cancer Res., 43: 2277-2282, 1983. tions of PALA could perturb nucleotide pools. The measure 10. Kafarski, P., Lejczak, B., Mastalerz, P., Dus, D., and Radzikowski, C. N- ment of inhibition by pyrazofurin-induced orotic aciduria/oro- (Phosphonacetyl)amino phosphonates. Phosphonate analogues of W-(phos- phonacetylH-aspartic acid (PALA). J. Med. Chem., 28: 1555-1558, 1985. tidinuria by PALA in Phase I studies showed that higher doses 11. Swyryd, E. A., Seaver, S. S., and Stark, G. R. JV-fPhosphonacetylH- were not more effective than lower doses in decreasing flux aspartate, a potent transition state analog inhibitor of aspartate transcar through the de novo pathway of pyrimidine synthesis. A new bamylase, blocks proliferation of mammalian cells in culture. J. Biol. Chem., wave of clinical trials utilizing the lowest "biochemically active" 2^9:6945-6950, 1974. 12. Yoshia, T., Stark, G. R., and Hoogenraad, N. J. Inhibition by W-(phos- dose of PALA in combination with the highest tolerable dose phonacetyl)-L-aspartate of aspartate transcarbamylase activity and drug- induced cell proliferation in mice. J. Biol. Chem., 249: 6951-6955, 1974. of the active drug has been initiated. 13. Martin, D. S., Stolli, R. L., Sawyer, R. C., Spiegelman, S., Casper, E. S., However, a variety of questions remain unresolved in the and Young, C. W. Therapeutic utility of utilizing low doses of iV-(phos- clinical setting. What is the effect of PALA on UTP and CTP phonacetyl)-L-aspartic acid in combination with 5-fluorouracil: a murine study with clinical relevance. Cancer Res., 43: 2317-2321, 1983. pools of normal and tumor tissue? Does administration of 14. Hunting, D., and Henderson, J. F. Relation between ribo- and deoxyribo- PALA 24 h prior to FUra result in enhanced incorporation of nucleotide concentrations and biological parameters in cultured Chinese FUra into RNA? Is the salvage of circulating pyrimidines a hamster ovary cells. Biochem. Pharmacol., 31: 1109-1115, 1982. 15. Mover, J. D., Smith, P. A., Levy, E. J., and Handschumacher, R. E. Kinetics clinically important mechanism of resistance to inhibitors of de of JV-(phosphonacetyl)-L-aspartateand pyrazofurin depletion of pyrimidine novo pyrimidine biosynthesis? Hopefully, these questions may ribonucleotide and deoxyribonucleotide pools and their relationship to nucleic acid synthesis in intact and permeabilized cells. Cancer Res., 42: be answered by clinical trials incorporating biochemical end 4525-4531, 1982. points. In view of tumor cell heterogeneity and the striking 16. Grant, S., Rauscher, F., Ill, and Cadman, E. Differential effect of N- intrinsic and acquired resistance to antineoplastic agents, a (phosphonacetyl)-L-aspartate on 1-/3-D-arabinofuranosylcytosine metabo lism and cytotoxicity in human leukemia and normal bone marrow progen single manipulation may not be sufficient to circumvent clinical itors. Cancer Res., 42:4007-4013, 1982. resistance to an agent such as FUra. Studies by Martin et al. 17. Chan, T. C. K., and Howell, S. B. Mechanism of synergy between N- have demonstrated that partial regressions in tumor-bearing (phosphonacetyl)-L-aspartate and dipyridamole in a human ovarian carci noma cell line. Cancer Res., 45: 3598-3604, 1985. mice occurred only with the concurrent administration of mul 18. Kensler, T. W., Mutter, G., Hankerson, J. G., Reck, L. J., Harley, C., Han, tiple modulating agents. If the activity of PALA, 250 mg/m2, N., Ardalan, B., Cysyk, R. L., Johnson, R. K., Jayaram. H. N., and Cooney, followed 24 h later by FUra administered as a 24-h CI is D. A. Mechanism of resistance of variants of the Lewis lung carcinoma to JV-(phosphonacetyl)-L-aspartic acid. Cancer Res., 41: 894-904, 1981. confirmed in Phase II trials, should further efforts be directed 19. Ardalan, B., Jayaram, H. N., and Johnson, R. K. Collateral sensitivity to towards building on this combination or towards definitive /V-(phosphonacetyl)-L-aspartic acid in a line of P388 leukemia cells selected for resistance to (L-(a5, 55)-a-amino-3-chloro-4,5-dihydro-5-isoxazole- Phase III trials designed to document unequivocally the benefit acetic acid (acivicin). Cancer Res., 43: 1598-1601, 1983. of the investigational agent as a prelude to commercial licen 20. Moyer, J. D., and Handschumacher, R.E. Selective inhibition of pyrimidine synthesis and depletion of nucleotide pools by /V-(phosphonacetyl)-L-aspar- sing? If the decision is to add other agents such as MTX, tate. Cancer Res., 39: 3089-3094, 1979. MMPR, or leucovorin to the two drug combination, then the 21. Clinical Brochure, A'-Phosphonacetyl-L-aspartate disodium (PALA), NSC opportunity to isolate the contribution of PALA may be lost. 224131. Bethesda, MD; Division of Cancer Treatment, National Cancer Institute, NIH, 1977. In summary, since PALA is inactive as a single agent, the 22. Laster, W. R., Jr., and Schabel, F. M., Jr. Collateral sensitivity of P388/ clinical development plans currently focus on its potential role Ara-C and P388/5-FU to iV-(phosphonacetyl)-L-aspartate (PALA). Proc. as a biochemical modulator. If effective, the low doses of PALA Am. Assoc. Cancer Res., 20: 95, 1979. 