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Biomedical subjects

J L Grem

Publications and source records attributed to J L Grem.

At least 55 records · Page 3Linked to original sources
PubMed1996

Severe neurotoxicity following 5-fluorouracil-based chemotherapy in a patient with dihydropyrimidine dehydrogenase deficiency.

Patients with decreased dihydropyrimidine dehydrogenase (DPD) activity are at increased risk for experiencing serious adverse reactions following 5-fluorouracil (5-FU)-based chemotherapy. Symptoms include severe and potentially life-threatening gastrointestinal toxicity, myelosuppression, and neurological toxicity. In the present study, we describe a 50-year-old Caucasian man who developed severe encephalopathy during his second cycle of 5-FU chemotherapy. The patient remained in a comatose state for 4 days but then showed dramatic improvement in his neurological status following continuous i.v. infusion of thymidine at 8 g/m2/day. Laboratory studies revealed the patient to be severely DPD deficient, as demonstrated by DPD enzyme activity from peripheral blood mononuclear cells being below the lower limit of the 95th percentile of a control population and by Western immunoblot analysis showing undetectable levels of DPD protein. Additional studies revealed a significant defect in pyrimidine catabolism with a 3.3- and 365-fold increase in the levels of uracil in plasma and urine, respectively, compared to normal subjects. Family studies suggest that the inheritance pattern of this syndrome is complex and most consistent with an autosomal recessive trait. This study demonstrates that cancer patients with DPD deficiency are at increased risk for developing severe neurological toxicity secondary to 5-FU chemotherapy, and that infusional thymidine should be considered as a potential rescue agent against this particular host toxicity.

Brain Diseases↗
PubMed1995

Determinants of sensitivity to 1-beta-D-arabinofuranosylcytosine in HCT 116 and NCI-H630 human colon carcinoma cells.

The cytotoxicity and metabolism of 1-beta-D-arabinofuranosylcytosine (AraC) and its effects on DNA synthesis and integrity were studied in HCT 116 and NCI-H630 human colon cancer cells. In 116 cells, 0.1 microM AraC decreased colony formation by approximately 50%, whereas 1 microM was required in H630 cells. AraCTP levels after a 24-hr AraC exposure were 2.3- to 3.5-fold lower in H630 cells due to increased ability to deaminate AraCMP. AraC DNA levels increased in proportion to AraCTP pools (r = 0.99) and were 2-fold higher in 116 cells after a 24-hr exposure to 0.1 and 1 microM AraC. Although the half-life of AraCTP was < 1 hr in both lines, > 80% of AraC DNA was retained at 24 hr after drug removal. Clonogenic capacity was inversely related to the extent of AraC DNA incorporation. Interference with nascent DNA chain elongation increased with increasing AraC concentration x time. A 24-hr AraC exposure produced a dramatic shift in the elution profile of nascent DNA during a 15-hr elution at pH 12.1; these effects were greater in 116 cells (DNA retained on filter [percentage of control]): 78%, 23%, and 9% with 0.1, 1, and 10 microM AraC versus 84%, 42%, and 18% in H630 cells, respectively. The extent of nascent DNA damage was proportional to AraC DNA content. Net DNA synthesis was potently inhibited during AraC exposure in both lines. H630 cells had partial recovery of DNA synthesis at 24 hr after drug removal, whereas persistent inhibition was noted in 116 cells. A slight excess of double-strand breaks in parental DNA was detected after a 24-hr exposure to 10 microM AraC in 116 cells. The extent of DNA fragmentation was more pronounced 16 hr after drug removal and was greater in 116 cells: 8.5%, 19%, and 21% with 0.1, 1, and 10 microM AraC DNA content, magnitude of nascent DNA damage, duration of DNA synthetic inhibition, and induction of double-stranded DNA fragmentation appeared to be the crucial determinants of lethality.

Cell Survival↗
PubMed1995

Effects of 9-aminocamptothecin on newly synthesized DNA in patient bone marrow samples.

