Search

The addition of oxaliplatin or CPT-11 to 5-FU has become common practice in the treatment of colorectal cancer. It is not known, however, which fluoropyrimidine drug (5-FU, FUdR, or FUR) will produce superior cytotoxicity when combined with either oxaliplatin or CPT-11. The purpose of the study was to determine the effects of oxaliplatin and CPT-11 on cytotoxicity and nucleic acid incorporation of all three fluoropyrimidines.HT-29 cells were exposed for 2 h to IC(10), IC(30), and IC(70) of oxaliplatin and CPT-11. Subsequently, cells were exposed for 24 h to IC(10), IC(30), and IC(70) of 5-FU, FUdR, and FUR. Cytotoxicity was measured by the MTT assay. Nucleic acid incorporation of [(3)H]fluoropyrimidine was then compared in the presence and absence of oxaliplatin or CPT-11 pretreatment.Synergistic cytotoxicity was displayed when IC(30) of oxaliplatin or CPT-11 was combined with IC(10) and IC(30) of the fluoropyrimidines. One fluoropyrimidine did not achieve superior cytotoxicity over the others. After pretreatment with oxaliplatin or CPT-11, cytotoxic antagonism was observed as the concentration of a fluoropyrimidine increased up to IC(70). The increasing cytotoxic antagonism correlated with decreases in fluoropyrimidine nucleic acid incorporation. The most significant incorporation difference existed within the 5-FU treated group.No single fluoropyrimidine is more cytotoxically effective over the others when combined with oxaliplatin or CPT-11. Correlation of cytotoxic antagonism to the inhibition of fluoropyrimidine nucleic acid incorporation implies difficulties in drug transport and/or metabolism only after oxaliplatin or CPT-11 pretreatment.

5-Fluorouracil (5-FU) is the most routinely administered drug in the treatment of colon cancer. The main mechanism of the drug is not completely understood and its method of administration has been strongly disputed. A 24-hour infusion of 5-FU has clinically yielded better response rates and lower toxicities in comparison to bolus administration, but an exploration into possible mechanisms needs to be performed. Experiments were conducted with two 5-FU resistant cell lines where cytotoxicity, thymidylate synthase (T.S.) activity, thymidine kinase (T.K.) activity, DNA and RNA incorporation, and T.S. expression were contrasted between a 10 microM/24 hour administration of 5-FU (simulating continuous exposure) and a 100 microM/1 hour schedule (simulating bolus administration). After 6 days from the initial exposure, the 10 microM/24 hour schedule (schedule A) inhibited more cell growth than the 100 microM/1 hour regimen (schedule B) by more than 38% and 17% in the two cell lines. After the 6-day observation, schedule A inhibited twice as much T.S. activity as schedule B. Incorporation of [14C]-5-FU into DNA and total RNA was higher in cells exposed to schedule A in comparison to schedule B over the 6 days. T.S. expression and T.K. activity patterns were variable over time. Thus, the exposure of 10 microM/24 hour 5-FU results in superior cytotoxicity when compared to a 100 microM/1 hour regimen and its effectiveness may be explained mechanistically by T.S. activity and DNA and RNA incorporation.

To study the combination of 5FUDR, recombinant leukocyte interferon (IFN), and doxorubicin in patients with unresectable hepatocellular carcinoma. IFN was administered at a dose of 6 miu/m2 subcutaneously followed in 2 h by doxorubicin 20 mg/m2 intravenously. After doxorubicin, 5FUDR was given as a 24-h infusion at a starting dose of 80 mg/kg. The dose of IFN was escalated to three times a week if tolerated. Both doxorubicin and 5FUDR were administered once weekly. There were 30 patients entered into the study. Among the 30 patients, there were two partial responses (7%) and one patient had stable disease. Toxicity was generally tolerable with fever, and chills, fatigue, and myelosuppression as the most common side effects. This chemotherapy combination was generally well tolerated, but has limited activity in unresectable, advanced hepatocellular carcinoma.