23. Johnson, R. K., Inouye, T., Goldin, A., and Stark, G. R. Antitumor activity used in the more recent clinical studies should permit admin of /V-(phosphonacetyl)-L-aspartate acid, a transition-state inhibitor of as istration of higher doses of the effector agent. It is hoped that partate transcarbamylase. Cancer Res., 36: 2720-2725, 1976. 4451 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1988 American Association for Cancer Research. BIOCHEMISTRY AND CLINICAL ACTIVITY OF PALA

24. Johnson, R. K., Swyryd, E. A., and Stark, G. R. Effects of iV-(phosphon- Enzymic capacities of purine de novo and salvage pathways for nucleotide acetyl)-L-aspartate on murine tumors and normal tissues in vivo and in vitro synthesis in normal and neoplastic tissues. Cancer Res., 44: 2475-2479, and the relationship of sensitivity to rate of proliferation and level of 1984. aspartate transcarbamylase. Cancer Res., 38: 371-378, 1978. 52. Ahmed, N. K. Enzymes of the de novo and salvage pathways for pyrimidine 25. Valeriote, F., and Grates, H. MOPC-315 murine plasmacytoma as a model biosynthesis in normal colon, colon carcinoma, and xenografts. Cancer anticancer screen for human multiple myeloma. J. Nati. Cancer Inst., 76: (Phila.), 54: 1370-1373, 1984. 61-65, 1986. 53. Harrison, S. D., Jr., Giles, H. D., and Denine, E. P. HÃ©matologieand 26. Corbett, T. H., Roberts, B. J., Leopold, W. R., Peckham, J. C, Wilkoff, L. histopathologic evaluation of A'-(phosphonacetyl)-L-aspartate (PALA) in J., fi al. Induction and chemotherapeutic response of two transplantable mice. Cancer Chemother. Pharmacol., 2: 183-187, 1979. ductal adenocarcinomas of the pancreas in C57BL/6 mice. Cancer Res., 44: 54. Sieber, S. M., Botkin, C. C., Soong, P., Lee, E. C., and Whang-Peng, J. 717-726, 1984. Embryotoxicity in mice of phosphonacetyl-u-aspartic acid (PALA), a new 27. Leung, S. W., Fung, K. P., Choy, Y. M., and Lee, C. Y. Inhibition by N- antitumor agent. 1. Embryo-lethal, teratogenic, and cytogenetic effects. (phosphonacetyl)-L-aspartate of Ehrlich ascites tumour growth and glucose Teratology, 22: 311-319, 1980. transport. Cancer Lett. 23: 183-188, 1984. 55. Sieber, S. M., Botkin, C. C., Leslie, K. A., and Cooney, D. A. Embryotox 28. Goldin. A., Venditti, J. M., MacDonald, J. S., Muggia, F. M., Henney, J. icity in mice of phosphonacetyl-L-aspartic acid (PALA), a new antitumor E., and DeVita, V. T., Jr. Current results of the screening program at the agent. II. Studies on its mechanism and reversibility. Teratology, 22: 321- Division of Cancer Treatment, National Cancer Institute. Eur. J. Cancer, 328, 1980. 17: 129-142. 1981. 56. Erlichman, C., Strong, J. M., and Chabner, B. A. Application of a simple 29. Sordillo, P. P., Helson, C., Lesser, M., and Helson, L. Effect of Phase I competitive protein-binding assay technique to the pharmacokinetics of \ and II chemotherapeutic agents against human lymphomas heterotrans- (phosphonacetyl)-u-aspartate in humans. Cancer Res., 40: 1902-1906, planted in nude mice. Oncology (Basel). 40: 15-17, 1983. 1980. 30. Houchens, D. P., Ovejera, A. A., Riblet. S. M., and Slagel, D. E. Human 57. Cooney, D. A., Karlowicz, M. G., Cubillan, J., Roettger, M., and Jayaram, brain tumor xenografts in nude mice as a chemotherapy model. Eur. J. H. N. An enzymatic technique for measuring iV-phosphonacetyl-L-aspartic Cancer Clin. Oncol., 19: 799-805, 1983. acid in tissues. Cancer Treat. Rep., 62: 1503-1507, 1978. 31. Shoaf, W. T., and Jones, M. E. Uridylic acid synthesis in Ehrlich ascites 58. Friedman, J., Moore, E. C., Hall, S. W., and Loo, T. L. Enzymatic assay carcinoma. Properties, subcellular distribution, and nature of enzyme com for the antitumor agent A'-(phosphonacetyl)-L-aspartic acid (PALA). Cancer plexes of the six biosynthetic enzymes. Biochemistry, 12:4039-4051, 1973. Treat. Rep., 63: 85-88, 1979. 32. Tatibana, M., and Ito. K. Control of pyrimidine biosynthesis in mammalian 59. Kensler, T. W., Jayaram, H. N., and Cooney, D. A. A facile enzymatic tissues. I. Partial purification and characterization of glutamine-utilizing technique for the estimation of nanomolar concentrations of /V-phosphon- carbamyl phosphate synthetase of mouse spleen and its tissue distribution. acetyl-L-aspartic acid in plasma. J. Biochem. Biophys. Methods, 2: 29-35, J. Biol. Chem., 244: 5403-5413, 1969. 1980. 