9-Aminocamptothecin (9-AC) inhibited cell growth and DNA synthesis in HCT 116 human colon cancer cells in a concentration- and time-dependent manner. Interference with nascent DNA chain elongation was monitored using pH step alkaline elution. After a 3-day 9-AC exposure, 38% (10 nM) and 53% (50 nM) of the total [3H]DNA eluted with pH steps 11.3-11.7, compared to 9% in control cells. Effects on nascent DNA integrity were also evaluated by fixed elution with pH 12.1 buffer. After a 3-day exposure to 9-AC, 27% (10 nM) and 82.5% (50 nM) of the total [3H]DNA eluted relative to control. Paired bone marrow samples were then obtained in 10 patients before treatment and between 42 and 72 h of a continuous i. v. infusion of 9-AC (35-74 microgram/m2/h for 72 h). The mononuclear cells were incubated with [3H]dThd for 2 or 4 h, and then analyzed using either pH step or fixed pH alkaline elution, respectively. In seven patients receiving >/=47 microgram/m2/h 9-AC, 4% +/- 1.5% (mean +/- SE) of the total [3H]DNA eluted with pH steps </= 11.7 in the pretreatment samples compared to 13% +/- 3.6% during 9-AC (P = 0. 037). An altered fixed pH elution profile of nascent DNA was noted in two patients treated with 59 and 74 microgram/m2/h 9-AC compared to baseline. DNA synthesis was inhibited by 89% +/- 5% during infusion of >/=59 microgram/m2/h 9-AC (n = 7). Since hematological toxicity is dose limiting on this 9-AC schedule, these cellular pharmacodynamic studies provide evidence of a DNA-directed cytotoxic effect of 9-AC in a sensitive host target tissue.

Adult↗
PubMed1994

Cytotoxicity and metabolism of 4-methoxy-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine in HCT 116 colon cancer cells.

We examined the cytotoxicity, biochemical effects and metabolism of 4-methoxy-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (MRPP), a synthetic nucleoside inhibitor of phosphoribosylpyrophosphate synthetase, in HCT 116 human colorectal cancer cells. A 4-hr exposure to 1 and 10 microM MRPP inhibited cell growth over a 72-hr period by 76 and 89%, and inhibited clonogenic capacity by 36 and 65%, respectively. MRPP was avidly metabolized to the 5'-monophosphate derivative (MRPP-MP), and MRPP-MP formation increased with increasing MRPP exposure (microM.hr). MRPP-MP was stable, and the intracellular half-life was in excess of 48 hr. A 4-hr exposure to 10 microM MRPP resulted in significant decreases in ATP, UTP, GTP, CTP, dATP, dTTP, and PRPP pools. Near maximal ribonucleotide triphosphate depletion was achieved with > or = 24 microM.hr MRPP, and growth inhibition as a function of MRPP microM.hr closely reflected the biochemical effects. Ribonucleotide triphosphate pools remained depleted for up to 48 hr after drug removal, apparently as a consequence of the prolonged retention of MRPP-MP. MRPP (10 microM) inhibited the salvage of [3H]guanine, [3H]adenine and [3H]guanosine, and concurrent exposure to MRPP and either 100 microM adenine, hypoxanthine, or guanine did not reverse ATP or GTP depletion. Concurrent exposure to 10 microM MRPP and either 10 microM adenosine, uridine or thymidine was accompanied by repletion of ATP, UTP, and dTTP pools, respectively, but depletion of other nucleotide pools was not corrected. In contrast, 10 microM guanosine did not correct GTP depletion in the presence of MRPP. The combination of 10 microM each of thymidine, uridine, adenosine and guanosine during and following a 24-hr exposure to MRPP provided partial protection against 0.1 or 1 microM MRPP, but did not affect the cytotoxicity associated with 10 microM MRPP. MRPP is a novel antimetabolite that inhibits both de novo and salvage pathways for purine synthesis and de novo pyrimidine synthesis.

Adenosine Triphosphate↗
PubMed1994

Analysis of the active lactone form of 9-aminocamptothecin in plasma using solid-phase extraction and high-performance liquid chromatography.

We have developed a sensitive HPLC assay to quantitate the active lactone form of 9-aminocamptothecin (9AC) in human plasma over the concentration range 10-0.25 nM (0.091 ng/ml). Solid-phase extraction separated 9AC lactone from its less active metabolite, 9AC carboxylate, allowing samples to be stored for up to two months prior to reversed-phase HPLC analysis. An acidic (pH 2.55) isocratic HPLC mobile phase was used to enhance 9AC fluorescence resulting in an over 50-fold increase in assay sensitivity compared to previous methods. This assay was able to measure steady-state 9AC lactone concentrations even at the lowest dose level of 9AC used in our Phase I clinical trial.