Fluoropyrimidine analogue 5-Flurorouracil (5-FU) is the mainstay of CRC treatment. It was designed as a competitive inhibitor of the enzyme thymidylate synthase [TS; TYMS]. All the new agents (Irinotecan, CPT-11, Avastin etc) are effective only in combination with 5-FU. The enzyme thymidylate synthase is pivotal for DNA replication. It controls cell survival and proliferation. Increases in TS level that follows 5-FU administration renders the treatment ineffective. Considerable efforts have been made to reverse 5-FU resistance by down-regulating TS expression. In this context, we demonstrated that arsenic trioxide (ATO) sensitized 5-FU resistant colorectal cancer cells by inhibiting TS mRNA expression both in vitro and in clinic. However, the precise mechanism of TS reduction by arsenic trioxide is not known. Many investigators, including ourselves, have shown a direct correlation between Glioma associated oncogene homolog (Gli) and TS expression in cultured cells as well in patient samples, who received arsenic trioxide and 5-FU combination therapy. However, the link between ATO, Gli and TS is not known. Gli is an important molecule in hedgehog (hh) signaling. It plays a vital role in development and cancer. As a transcription factor, Gli binds the sequence 5’ GACCACCCA 3’ a conserved 9 bp DNA motif. We hypothesized that TS promoter may harbor one or more of the 9 bp Gli motifs. We queried for Gli motifs in the TS gene regulatory region. Position specific base frequencies for the TF consensus sequence for Gli1-3, Ci, cECF, TGM were prepared using the data from Hallikas et al. (2006). We used FIMO (Find Individual Motif Occurrences) tool in the Meme Suite (http://meme.nbcr.net) to search for Gli motifs in the query sequence. Anti-Gli antibodies and non-specific IgG were used to precipitate chromatin immuno complex. The immuno precipitates were probed with TS specific primer. This search yielded 35 possible Gli motifs in TS promoter. From among the 35 motifs with significant homology (p≤0.001), only top eight matches had passed FDR threshold (q≤0.05). However, these eight matches represented three distinct binding sites on TS promoter. Thus we computationally determined that TS promoter has three unique Gli motifs viz BS1, BS2 and BS3. BS-2 in TS promoter is identical to BS-3 on the Jun and BS1 is identical to the BS 1 of Fgf15 which had been experimentally confirmed to be regulated by Gli1. The binding site was subsequently confirmed by ChIP assays. It appears that the inhibition of Gli1 by ATO down regulates TS expression. This cascade of reactions initiated by ATO reverts refractory tumors, once again sensitive to 5-FU. Taken together the results strongly indicate a direct role of Gli in TS transcription. Citation Format: Subbarayan R. Pochi, Nikesh Doshi, Xiaoqing Han, Zhiqiang Wang, Anthony Capobianco, David Robbins, Bach Ardalan. Regulatory role of Gli motifs in thymidylate synthase expression . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4019. doi:10.1158/1538-7445.AM2013-4019

A Phase II trial of combination therapy with recombinant leukocyte interferon (alpha IFN) and doxorubicin was performed in patients with unresectable hepatocellular carcinoma. alpha IFN was administered at a starting dose of 20 x 10(6) U/m2 intramuscularly or subcutaneously with doxorubicin 20 mg/m2 intravenously weekly x 3 weeks followed by a 2-week period rest. There were 22 patients entered into the study. Among the 21 patients, there were 2 partial responses (10%), one minor response, and one patient had stable disease. Toxicity was generally tolerable, with fever, fatigue, and myelosuppression being the most common side effects. This combination of weekly recombinant leukocytic interferon and doxorubicin has modest and limited activity in hepatocellular carcinoma.

e14081 Background: In vitro, prolonged exposure of colon cancer cells to 5-FU induces resistance and is associated with increased synthesis of thymidylate synthase (TS). 5-FU resistance is well correlated with the over expression of intratumoral TS mRNA which if down regulated may reverse 5FU resistance. Earlier we demonstrated that ATO significantly down regulated TS in vitro and in patients thereby sensitizing them back to 5-FU. Methods: A treatment cycle consisted of four weeks of therapy and one week of rest. On days one through five, 8, 11, 15 and 22, the patients (pts.) received a 4-hour ATO infusion; on days 8, 15 and 22 a 24-hour 5 FU/ leucovorin infusion. To date 12 pts with refractory metastatic colorectal carcinoma were enrolled. In the 5-FU dose escalation phase, patient one received 1,600 mg/ m2; patient two 2,000 mg/m2; patient three 2,300 mg/m2; patient four and all the subsequent patients 2,600 mg/m2 of 5-FU. ATO was infused at 0.15 mg/kg in patient one through seven and at 0.20 mg/kg in p...