33. Levine, R. L., Hoogenraad, N. J., and Kretchmer, N. Regulation of activity 60. Lankelma, J., Fenders, P. G. M., Leyva, A., and Pinedo, H. M. Determi of carbamoyl phosphate synthetase from mouse spleen. Biochemistry, Iti: nation of jV-(phosphonacetyl)-L-aspartic acid (PALA) in plasma and urine 3694-3699, 1971. by high pressure liquid chromatography. Eur. J. Cancer, 26: 1483-1487, 34. Tatibana, M., and Shigesada, K. Control of pyrimidine biosynthesis in 1980. mammalian tissues. V. Regulation of glutamine-dependent carbamyl phos 61. Branfman, A. R., Valia, K. H., and Bruni, R. J. Gas chromatography and phate synthetase: Activation by 5-phosphoribosyl 1-pyrophosphate and in mass spectrometry of yV-(phosphonacetyl)-L-aspartic acid. J. Chromatogr., hibition by uridine triphosphate. J. Biochem. (Tokyo), 72: 549-560, 1972. 151:71-14, 1978. 35. Ito, K., and Uchino, H. Control of pyrimidine biosynthesis in human 62. Strong, J. M., Kinney, Y. E., Branfman, A. R., and Cysyk, R. L. Determi lymphocytes. J. Biol. Chem., 248: 389-392, 1973. nation of A/-(phosphonacetyl)-L-aspartic acid (PALA) in dog serum and 36. Kempe, T. D., Swyryd, E. A., Bruist, M., and Stark, G. R. Stable mutants urine by gas chromatography and selected ion monitoring. Cancer Treat. of mammalian cells that overproduce the first three enzymes of pyrimidine Rep., 63: 775-780, 1979. nucleotide biosynthesis. Cell, 9 (Part 1): 541-550, 1976. 63. Roboz, J., Suzuki, R., and Rose, E. Mass spectrometric technique for the 37. Padgett, R. A., Wahl, G. M., Coleman, P. F., and Stark, G. R. /V-(Phos- determination of A"-phosphonacetyl-L-aspartic acid in serum. J. Chroma phonacetyl)-L-aspartate-resistant hamster cells overaccumulate a single togr., 181: 195-205, 1980. mRNA coding for the multifunctional protein that catalyzes the first steps 64. Branfman, A. R., Bruni, R. J., Merrill, Y. N., and Chadwick, M. Quanti of UMP synthesis. J. Biol. Chem., 254:974-980, 1979. talion of the antitumor agent N-(phosphonacetyl)-L-aspartic acid in human 38. Wahl, G. M., Padgett, R. A., and Stark, G. R. Gene amplification causes plasma and urine by gas chromatography-mass spectrometry-selected ion overproduction of the first three enzymes of UMP synthesis in jV-(phos- monitoring. J. Chromatogr., 182: 163-169, 1980. phonacetyl)-L-aspartate-resistant hamster cells. J. Biol. Chem., 254: 8679- 65. Chadwick, M., Silveira, D. M., MacGregory, J. A., Branfman, A. R., Liss, 8689. 1979. R. H., and Yesair, D. W. Comparative physiological disposition of N- 39. Jayaram, H. N., Cooney, D. A., Vistica, D. T., Kariya, S., and Johnson, R. (phosphonacetyl)-L-aspartate in several animal species after intravenous and K. Mechanisms of sensitivity or resistance of murine tumors to A'-fphos- oral administration. Cancer Res., 42:627-632, 1982. phonacetyl)-L-aspartate (PALA). Cancer Treat. Rep., 63:1291-1302,1979. 66. Chadwick, M., Silveira, D. M., McGarrigle, P. L., Liss, R. H., and Schepis, 40. Leyva, A., Appel, H., Smith, P., Lankelma, J., and Pinedo, H. M. Inhibition P. J. Distribution of PALA in tissues and tumors of mice after intravenous of cell growth by /V-(phosphonacetyl)-L-aspartate in human and murine cells administration. Proc. Am. Assoc. Cancer Res., 20: 241, 1979. in vitro. Cancer Lett., 12: 169-173, 1981. 67. Ardalan, B., Kensler, T. W., Jayaram, H. N., Morrison, W., Choie, D. D., 41. Karle, J. M., Cowan, K. H., Chisena, C. A., and Cysyk, R. L. Uracil Chadwick, M., Liss, R., and Cooney, D. A. Long-term association of N- nucleotide synthesis in a human breast cancer cell line (MCF-7) and in two (phosphonacetyl)-L-aspartate with bone. Cancer Res., 41: 150-156, 1981. drug-resistant sublines that contain increased levels of enzymes of the de 68. Loo, T. L., Friedman, J., Moore, E. C., Valdivieso, M., Marti, J. R., and novo pyrimidine pathway. Mol. Pharmacol., 30: 136-141, 1986. Stewart, D. Pharmacological disposition of A'-iphosphonacetylJ-L-aspartate 42. Christopherson, R. I., and Jones, M. E. The overall synthesis of L-5,6- in humans. Cancer Res., 40: 86-96, 1980. dihydroorotate by multienzymatic protein pyrl-3 from hamster cells. J. 69. Ohnuma, T., Hart, R., Roboz, J., and Andrejczuk, A. Clinical and phar Biol. Chem., 255: 11381-11395, 1980. macologie studies with phosphonacetyl L-aspartate (PALA). Proc. Am. 43. Monks, A., Anderson, L. W., Strong, J., and Cysyk, R. L. Flux through the Assoc. Cancer Res./Am. Soc. Clin. Oncol., 20: 344, 1979. de novo pyrimidine pathway in vivo. Effect of A'-(phosphonacetyl)-L-aspar- 70. Lankelma, J., Penders, P. G. M., Leyva, A., Kleeberg, U. R., Kenny, J. B., tate, a potent inhibitor of aspartate transcarbamylase. J. Biol. Chem., 258: Bramwell, V., McVie, G., and Pinedo, H. M. Concentrations of/V-(phos- 13564-13569,1983. phonacetyl)-L-aspartate (PALA) in plasma and tears in man. Eur. J. Cancer 44. Anderson, E. P., and Brockman, R. W. Feedback inhibition of uridine Clin. Oncol., 17: 1199-1204, 1981. kinase by cytidine triphosphate and uridine triphosphate. Biochim. Biophys. 