Antineoplastic Agents↗
PubMed1994

Determinants of trimetrexate lethality in human colon cancer cells.

We examined the cytotoxicity and biochemical effects of the lipophilic antifol trimetrexate (TMQ) in two human colon carcinoma cell lines, SNU-C4 and NCI-H630, with different inherent sensitivity to TMQ. While a 24 h exposure to 0.1 microM TMQ inhibited cell growth by 50-60% in both cell lines, it did not reduce clonogenic survival. A 24 h exposure to 1 and 10 microM TMQ produced 42% and 50% lethality in C4 cells, but did not affect H630 cells. Dihydrofolate reductase (DHFR) and thymidylate synthase were quantitatively and qualitatively similar in both lines. During drug exposure, DHFR catalytic activity was inhibited by > or = 85% in both cell lines; in addition, the reduction in apparent free DHFR binding capacity (< or = 20% of control), depletion of dTTP, ATP and GTP pools and inhibition of [6-3H]deoxyuridine incorporation into DNA were similar in C4 and H630 cells. TMQ produced a more striking alteration of the pH step alkaline elution profile of newly synthesised DNA in C4 cells compared with 630 cells, however, indicating greater interference with DNA chain elongation or more extensive DNA damage. When TMQ was removed after a 24 h exposure to 0.1 microM, recovery of DHFR catalytic activity and apparent free DHFR binding sites was evident over the next 24-48 h in both cell lines. With 1 and 10 microM, however, persistent inhibition of DHFR was evident in C4 cells, whereas DHFR recovered in H630 cells. These data suggest that, although DHFR inhibition during TMQ exposure produced growth inhibition, DHFR catalytic activity 48 h after drug removal was a more accurate predictor of lethality in these two cell lines. Several factors appeared to influence the duration of DHFR inhibition after drug removal, including initial TMQ concentration, declining cytosolic TMQ levels after drug removal, the ability to acutely increase total DHFR content and the extent of TMQ-mediated DNA damage. The greater sensitivity of C4 cells to TMQ-associated lethality may be attributed to the greater extent of TMQ-mediated DNA damage and more prolonged duration of DHFR inhibition after drug exposure.

Biological Transport↗
PubMed1994

Chemotherapy of advanced gastrointestinal cancer.

Pancreatic, gastric, and colorectal carcinomas are diagnosed in 200,000 Americans each year. Therapeutic options for patients with advanced disease are limited; conventional chemotherapy is palliative and produces complete responses in only a few patients. Clinical research has focused on the evaluation of investigational new drugs, combination regimens, and biochemical modulation of fluorouracil. Unfortunately, the results of recent phase II studies of new agents have been disappointing. The exception is CPT-11, a topoisomerase I inhibitor, which showed promising activity in colorectal cancer (in including patients who had failed prior fluorouracil therapy). Modified regimens of fluorouracil and methotrexate with either doxorubicin alone or with epirubicin and cisplatin were associated with response rates approaching 50% in patients with gastric cancer, but appeared to be less toxic than previously published regimens. A randomized trial comparing fluororacil alone or with oral leucovorin allowed early dose escalation according to individual tolerance; the response rate to fluorouracil alone (23%) was higher than that reported in previous phase III trials, suggesting the importance of using adequate doses to produce toxicity in terms of clinical response. Hepatic arterial infusion of floxuridine was associated with a 39% response rate in colorectal cancer patients with disease confined to the liver for whom systemic fluorouracil therapy had failed, suggesting this approach is a reasonable therapeutic option in carefully selected patients.

Antineoplastic Agents↗
PubMed1994

Phase I and pharmacokinetic study of recombinant human granulocyte-macrophage colony-stimulating factor given in combination with fluorouracil plus calcium leucovorin in metastatic gastrointestinal adenocarcinoma.