e15114 Background: In a previous study, IFL was used in patients (pt) with untreated metastatic colon cancer and a median overall survival (MOS) of 31 months (m). In the current study we added B to IFL to determine toxicity profiles (TP) and the response rate (RR) to the B-IFL regimen. The primary end-point is MOS Methods: Each cycle involved 6 weeks (wks) of treatment. The treatment cycle consisted of a 90 minute infusion of I (110 mg/m2), followed by a 24 hour infusion of F (120mg/kg) and L (500 mg/m2) on wks 1, 2, 4, 5. Pt received B (7.5mg/kg) over 90 minutes on wks 1 and 4 prior to IFL. No therapy was given on wks 3 and 6. Eligible pt received 2 cycles of B-IFL followed by CT scan. Quality of life data and thymidylate synthase expression in peripheral blood mononuclear cells was monitored Results: 22 pt with a median age of 57 (38–82), 11 males and 11 females were enrolled. Median KPS was 90% (50–100). 8 pt (36%) had bilobar liver disease and involvement of 1 other organ, 6 pt (27%) had bilobar liver disease with involvement of ≥ 2 other organs; 5 pt (23.5%) had bilobar liver disease; 2 pt (9%) had abdominal carcinomatosis; 1 pt (4.5 %) had involvement in one liver lobe. Grade (Gr) 4 toxicity: pulmonary embolus 1 pt (5%) incidental CT finding. Gr 3 occurred in 11 pt (50%); DVT, diarrhea (Drh) 3 pt (14%); fatigue (Ftg), infection, port site thrombosis (Pst), small bowel obstruction 2 pt (9%); wound dehiscence 1pt (5%); Gr 2 and 1: constipation; Drh; Ftg; nausea; neutropenia; Pst; alopecia; anorexia; mucositis. 17 pt remain alive with median follow up of 15 m (2–28). 5 pt have died due to progression of disease. The estimated median time to progression was 13 m with corresponding lower 95% confidence bound of 8.4 m. Kaplan-Meier estimate at 2 yr survival is 61% (95% CI 27–83%). RR in 21 pt was 67% (95% CI: 43–85%). Conclusions: B-IFL regimen has a manageable TP. RR and progression free interval were promising. Pt accrual is ongoing. Supported by Pfizer. No significant financial relationships to disclose.

15503 Background: A pathological complete response (pCR) to NAC, without the use of radiation therapy (RT), has not been previously reported in operable chemo-naive stage III EAC. Toxicity profiles (TP) to FLOD are explored in this ongoing study. Methods: Patients (Pt), who had been diagnosed with operable stage III EAC, received 2 cycles of NAC. Each cycle consisted of a 30 min infusion of D (25 mg/m2), followed by F (110 mg/kg) and L (500 mg/m2) as a 24 hour infusion on Week (Wk) 1, 2 and 3. O (85 mg/m2) was infused over 2 hours on Wk 1 and 3. Wk 4 was rest. Dose reductions (DR) were allowed: 100 and 90 mg/kg for F, 20 and 15 mg/m2 for D and 65 mg/m2 only for O. NAC delays up to 2 Wk were allowed. EUS and PET/CT were performed prior and after NAC. Patients underwent surgical resection after 2 cycles of NAC. TP were graded per NCI CTCAE v. 3. The pCR rate is set as a primary endpoint. Results: 29 Pt, median age 63 (50–80), 23 males and 6 females were accrued. KPS ranged from 70 to 100. TP were: Grade 4: ...

4054 Background: A Phase II trial to evaluate neoadjuvant (NAD) and adjuvant (AD) combination chemotherapy (CT) without radiation therapy (RT), for Stage III esophageal adenocarcinoma. Methods: Stage III disease by CAT scan or EUS and ECOG performance status 0–1. The CT cycles included Cisplatin, Taxol, FUDR and Leucovorin. The chemotherapy was given for 16 weeks prior to surgery, followed by adjuvant chemotherapy for patients whose pathology at the time of surgery demonstrated microscopic disease. Survival was estimated via Kaplan Meier. Results: 33 patients were enrolled: 28 completed NAD, 15 received AD. Of 33 pts given NAD, 16 had grade I/II toxicity and 14 had grade III/IV. In addition, 6 of 15 pts receiving AD had grade I/II toxicity and 3 had grade III/IV during that phase of treatment. Thirty-two (32) patients went to surgery, 30 patients were resected: 24 transhiatial and 6 transthoracic. Of the 28 patients who completed NAD, 19 (68%) demonstrated improvement in dysphagia; 11 (41%) gained weight, and 7 (26%) had no weight change. Pathologic response to NAD: 30 patients had partial response and 2 patients had progressive disease, one died prior to surgery, post carotid endarterectomy. Ten (10) patients showed no gross disease. Twenty-three (23) patients have expired, 17 disease related with distant metastasis, 1 with local recurrence and distant metastasis, 3 non-disease related and 2 unresectable cases. Kaplan-Meier estimates of overall survival at 1, 3, and 5 years were 73% (95% CI: 58 to 88%), 52% (95%CI: 34 to 69%), and 29% (95% CI: 13 to 45%), respectively. Median survival was 42 months (95% CI: 14 to 52 months). Ten (10) patients are alive after a median follow up of 73 months (range 45 to 89). Seven (70%) of the surviving patients received both NAD (2 cycles) and AD (1 to 2 cycles) and all had no evidence of disease as of last clinical and radiological evaluation. Conclusions: This regimen of combination CT for locally advanced esophageal adenocarcinoma is safe and comparable with those regimens that contain RT. There has been 1 local recurrence; 17 patients have recurred distantly. Omission of RT may allow for a more aggressive CT and reduction in the local complications post-surgery. This study needs to be confirmed in a larger phase II or in randomized phase III trial. No significant financial relationships to disclose.