71. Stewart, D. J., Leavens, M., Friedman, J., Benjamin, R. S., Moore, E. C., Acta, 91: 380-386, 1964. Bodey, G. P., Valdivieso, M., Burgess, M. A., Wiseman, C., and Loo, T. L. 45. Johnson, R. K. Reversal of toxicity and antitumor activity of /V-(phosphon- Penetration of jV-(phosphonacetyl)-L-aspartate into human central nervous acetyl)-L-aspartate by uridine or carbamyl-DL-aspartate in vivo. Biochem. system and intracerebral tumor. Cancer Res., 40: 3163-3166, 1980. Pharmacol., 26: 81-84, 1977. 72. Karle, J. M., Anderson, L. W., Dietrick, D. D., and Cysyk, R. L. Effect of 46. Chan, T. C. K., Young, B., King, M. E., Taetle, R., and Howell, S. B. inhibitors of the de novo pyrimidine biosynthetic pathway on serum uridine Modulation of the activity of PALA by dipyridamole. Cancer Treat. Rep., levels in mice. Cancer Res., 41:4952-4955, 1981. 69:425-430, 1985. 73. Karle, J. M., Anderson, L. W., Erlichman, C., and Cysyk, R. L. Serum 47. Mclvor, R. S., Wohlhueter, R. M., and Plagemann, P. P. G. Uridine uridine levels in patients receiving JV-(phosphonacetyl)-L-aspartate. Cancer phosphorylase from Novikoff rat hepatoma cells: purification, kinetic prop Res., 40: 2938-2940, 1980. erties, and its role in uracil anabolism. J. Cell. Physio!., 122:397-404,1985. 74. Kensler, T. W., Erlichman, C., Jayaram, H. N., Tyagi, A. K., Ardalan, B., 48. Karle, J. M., Anderson, L. W., and Cysyk, R. L. Effect of plasma concen and Cooney, D. A. Peripheral leukocytes as indicators of the enzymatic trations of uridine on pyrimidine biosynthesis in cultured 1.1210 cells. J. effects of iV-(phosphonacetyl)-L-aspartic acid (PALA) on human L-aspartate Biol. Chem., 259:67-72, 1984. transcarbamoylase (ATCase) activity. Cancer Treat. Rep., 64: 967-973, 49. Weber, G. Biochemical strategy of cancer cells and the design of chemo 1980. therapy. Cancer Res., 43: 3466-3492, 1983. 75. Casper, E. S., Vale, K., Williams, L. J., Martin, D. S., and Young, C. W. 50. Weber, G., Lui, M. S., Natsumeda, Y., and Faderan, M. A. Salvage capacity Phase I and clinical pharmacological evaluation of biochemical modulation of hepatoma 3924A and action of dipyridamole. Adv. Enzyme Regul.. 21: of 5-fluorouracil with A'-(phosphonacetyl)-L-aspartic acid. Cancer Res., 43: 53-69, 1983. 2324-2329, 1983. 51. Natsumeda, Y., Prajda, N., Donohue, J. P., Glover, J. L., and Weber, G. 76. Chen, J-J., and Jones, M. E. Effect of 6-azauridine on de novo pyrimidine 4452 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1988 American Association for Cancer Research. BIOCHEMISTRY AND CLINICAL ACTIVITY OF PALA

biosynthesis in cultured Ehrlich ascites cells. J. Biol. Chem., 254: 4908- psoriasis to A'-(phosphonacetyl)-L-aspartate. Lancet, /: 1257-1258, 1981. 4914, 1979. 104. Wiley, R. G., Gralla, R. J., Casper, E. S., and Kemeny, N. Neurotoxicity of 77. Gralla, R. J., Casper, E. S., Natale, R. B., Yagoda. A., and Young, C. W. the pyrimidine synthesis inhibitor *V-(phosphonacetyl)-L-aspartate. Ann. Phase I trial of PALA. Cancer Treat. Rep.. 64: 1301-1305, 1980. Neurol., 12: 175-183, 1982. 78. Valdivieso, M., Moore, E. C, Burgess, A. M., Marti, J. R., Russ, J., 105. Chabner, B. A. Pyrimidine antagonists. In: B. A. Chabner (Ã©d.).Pharma Plunkett, W., et al. Phase I clinical study of vV-fphosphonacetyO-L-aspartate cologie Basis of Cancer Treatment, pp. 183-212. Philadelphia: W. B. acid (PALA). Cancer Treat. Rep., 64: 285-292, 1980. SaundersCo., 1981. 79. Kovach, J. S., Schutt, A. J., Moertel, C. G., and O'Connell, M. J. Phase I 106. Heidelberger, C., Danenberg, P. V., and Moran, R. G. Fluorinated pyrimi- study of A'-(phosphonacetyl)-L-aspartic acid (PALA). Cancer Treat. Rep., dines and their nucleosides. In: A. Meister (ed.). Advances in Enzymology 63: 1909-1912, 1979. and Related Areas in Molecular Biology, pp. 57-119. New York: John 80. Hart, R. D.. Ohnuma, T., and Holland, J. F. Initial clinical study with N- Wiley and Sons. Inc., 1983. (phosphonacetyl)-L-aspartic acid (PALA) in patients with advanced cancer. 107. Ardalan, B., and Glazer, R. An update on the biochemistry of 5-fluorouracil. Cancer Treat. Rep., 64: 617-624, 1980. Cancer Treat. Rev., 8: 157-167, 1981. 81. Ervin, T. J., Blum. R. H., Meshad, M. W., Kufe, D. W., Johnson, R. K., 108. Major, P. P., Egan, E., Herrich, D., and Kufe, D. W. 5-Fluorouracil and Canellos, G. P. Phase I trial of A'-(phosphonacetyl)-L-aspartic acid incorporation in DNA of human breast carcinoma cells. Cancer Res., 42: (PALA). Cancer Treat. Rep., 64: 1067-1071, 1980. 3005-3009, 1982. 