PURPOSE: To determine the toxicities and potential for dose escalation of intravenous (IV) bolus fluorouracil (5-FU) given with 500 mg/m2/d leucovorin (LCV) and granulocyte-macrophage colony-stimulating factor (GM-CSF). PATIENTS AND METHODS: Thirty-seven patients received escalating doses of 5-FU/LCV on days 1 to 5 with subcutaneous GM-CSF either 5 or 10 micrograms/kg/d starting on day 6 or 3 micrograms/kg/d starting on day 1. 5-FU was escalated from 370 mg/m2/d by 15% increments between patient cohorts and within patients according to tolerance. RESULTS: With GM-CSF starting on day 6, dose-limiting toxicity occurred during cycle no. 1 in all three patients entered at 5-FU 490 mg/m2/d. However, individual patients tolerated 5-FU doses up to 644 mg/m2/d. When all cycles were analyzed, grade 3 to 4 mucositis and grade 4 granulocytopenia complicated < or = 15% and < or = 6% of cycles with 5-FU doses < or = 560 mg/m2/d (115 cycles). With GM-CSF starting on day 1, dose-limiting granulocytopenia occurred during cycle no. 1 in five of 10 patients entered at 5-FU 490 mg/m2/d. Although the granulocyte nadirs were significantly lower at each 5-FU dose level with the concurrent GM-CSF schedule (eg, 490 mg/m2/d: median, 879/microL v3,286/microL; two-tailed P [P2] < .001), dose-limiting granulocytopenia complicated < or = 16% of cycles with 5-FU < or = 560 mg/m2/d (99 cycles); > or = grade 3 mucositis occurred in < or = 20% of cycles. Grade 3 to 4 diarrhea was unusual with either GM-CSF schedule. Most patients treated with GM-CSF > or = 5 micrograms/kg/d required dose reductions for constitutional toxicity; 3.0 to 3.8 micrograms/kg/d was better tolerated. Venous thrombosis occurred in 17% of patients (concurrent v sequential GM-CSF, 29% v 5%; P2 = .08). The median delivered 5-FU dose-intensity for GM-CSF starting either on day 6 or on day 1 was 615 and 647 mg/m2/wk (P2 = .41), respectively. Pharmacologic exposure to 5-FU increased with higher doses of 5-FU, and concurrent GM-CSF administration did not affect 5-FU clearance. CONCLUSION: A starting dose of 425 mg/m2/d of 5-FU with LCV on days 1 to 5 could be safely combined with GM-CSF starting either on day 1 or day 6, with further 5-FU dose escalation according to individual tolerance.

Adenocarcinoma↗
PubMed1994

Antimetabolites.

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Animals↗
PubMed1994

Enhanced cytotoxicity with interleukin-1 alpha and 5-fluorouracil in HCT116 colon cancer cells.

Recombinant human interleukin-1 alpha (rIL-1 alpha), at concentrations that were not growth-inhibitory when given alone (100-10,000 U/ml), enhanced the growth inhibition resulting from a 72-h fluorouracil (FUra) exposure in HCT116 colon cancer cells. Median-effect analysis of clonogenic assays indicated that rIL-1 alpha, given 24 h prior to and following a 24-h exposure to FUra, increased lethality in a more than additive fashion. rIL-1 alpha did not appear to significantly affect [3H]-FUra metabolism, total [3H]-FUra-RNA incorporation or RNA retention after drug removal, inhibition of thymidylate synthase, or thymidine triphosphate pool depletion. During continuous exposure to rIL-1 alpha, transient stimulation of RNA and DNA synthesis was observed at 72 h, with a return to normal by 96 h. A 24-h exposure to 10 microM FUra altered the elution profile of newly synthesized DNA as monitored by pH step alkaline elution. An accumulation of lower-MW single-stranded DNA species was noted with FUra compared to control, accompanied by a significantly decreased proportion of DNA retained on the polycarbonate filter: 10% retained vs. 32% for control (P = 0.01). A 48-h exposure to rIL-1 alpha alone did not affect the elution profile of nascent DNA species, nor did it enhance the effects of FUra. Although FUra did not appreciably affect pulse [3H]-uridine incorporation into RNA for the initial 8-24 h of FUra exposure, progressive inhibition of net RNA synthesis was observed thereafter. FUra prevented the stimulatory effect of rIL-1 alpha on RNA synthesis, and net RNA synthesis was significantly inhibited (by 64-79% after 72 and 96 h) with the combination compared to rIL-1 alpha alone. Continuous exposure to 10 microM thymidine did not rescue cells from the lethality of FUra alone or the combination of FUra plus rIL-1 alpha, suggesting that depletion of deoxythymidine triphosphate as a consequence of thymidylate synthase inhibition was not the most important component of FUra toxicity. In contrast, 1 mM uridine provided partial protection against the toxicity of FUra alone or with rIL-1 alpha. Although uridine did not affect FUra metabolism, it decreased FUra-RNA incorporation by 42-60%, presumably as a consequence of the 2-fold expansion of UTP pools. [125I]-rIL-1 alpha binding was nonspecific; with a 24-h exposure, however, internalized [125I]-rIL-1 alpha exceeded cell surface-bound material by 2-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotransformation↗
PubMed1993