4196 Background: Endoscopic ultrasound (EUS) plays a central role in the initial staging and treatment of esophageal cancer (EC). Use of EUS for re-staging patients (pts) after neoadjuvant therapy is controversial. Few studies have suggested that EUS post-neoadjuvant therapy can accurately predict tumor response and overall survival in pts with locally advanced disease. Methods: EUS data from an ongoing study on neoadjuvant chemotherapy for locally advanced EC with Paclitaxel 125 mg/m2, Fluorodeoxyuridine 150 m/Kg or 80 mg/Kg, Leucovorin 500 mg/m2 and Cisplatin 100 mg/ m2 was collected. EUS performed pre- and post-chemotherapy in all pts. Maximum tumor thickness was measured on EUS pre- and post-chemotherapy and EUS stage compared to surgical pathology (AJCC TNM stage). More than 50% wall thickness reduction was classified as a response by EUS. Results: 38 pts were included, mean age 62 yrs. There were 35 adenocarcinomas of the gastroesophageal junction and three squamous cell carcinomas. The median follo...

4147 Background: Patients (Pts) with localized esophageal carcinoma were evaluated for toxicity, operative mortality/morbidity, and overall survivorship in a regimen that did NOT contain radiation therapy. Methods: Pts with T3N1M0 esophageal carcinoma confirmed by endoscopic ultrasound and CAT scan were eligible for this study. Our therapy consisted of combined Cisplatin100 mg/m2, Paclitaxel (Taxol) 125mg/ m2, Fluorodeoxyuridine (FUdR) 150 mg /Kg or 80 mg/Kg, and Leucovorin 500mg /m2. Chemotherapy was given for 16 weeks prior to surgery. Adjuvant chemotherapy was administered for the patients whose pathology at the time of the surgery demonstrated microscopic disease. Results: 33 pts (29 males, 4 females) with adenocarcinoma (n=30) or squamous cell carcinoma (n=3) were enrolled. 26 pts (78%) were symptomatic with dysphagia at the time of enrollment on the study. 28 pts completed two cycles of neoadjuvant chemotherapy, 19 (68%) showed improvement of dysphagia. There were 9 instances of grade 3 or 4 hematol...

Fifty-two patients with advanced gastrointestinal (GI) malignancies who had not received previous chemotherapy or radiation therapy were randomized to be treated either with 24-hour infusion of weekly fluorouracil (5-FU) or the same plus N-(phosphonacetyl)-L-aspartic acid (PALA). Forty-seven patients were evaluable for the assessment of toxicity and antitumor activity. PALA was administered as an intravenous (IV) bolus over 15 minutes at a fixed dose, 250 mg/m2. The latter agent was administered 24 hours before the start of 5-FU infusion. 5-FU was initially administered at 750 mg/m2 and was incrementally increased to 3,400 mg/m2. In both arms of the randomized study, the courses were repeated every week. In both arms of the study, ataxia and myelosuppression were the dose-limiting toxic effects. At 5-FU dose of 3,400 mg/m2, one patient in each arm developed grade 3 hematologic toxicity. Other reversible side effects included grade 2 skin changes, nausea, and vomiting. During the administration of 2,600 mg/m2 of 5-FU over 24 hours, the steady state plasma 5-FU concentration was approximately 20 mumol/L. The maximum tolerated dose (MTD) for 5-FU for protracted treatment is 2,600 mg/m2 in either arm of the study. Therapeutic response was predominantly seen in the combination arm: there were two patients with complete response (CR) and 11 patients with partial response (PR) of 28 patients in the study. In the 5-FU alone arm there were four PR and 19 patients in the study.