82. Erlichman, C., Strong, J. M., Wiernik, P. H., McAvoy, L. M., Cohen, M. 109. Sawyer, R. C., Stolfi, R. L., Martin, D. S., and Spiegelman, S. Incorporation H., Levine, A. S., Hubbard, S. M., and Chabner, B. A. Phase I trial of N- of 5-fluorouracil into murine bone marrow DNA in vivo. Cancer Res., 44: (phosphonacetyl)-L-aspartate. Cancer Res., 39: 3992-3995, 1979. 1847-1851, 1984. 83. Natale, R. B., Yagoda, A., Kelsen, D. P., Gralla, R. J., and Watson, R. C. 110. Schuetz. J. D., Collins, J. M., Wallace, H. J., and Disasio, R. B. Alteration Phase II trial of PALA in hypernephroma and urinary bladder cancer. of the secondary structure of newly synthesized DNA from murine bone Cancer Treat. Rep., 66: 2091-2092, 1982. marrow cells by 5-fluorouracil. Cancer Res., 46: 119-123, 1986. 84. Taylor. S. G., Davis, T. E., Falkson. G., and Keller. A. M. PALA in 111. Ardalan, B., Glazer, R. I., Kensler, T. W., Jayaram, H. N., Pham, T. V., advanced breast cancer. A phase II pilot study by the ECOG. Am. J. Clin. MacDonald, J. S., and Cooney, D. A. Synergistic effect of 5-fluorouracil Oncol., 5:627-629, 1982. and iV-(phosphonacetyl)-L-aspartate on cell growth and ribonucleic acid 85. Paridaens, R., Mouridsen, H. T., Palshof, T., Cocconi, G., Van Oosterom, synthesis in human mammary carcinoma. Biochem. Pharmacol., 30: 2045- A., Rotmensz, N., Sylvester, R., Heuson, J. C., and Rozencweig, M. Ad 2049, 1981. vanced breast cancer: a phase II trial of the EORTC Breast Cancer Coop- 112. Peters, G. J., Laurensse, E.. Lankelma, J., Leyva, A., and Pinedo, H. M. erative Group. Eur. J. Cancer Clin. Oncol., 18:67-71, 1982. Separation of several 5-fluorouracil metabolites in various melanoma cell 86. Muss, H. B., Bundy, B., DiSaia, P. J., Stehman, F. B., and Beecham, J. lines. Evidence for the synthesis of 5-fluorouracil-nucleotide sugars. Eur. J. PALA(NSC-224131)in advanced carcinoma of the cervix. A phase II study Cancer Clin. Oncol., 20: 1425-1431, 1984. of the Gynecologic Oncology Group. Am. J. Clin. Oncol., 7:741-744,1984. 113. Major, P. P., Egan, E. M., Sargent, L., and Kufe, D. W. Modulation of 5- 87. Rubin, J., Purvis, J., Britell, J. C., Hahn, R. G., Moertel, C. G., and Schutt, FU metabolism in human MCF-7 breast carcinoma cells. Cancer Chemo- A. J. Phase II study of PALA in advanced large bowel carcinoma. Cancer ther. Pharmacol., Â«:87-91, 1982. Treat. Rep.. 65: 335-336, 1981. 114. Holstege, A.. Herrmann, B., and Keppler. D. O. R. Increased formation of 88. Van Echo, D. A., Diggs. C. H., Scoltock, M., and Wiernik, P. H. Phase II nucleotide derivatives of 5-fluorouridine in hepatoma cells treated with evaluation of A'-(phosphonacetyl)-L-aspartic acid (PALA) in metastatic ad- inhibitors of pyrimidine synthesis and D-galactosamine. FEBS Lett., 95: enocarcinoma of the colon or rectum. Cancer Treat. Rep., 64: 339-342, 361-365, 1978. 1980. 115. Liang, C-M., Donehower, R. C., and Chabner, B. A. Biochemical interac 89. Carroll, D. S., Gralla. R. J., and Kemeny, N. E. Phase II evaluation of N- tions between yV-(phosphonacetyl)-L-aspartate and 5-fluorouracil. Mol. (phosphonacetyl)-L-aspartic acid (PALA) in patients with advanced colorec- Pharmacol., 21: 224-230, 1982. tal carcinoma. Cancer Treat. Rep., 64: 349-351, 1980. 116. Anukarahanonta, T., Holstege, A., and Keppler, D. O. R. Selective enhance 90. Kaplan, B. H., Vogl, S. E., Amato, D., Earhart, R., and Leerner, H. Single ment of 5-fluorouridine uptake and action in rat hepatomas in vivo following agent chemotherapy of advanced head and neck cancer: methylglyoxal pretreatment with D-galactosamine and 6-azauridine or A'-(phosphonacetyl)- bisguanylhydrazone (MGBG) and /V-(phosphonacetyl)-L-aspartate (PALA). L-aspartate. Eur. J. Cancer, 16: 1171-1180, 1980. Proc. Am. Soc. Clin. Oncol., 2: 165, 1983. 117. Miller, A. A., Moore, E. C., Hurlbert, R. B., Benvenuto, J. A., and Loo, T. 91. Creagan, E. T., Nichols, W. C.. and O'Fallon, J. R. Phase II evaluation of L. Pharmacological and biochemical interactions of yV-(phosphonacetyl)-L- PALA in patients with advanced head and neck cancer. Cancer Treat. Rep-, aspartate and 5-fluorouracil in beagles. Cancer Res., 43: 2565-2570, 1983. 05:827-829,1981. 118. Ardalan, B., Jasmin, D., Jayaram, H. N., and Presant, C. A. Phase I study 92. Ettinger, D. S., Tritchler, D., Earhart, R., and Creech. R. H. Phase II study of continuous-infusion PALA and 5-FU. Cancer Treat. Rep., 68: 531-534, of PALA and PCNU in the treatment of non-small cell lung cancer (EST 1984. 