A phase I study of continuous infusion 5-fluorouracil plus calcium leucovorin in combination with N-(phosphonacetyl)-L-aspartate in metastatic gastrointestinal adenocarcinoma.

Preclinical studies suggest that the biochemical effects of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate carbamoyltransferase (ACTase), may increase the metabolic activation of 5-fluorouracil (5-FU) and enhance its cytotoxicity through both RNA- and DNA-directed mechanisms. In this Phase I trial, 22 evaluable patients with adenocarcinoma of the gastrointestinal tract were entered at escalating doses of 5-FU starting at 1150 mg/m2/day given as a concurrent 72-h i.v. infusion with a fixed dose of leucovorin (LCV), 500 mg/m2/day. The dose of 5-FU was escalated within patients according to individual tolerance, and then PALA at 250 mg/m2 was added 24 h prior to the initiation of the 5-FU/LCV infusion of the subsequent cycle. Dose-limiting mucositis and myelosuppression occurred during the initial cycle in 3 of 5 patients treated with 2300 mg/m2/day 5-FU; therefore, the recommended dose of 5-FU with concurrent LCV is 2000 mg/m2/day. Twenty-seven additional patients were then treated with escalating doses of PALA ranging from 375 to 2848 mg/m2, i.v., followed 24 h later by 2000 mg/m2/day 5-FU with high-dose LCV. Dose-limiting mucositis and myelosuppression occurred during the initial cycle in 2 of 3 patients entered at 2848 mg/m2 PALA. Dose-limiting mucositis and skin rash ultimately required both PALA and 5-FU dose reductions in 4 of 6 patients treated with 1899 mg/m2 PALA. Toxicity was similar, however, in patients receiving PALA at doses ranging from 375 to 1266 mg/m2. The mean steady-state plasma concentration of 5-FU at 2000 mg/m2/day was 6.5 +/- 0.9 microM; patients with 5-FU levels > 9 microM had a significantly higher incidence of serious gastrointestinal and hematological toxicity. Compared to each patient's own baseline, a significant trend for decreasing ACTase activity with increasing PALA dose was evident using cytosol isolated from peripheral blood mononuclear cells 24 h after PALA treatment (P2 = 0.01). PALA < or = 844 mg/m2 failed to appreciably inhibit ACTase activity at 24 h in most patients; furthermore, a decrease in ACTase activity by > 50% from baseline was seen in only 29% of cycles. More consistent inhibition of ACTase activity was seen with PALA > or = 1266 mg/m2. Even with the highest PALA doses, however, ACTase activity returned to baseline by 96 h in most patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenocarcinoma↗
PubMed1993

Measurement of cyclopentenyl cytosine 5'-triphosphate in vitro and in vivo by multidimensional high-performance liquid chromatography.

Cyclopentenyl cytosine 5'-triphosphate (CPEC-TP) is the active metabolite of the investigational drug cyclopentenyl cytosine (CPEC), a nucleoside analogue which exhibits noteworthy antineoplastic activity against several murine and human tumors in tissue culture, and which is now undergoing Phase I clinical trials. This study describes a method to measure the intracellular CPEC-TP levels in peripheral blood mononuclear cells (PBM cells) from patients treated with CPEC, without using radiolabeled drug. The method utilizes on-line multidimensional high-performance liquid chromatography (HPLC) with two columns of different retention mechanisms connected via an automated programmable switching valve. The elution fraction containing CPEC-TP is initially separated from cellular components using a gel sizing column (TSK-G2000-SW) and then rechromatographed by means of a reversed-phase column operated in an ion-pairing mode (YMC-A-312-ODS). The limit of quantitation of CPEC-TP by this method is 2.5 pmol per injection. When CPEC-TP levels were measured in PBM cells from 12 cancer patients after 20 h continuous infusion of CPEC at doses ranging from 3.5 to 5.9 mg/m2/h, the levels attained ranged from 1.4 to 13.4 microM (3.6 to 33.5 pmol/10(7) cells). However, wide variability in the concentrations of CPEC-TP achieved were evident at each dose and did not appear to correlate either with the CPEC dose or with CPEC plasma levels. This method was validated by a comparison of the quantitation of CPEC-TP in cultured PBM cells and Molt-4 cells (a human T-cell line adapted for growth in tissue culture) after incubation with both unlabeled and radiolabeled CPEC.