2580): an Eastern Cooperative Oncology Group study. Cancer Treat. Rep., 119. Erlichman, C., Donehower, R. C., Speyer, J. L., Klecker, R., and Chabner, 68:1297-1298, 1984. B. A. Phase I-Phase II trial of /V-phosphonacetyl-L-aspartic acid given by 93. Casper, E. S., Gralla, R. J., Kelsen, D. P., Houghton, A., Golbey, R. B., intravenous infusion and 5-fluorouracil given by bolus injection. J. Nati. and Young, C. W. Phase II evaluation of A'-(phosphonacetyl)-L-aspartic Cancer Inst., 68: 227-231, 1982. acid (PALA) in patients with non-small cell carcinoma of the lung. Cancer 120. Meshad, M. W., Ervin, T. J., Kufe, D., Johnson, R. K., Blum, R. H., and Treat. Rep., 64: 705-707, 1980. Frei, E., III. Phase I trial of combination therapy with PALA and 5-FU. 94. Creagan, E. T., Eagan, R. T., Fleming, T. R., Frytak, S., Nichols, W. C., Cancer Treat. Rep., 65: 331-334, 1981. Ingle, J. N., and Kvols, L. K. Phase II evaluation of PALA in patients with 121. Weiss, G. R., Ervin, T. J., Meshad, M. W., Schade, D., Branfman, A. R., metastatic lung cancer. Cancer Treat. Rep., 65: 356-357, 1981. Bruni, R. J., Chadwick, M., and Kufe, D. W. A Phase I trial of combination 95. Creech, R. H., Tritchler. D., Ettinger, D. S., Ferrare, J. A.. Ruckdeschel, J. therapy with continuous-infusion PALA and continuous-infusion 5-FU. C., Vogl, S. E., and Woll, J. Phase II study of PALA, amsacrine, teniposide, Cancer Chemother. Pharmacol., 8: 301-304, 1982. and zinostatin in small cell lung carcinoma (EST 2579). Cancer Treat. Rep., 122. O'Connell, M. J., Powis, G., Rubin, J., and Moertel, C. G. Pilot study of 68: 1183-1184, 1984. PALA and 5-FU in patients with advanced cancer. Cancer Treat. Rep., 66: 96. Muggia. F. M., Tsiatis, A. A., O'Connell, M. J., Click, J. H.. Opfell, R. 77-80, 1982. W., and Coren, A. Phase II trial of PALA in lymphoma: an Eastern 123. Bedikian, A. Y., Stroehlein, J. R., Karlin. D. A., Bennetts, R. W., Bodey, Cooperative Oncology Group study. Cancer Treat. Rep., 68:551-553,1984. G. P., and Valdivieso, M. Chemotherapy for colorectal cancer with a 97. Creagan, E. T., Ahmann, D. L., Ingle, J. N., Purvis, J. D., and Green, S. J. combination of PALA and 5-FU. Cancer Treat. Rep., 65: 747-753, 1981. Phase II evaluation of PALA and AMSA for patients with disseminated 124. Bedikian, A. Y., Valdivieso, M., Stroehlein, J. R., Karlin, D. A., Bennet, R. malignant melanoma. Cancer Treat. Rep., 65:169, 1981. W., and Bodey, G. P. Phase I-II evaluation of PALA-5-fluorouracil com 98. Kleeberg, U. R., Mulder, J. H., Rumke, P., Thomas, D., and Rozencweig, bination in patients with colorectal carcinoma. Proc. Am. Assoc. Cancer M. AL(Phosphonacetyl)-L-aspartate (PALA) in advanced malignant mela Res./Am. Soc. Clin. Oncol., 21:415, 1980. noma: a phase H trial of the EORTC Malignant Melanoma Cooperative 125. Ardalan, B.. and Singh, G. A randomized Phase I-II study of short-term Group. Eur. J. Cancer Clin. Oncol., /*: 723-726, 1982. infusion of high-dose 5-fluorouracil with or without A'-(phosphonacetyl)-L- 99. Muss, H. B., Slavik, M., Bundy. B., Stehman, F. B., and Creasman, W. T. aspartic acid in patients with advanced pancreatic and colorectal cancers. A phase II study of PALA (NSC 224131) in patients with advanced ovarian Proc. Am. Soc. Clin. Oncol., 6: 81, 1987. carcinoma. A Gynecologic Oncology Group study. Am. J. Clin. Oncol., 7: 126. Weiss, G. R., Ervin, T. J., Meshad, M. W., and Kufe, D. W. Phase II trial 257-260, 1984. of combination therapy with continuous-infusion PALA and bolus-injection 100. Earhart, R. H., Elson, P. J., Rosenthal, S. N., Hahn, R. G., and Slayton, 5-FU. Cancer Treat. Rep., 66: 299-303, 1982. R. E. Phase II study of PALA and AMSA in advanced renal cell carcinoma. 127. Presant, C. A., Ardalan, B., Multhauf, P., Chan, C., Staples, R., Green, L., Am. J. Clin. Oncol., 6: 555-560, 1983. Browning, S., Carr, B. I., Chang, F-F., and Thayer, W. Continuous five-day 101. Kurzrock, R., Yap, B-S., Plager, C., Papdopoulos, N., Benjamin, R. S., infusion of PALA and 5FU: a pilot phase II trial. Med. Pediatr. Oncol., / / Valdivieso, M., and Bodey, G. P. Phase H evaluation of PALA in patients 162-163, 1983. with refractory metastatic sarcomas. Am. J. Clin. Oncol., 7:305-307,1984. 128. Muggia, F. M., Camacho, F. J., Kaplan, B. H., Green, M. D., Greenwald, 102. Bramwell, V., Van Oosterom, A., Mouridsen, H. T., Cheix, F., Somers, R., E. S., Wernz, J. C., and Engstrom, P. F. Weekly 5-fluorouracil combined Thomas, D., and Rozencweig, M. A'-(Phosphonacetyl)-L-aspartate (PALA) with PALA: toxic and therapeutic effects in colorectal cancer. Cancer Treat. in advanced soft tissue sarcoma: a phase II trial of the EORTC Soft Tissue Rep., 71: 253-256, 1987. Sarcoma Group. Eur. J. Cancer Clin. Oncol., 18: 81-84, 1982. 129. Mann, G , Hortobagyi, G., Yap, H., Buzdar, A., Schell, F., Valdivieso, M., 103. Earhart. R. H., DeConti, R. C., Rubin, J., and Ohnuma, T. Response of and Maddox, M. A comparative study of PALA, PALA/5-FU, and 5-FU 4453 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1988 American Association for Cancer Research. BIOCHEMISTRY AND CLINICAL ACTIVITY OF PALA