Animals↗
PubMed1993

A phase II study of continuous infusion 5-fluorouracil and leucovorin with weekly cisplatin in metastatic colorectal carcinoma.

BACKGROUND: Prolonged infusional 5-fluorouracil (5-FU) and bolus 5-FU modulated by leucovorin are associated with higher response rates than bolus 5-FU alone. Cisplatin enhances 5-FU cytotoxicity in some preclinical models. METHODS: The authors tested the feasibility of combining concurrent infusional leucovorin (500 mg/m2/d) with protracted infusional 5-FU (200 mg/m2/d) and weekly bolus cisplatin (20 mg/m2) in 22 patients with metastatic colorectal cancer. RESULTS: Four partial responses (PR) were noticed among 21 evaluable patients (19%). The median time to treatment failure and median survival were 6 months and 11 months, respectively. All but two patients required 5-FU dose reduction after a median of 2 weeks because of mucositis. However, severe mucositis and diarrhea occurred in only 18% and 5% of the patients, respectively. Palmar-plantar erythrodysesthesia of mild to moderate severity occurred in 55% of patients. Megaloblastic changes were evident in the peripheral blood during therapy, and may reflect prolonged DNA-directed toxicity of 5-FU. The median tolerated dose level of 5-FU was 113 mg/m2/d (range, 64-150 mg/m2/d). Mean steady-state plasma concentrations (Cpss) of 5-FU appeared to increase linearly from 0.19 microM to 0.39 microM over the dose range 64 to 200 mg/m2/d. Patients with grade 2 gastrointestinal toxicity had significantly higher 5-FU Cpss than patients with grade 0 or 1 toxicity. CONCLUSIONS: The early onset of toxicity with this regimen of protracted infusional 5-FU/high-dose leucovorin and weekly cisplatin required marked attenuation of the 5-FU dose intensity, and the results were no better than that expected with infusional 5-FU alone.

Adult↗
PubMed1993

Specific binding of human dihydrofolate reductase protein to dihydrofolate reductase messenger RNA in vitro.

Dihydrofolate reductase (DHFR) is a critical enzyme in de novo purine and thymidylate biosynthesis. An RNA gel mobility shift assay was used to demonstrate a specific interaction between human recombinant DHFR protein and its corresponding DHFR mRNA. Incubation of DHFR protein with either its substrates, dihydrofolate or NADPH, or with an inhibitor, methotrexate, repressed its ability to interact with DHFR mRNA. An in vitro rabbit reticulocyte lysate translation system was used to show that the addition of exogenous human recombinant DHFR protein to in vitro translation reactions specifically inhibited DHFR mRNA translation. These studies suggest that the direct interaction between DHFR protein and its mRNA may be a mechanism for regulation of DHFR synthesis.

Base Sequence↗
PubMed1993

Mechanism of resistance to cyclopentenyl cytosine (CPE-C) in Molt-4 lymphoblasts.