in advanced breast carcinoma (BC). Proc. Am. Soc. Clin. Oncol.. /: 76. 148. Plunkett. W., Adams. T., and Keating, M. Modulation of Ara-CTP metab 1982. olism in leukemia cells during high-dose ARA-C (HD ARA-C) therapy by 130. Buroker. T. R., Moertel, C. G., Fleming, T. R., Everson, L. K., Cullinan, thymidine and PALA. Proc. Am. Soc. Clin. Oncol., 6: 30, 1987. S. A. ft al. A controlled evaluation of recent approaches to biochemical 149. Grant, S., Rauscher, F., Ill, Jakubowski, A., and Cadman, E. Effect of jV- modulation or enhancement of 5-fluorouracil therapy in colorectal carci (phosphonacetyl)-u-aspartate on 5-azacytidine metabolism in P388 and noma. J. Clin. Oncol., 3: 1624-1631, 1985. L12IO cells. Cancer Res., 41: 410-418, 1981. 131. Martin. D. S.. Stolfi, R. L., Sawyer, R. C., Spiegelman, S., and Young, C. 150. Erlichman, C., and Vidgen, D. Antitumor activity of jV-(phosphonacetyl)- W. Improved therapeutic index with sequential A"-(phosphonacetyl)-L-as- L-aspartic acid in combination with nitrobenzylthioinosine. Biochem. Phar- partate plus high-dose methotrexate plus high-dose 5-fluorouracil and ap macol., .?.?.â€¢3177-3181.1984. propriate rescue. Cancer Res., 43:4653-4661, 1983. 151. Markman, M., Chan, T. C. K., Cleary, S., and Howell, S. B. Phase I trial 132. Reichman, B., Kemeny, N., Gordon, C., Ellison, S., Deonarine, S., Parenti, of combination therapy of cancer with /V-phosphonacetyl-L-aspartic acid R., Martin, D., and Young, C. Phase I trial of A'-phosphonacetyl-L-aspartate and dipyridamole. Cancer Chemother. Pharmacol., 19: 80-83, 1987. (PALA), methotrexate (MTX), 5-fluorouracil (5FU), and leucovorin (LV) 152. Chan, T. C. K., Markman, M., Cleary, S., and Howeli, S. B. Plasma uridine in metastatic colorectal cancer. Proc. Am. Soc. Clin. Oncol., 5:47, 1986. changes in cancer patients treated with the combination of dipyridamole 133. Kemeny, N., Reichman, B.. Gordon, C., Ellison, S., Deonarine, S., Slavik, and A'-phosphonacetyl-L-aspartate. Cancer Res., 46: 3168-3172, 1986. B., Martin. D., and Young. C. Phase I trial of A'-phosphonacetyl-L-aspartate 153. Levy, E. J., Gasser, T., Dreyer, R. N., and Handschumacher, R. E. Control (PALA), methotrexate (MTX), 5-fluorouracil (FU), and leucovorin (LV) In of the turnover and utilization of circulating uridine by liver bypass, nitro advanced colorectal cancer (CRC). Proc. Am. Assoc. Cancer Res., 28: 187, benzylthioinosine-PO4 or benzyl acyclouridine. Proc. Am. Assoc. Cancer 1987. Res., 23: 836, 1982. 134. Martin, D. S., Nayak, R., Sawyer, R. C, Stolfi, R. L., Young, C. W., 154. Jayaram, H. N., and Cooney, D. A. Analogs of L-aspartic acid in chemo Woodcock, T., and Spiegelman, S. Enhancement of 5-fluorouracil chemo therapy for cancer. Cancer Treat. Rep., 63: 1095-1108, 1979. therapy with emphasis on the use of excess thymidine. Cancer Bull., 30: 155. Creagan, E. T., O'Connell, M. J., and Kovach, J. S. A phase I study of the 219-224, 1978. combination A'-(phosphonacetyl)-L-aspartate (PALA, NSC-224131) and L- 135. Martin, D. S., Stolfi, R. L., Sawyer, R. C., Nayak, R.. Spiegelman, S., alanosine (NSC-153353) in patients with advanced cancer. Cancer (Phila.), Schmid. F., Heimer, R., and Cadman, E. Biochemical modulation of 5- 52:615-618, 1983. fluorouracil and cytosine arabinoside with emphasis on thymidine, Pala, 156. Trader, M. W., Schabel, F. M., Jr., Laster, W. R., Jr., Brockman, R. W., and 6-methylmercaptopurine riboside. In: M. H. N. Tattersall and R. M. Kovach, J. S., and Lieber, M. M. Therapeutic synergism (TS) with L- Fox (eds.), Nucleosides and Cancer Treatment, pp. 339-382. New York: alanosine (A) plus A'-phosphonacetyl-L-aspartate (PALA) against ara-C Academic Press, 1981. resistant leukemia P388 (P388/Ara-C). Proc. Am. Assoc. Cancer Res., 23: 136. Woodcock, T. M., Martin, D. S., Damin, L. A. M., Kemeny, N. E., and 187, 1982. Young, C. W. Combination clinical trials with thymidine and fluorouracil: 157. O'Connell, M. J., Rubin, J., Schutt, A. J., Moertel, C. G., and Kvols, L. K. a Phase I and clinical pharmacologie evaluation. Cancer. 45: 1135-1143, Clinical trial of PALA and L-alanosine in advanced colorectal carcinoma. 1980. Cancer Treat. Rep., 67: 1141-1142, 1983. 137. Au, J.L.-S., Rustum, Y. M., Ledesma, E. S., Mittelman, A., and Creaven, 158. Morton, R. F., Creagan, E. T., Cullinan, S. A., Mailliard, J. A., Ebbert, L., P. J. Clinical pharmacological studies of concurrent infusion of 5-fluorour Veeder, M. H., and Chang, M. Phase II studies of single-agent cimetidine acil and thymidine in the treatment of colorectal carcinomas. Cancer Res., and the combination A'-phosphonacetyl-L-aspartate (NSC-224131) plus L- Â«.