Cyclopentenyl cytosine (CPE-C), a carbocyclic analogue of cytidine, has preclinical antineoplastic activity against ara-C resistant murine leukemias and a broad spectrum of human tumor xenografts. CPE-C is a prodrug and requires intracellular phosphorylation to cyclopentenyl cytosine triphosphate (CPE-CTP) which depletes endogenous CTP pools. The initial step in this activation process is catalyzed by uridine/cytidine kinase. We studied the mechanism of resistance to CPE-C in a Molt-4 T-cell leukemia line made resistant to CPE-C (Molt-4R) by culturing it in the continuous presence of increasing concentrations of CPE-C. Using a tetrazolium based colorimetric assay to assess cytotoxicity, the IC90 for the parent Molt-4 cells (Molt-4WT) was 0.5 microM after a 24 hr drug exposure. In contrast, cytotoxicity was not observed at concentrations as high as 1 mM in the Molt-4R cells. Following a brief exposure to 1 microM CPE-C, parent drug could be detected intracellularly in the resistant and sensitive cell lines. However, CPE-CTP formation was reduced markedly in the resistant cell line. Measurement of the activity of anabolic and catabolic enzymes in the Molt-4WT and Molt-4R cells revealed equivalent activities of alkaline and acid phosphatases as well as cytidine and dCMP deaminase but there was a significant reduction in uridine/cytidine kinase activity in Molt-4R cells. Endogenous ribonucleotide pools and CPE-CTP pools were measured in the absence and presence of CPE-C. CTP pools were reduced markedly in Molt-4WT cells following exposure to CPE-C. However, CTP pools in Molt-4R cells exposed to 100 microM CPE-C were two times greater than in the untreated Molt-4WT cells. At high concentrations of CPE-C (10 and 100 microM), Molt-4R cells were able to generate amounts of CPE-CTP equivalent to that seen in Molt-4WT cells exposed to 1 microM CPE-C (a cytotoxic concentration of drug in Molt-4WT cells), but no cytotoxic effect was seen in Molt-4R cells. Therefore, in addition to decreased uridine/cytidine kinase activity, a second mechanism of resistance that is the result of alterations in CTP synthetase activity also appears to be operative. Elucidation of the mechanism of resistance in vitro may provide insight into the mechanism of action of the drug and potential mechanisms of resistance in vivo.

Antineoplastic Agents↗
PubMed1993

Dose-dependent inhibition of aspartate carbamoyltransferase in peripheral blood mononuclear cells in patients receiving N-(phosphonacetyl)-L-aspartate.

Forty-eight patients with adenocarcinoma of the gastrointestinal tract were treated on this trial. The MTD of 5-FU given as a 72 hour infusion with high-dose leucovorin was initially determined to be 2000 mg/m2/d. Patients were treated at PALA dose levels ranging from 250 to 2848 mg/m2. Biochemical assessment of target enzyme activity was performed at each PALA dose level. We conclude that compared to each patient's own baseline, PALA at 250 mg/m2 failed to appreciably inhibit ACTase activity at 24 hours in most patients. More consistent inhibition of ACTase activity was seen with PALA at or above 1266 mg/m2, but toxicity was prohibitive with 2848 mg/m2 PALA. Even with the highest PALA doses, ACTase activity was back to baseline by 96 hours in most patients. PALA at 1266 mg/m2 given 24 hours prior to the start of 72 hour infusional 5-FU plus high-dose leucovorin was associated with acceptable toxicity and did not appear to compromise 5-FU dose-intensity. Finally, because of interpatient variability in the degree of ACTase inhibition following PALA, biochemical monitoring of target enzyme activity may permit more rational adjustment of the PALA dose in individual patients.

Antineoplastic Agents↗
PubMed1993

Measurement of aspartate carbamoyltransferase activity by high performance liquid chromatography.

We developed an assay which permits measurement of aspartate carbamoyltransferase (ACTase) activity. Cytosol from human peripheral blood mononuclear cells was used as the enzyme source. Using [14C]carbamoyl phosphate as the radiolabeled substrate, the formation of [14C]carbamoyl aspartate was quantitated by high performance liquid chromatography (HPLC) using an anion-exchange column with UV detection at 200-280 nm and an on-line liquid scintillation detector. A gradient method from an initially low concentration of ammonium phosphate, 1 mM (pH 3.0), to a higher concentration, 38 mM (pH 4.5), was used. The apparent Km values of carbamoyl phosphate and aspartate were 58 microM and 1.9 mM, respectively. ACTase inhibition by N-(phosphonacetyl)-l-aspartate (PALA) was consistent with a competitive model with respect to carbamoyl phosphate. The assay conditions were optimized to permit measurement of ACTase activity prior to and following therapy with PALA; ACTase was inhibited in a dose-dependent manner. This HPLC method permits direct quantitation of both the product of the reaction and the initial integrity of the substrate, [14C]carbamoyl phosphate, which is unstable in aqueous solutions.

Antimetabolites, Antineoplastic↗