â€¢2930-2937,1982. alanosine (NSC-153353) in advanced malignant melanoma. J. Clin. Oncol., 138. Kirkwood. J. M., Ensminger, W., Rosowsky. A., Papathanasopoulos, N., 5: 1078-1082, 1987. and Frei, E.. III. Comparison of pharmacokinetics of 5-fluorouracil and 5- 159. Trader, M. W.. Schabel, F. M., Jr., Laster, W. R., Jr., and Brockman, R. fluorouracil with concurrent thymidine infusions in a phase I trial. Cancer W. Combination chemotherapy of Ara-C-resistant leukemia P388 (P388/ Res., 40: 107-113, 1980. Ara-C) with inhibitors of purine and pyrimidine biosynthesis. Proc. Am. 139. O'Dwyer, P. J., King, S. A., Hoth, D. F., and Leyland-Jones. B. Role of Assoc. Cancer Res., 24: 265, 1983. thymidine in biochemical modulation: a review. Cancer Res., 47: 3911- 160. O'Dwyer, P. J., Alonso, M. T., and Leyland-Jones, B. Acivicin: a new 3919, 1987. glutamine antagonist in clinical trials. J. Clin. Oncol., 2: 1064-1071, 1984. 140. Schornagel.J. H., Leyva, A., and Pinedo, H. M. Is there a role for thymidine 161. Loh, E., and Kufe, D. W. Synergistic effects with inhibitors of de novo in cancer chemotherapy? Cancer Treat. Rev., 9: 331-352, 1982. pyrimidine synthesis, acivicin, and A'-(phosphonacetyl)-L-aspartic acid. Can 141. Casper, E. S., Michaelson. R. A., Kemeny, N., Martin, D. S., and Young, cer Res., 41: 3419-3423, 1981. C. W. Phase I evaluation of a biochemically designed combination: PALA, 162. Kensler, T. W., Reck, L. J., and Cooney, D. A. Therapeutic effects of thymidine, and 5-FU. Cancer Treat. Rep., 68: 539-541, 1984. acivicin and A'-(phosphonacetyl)-L-aspartic acid in a biochemically designed 142. Chiuten, D. F., Valdivieso, M., Bedikian, A., Benvenuto, J. A., Miller, A., trial against a A'-(phosphonacetyl)-L-aspartic acid-resistant variant of the Loo, T. L., Bodey. G. P., and Freireich, E. J Sequential administration Lewis lung carcinoma. Cancer Res., 41: 905-909, 1981. of thymidine, 5-fluorouracil. and PALA. A Phase I-II study. Am. J. Clin. 163. Stolfi, R. L., Martin, D. S., and Sawyer, R. C. Combination chemotherapy Oncol.. 8: 332-335, 1985. with acivicin, A'-(phosphonacetyl)-L-aspartic acid (PALA) and Ara-C against 143. Chiuten, D. E.. Valdivieso, M., Bedikian, A., Bodey, G. P., and Freireich, CD8F, breast tumors. Proc. Am. Assoc. Cancer Res., 26: 241, 1985. E. J Phase I-II clinical trial of thymidine, 5-FU, and PALA given in 164. Sawyer, R. C., Stolfi, R. L., and Martin, D. S. In vivobiochemical evaluation combination. Cancer Treat. Rep.. 69:611-613, 1985. of acivicin (ACIV) in combination with PALA plus 5-fluorouracil (FUra) 144. O'Connell. M. J., Moertel, C. G., Rubin, J., Hahn, R. G., Kvols, L. K., and or with 6-thioguanine (6-TG). Proc. Am. Assoc. Cancer Res., 27: 19, 1986. Schutt. A. J. Clinical trial of sequential A'-phosphonacetyl-L-aspartate. 165. Riva, C. M., Marque!, J., Suberville, A. M., Ziltoun, J., and Zittoun, R. thymidine. and 5-fluorouracil in advanced colorectal carcinoma. J. Clin. Modulation of I-/3-D-arabinofuranosyl (ara-C) by A'-fphosphonacetylJ-L- Oncol., 2: 1133-1138, 1984. aspartic acid (PALA) and acivicin (AC) in L1210 cells. Proc. Am. Assoc. 145. Benvenuto, J. A., Chiuten, D. F., Miller, A. A., Moore, E. C., and Loo, T. Cancer Res., 27:301, 1986. L. Clinical and biochemical pharmacologie study of the combination of 166. Taylor, S., and Eyre, H. J. Randomized phase II trials of acivicin (AT-125, PALA, 5-fluorouracil. and thymidine. Proc. Am. Assoc. Cancer Res., 24: NSC I6350I) and fludarabine (2-fluoro-ara-AMP, NSC 312887) 130. 1983. (2FLAMP) in recurrent malignant gliomas: a SWOG study. Proc. Am. Soc. 146. Basseches. P.. Appel, P. L., O'Connell, M. J., Moertel, C. G., and Powis, Clin. Oncol., 6:71, 1987. G. Increased toxicity of thymidine, 5-fluorouracil combinations in patients 167. Rubin, J., Schutt, A. J., O'Connell, M. J., Gertz, M. A., and Moertel, C. with impaired renal functionâ€”a pharmacokinetic study. Proc. Am. Assoc. G. A phase II study of the combination l,3-bis(2-chloroethyl)-l-nitrosourea Cancer Res., 23: 130, 1982. (BCNU) and A'-(phosphonacetyl)-L-aspartate (PALA), in patients with ad 147. Blumenreich, M., Andreeff, M., Chou, T.-C., Wittes, R., Clarkson, B., and vanced large bowel cancer. Am. J. Clin. Oncol., 7: 523-525, 1984. Young. C. Kinetic and biochemical modulation of pyrimidine antimetabo- 168. Voigt, H., and Kleeberg, U. R. PALA, vindesine, and cisplatin combination lites in therapy of acute leukemia. Proc. Am. Assoc. Cancer Res., 22: 193, chemotherapy in advanced malignant melanoma. A pilot study. Cancer 1981. (Phila.), S3: 2058-2062. 1984.

Jean L. Grem, Susan A. King, Peter J. O'Dwyer, et al.

Cancer Res 1988;48:4441-4454.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/48/16/4441.citation

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/48/16/4441.citation. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.