A comparison was made of the severity of chronic doxorubicin cardiotoxicity in adult male spontaneously hypertensive rats (SHR) and in genetically related normotensive Wistar-Kyoto rats (WKY). Groups of SHR and WKY were given 12 weekly iv injections of doxorubicin at 0.25, 0.5, or 1.0 mg/kg. When the study was concluded, mean arterial pressure was 127 to 161 nm Hg in doxorubicin-treated SHR compared with 74 to 87 mm Hg in similarly treated WKY. Lesions, consisting mainly of cytoplasmic vacuolization and myofibrillar loss, were noted in the hearts from both types of rats given the 1.0-mg/kg dose and were considerably more severe in SHR than in WKY (average scores 3.8 and 2.0). Renal lesions (glomerular vacuolization and dilatation of tubules with accumulations of proteinaceous material) were of comparable severity in both types of rats at the 9- and 12-mg/kg cumulative doses; however, they were more severe in SHR at the 6-mg/kg cumulative dose. Moderate cardiac alterations were present in all SHR (average score 1.6) given 0.5 mg doxorubicin/kg; at the same dose, lesions were minimal in two and absent in three WKY. In a second study, groups of rats were killed 1 week after 3,6,9, or 12 weekly iv injections of doxorubicin (1.0 mg/kg). Myocardial lesions were noted initially in SHR after six doses and in WKY after nine doses. Three of five SHR were dead by the 12th dose. These results indicate that spontaneously hypertensive rats are much more sensitive than normotensive rats to the cardiotoxic effects of doxorubicin.

A simple procedure for separation of ribose-5-phosphate, deoxyribose-1-phosphate and ribose-1-phosphate is based on high performance liquid chromatography using reversed phase 4 × 300 mm “μ Bondapak/NH2” column. The column is equilibrated with 0.13 M borate buffer (pH 7.5) followed by gradient elution of ribose-5-phosphate, deoxyribose-1-phosphate and ribose-1-phosphate using water, 0.05 M borate buffer containing 0.1 M MgCl2 (pH 9.6) and 0.05 M sodium acetate-acetic acid buffer containing 0.1 M MgCl2 (pH 5.0) as eluants respectively. Eluates of borate complex “μ Bondapak/NH2” column are brought to pH 9.6 by the addition of 1 N KOH and enzymatically hydrolysed with alkaline phosphatase (EC 3.1.3.1) to release the free pentoses. The free pentoses are mixed with a reagent solution prepared from aqueous solution of 2% cyanoacetamide and 0.6 M borate buffer (pH 9.6), and the mixture is boiled for 10 minutes and the absorbance of the product is measured at 276 nm using a spectrophotometer.

The biological effects of N-( phosphonacetyl )- l - aspartate (PALA) and 5-fluorouracil (5-FU) were examined singly, and in combination, on the growth of a human mammary carcinoma (MDA) cell line in culture. All combinations of 5-FU (2.5 × 10−7 to 1.5 × 10−5M) and PALA (6.0 × 10−5 to 3.6 × 10−3 M) resulted in synergistic inhibition of cell growth as revealed by a 50 per cent isobologram.To examine the biochemical basis for the synergism, measurements of the incorporation of [3H]-5-FU into total non-poly(A)- and poly(A)-RNA, and of the simultaneous incorporation of [14C]deoxyguanosine and [3H]deoxyuridine into DNA, were determined. The combination of 3.7 × 10−5M PALA and 1 × 10−6 M 5-FU produced 65–85 per cent inhibition of cell growth after continuous treatment for 13 days. Treatment of the cells for 3 or 24 hr with the same drug regimen produced approximately a 170 per cent increase in the incorporation of 1 × 10−6M [3H]-5-FU into poly(A)RNA in comparison to [3H]-5-FU treatment alone; exposure for 24 hr to 3.7 × 10−5 M PALA and 1 × 10−6 M [3H]-5-FU resulted in a 285 per cent increase in the incorporation of [3H]-5-FU into non-poly(A)RNA. The incorporation of either [14C]deoxyguanosine or [3H]deoxyuridine into DNA was not inhibited by this drug regimen; however, the incorporation of [3H]deoxyuridine into DNA was elevated significantly upon 12 or 24 hr of exposure to PALA alone. PALA and 5-FU treatment resulted in a 75 per cent reduction in the concentration of UTP and no change in the concentration of 5-fluorouridine-5′triphosphate 5-FUTP) versus 5-FU treatment alone. Thus, the proportion of 5-FUTP in the total 5FUTP + UTP pool was enhanced more than 3-fold by the combination regimen. These results indicate that the synergistic effect of the combination of PALA and 5-FU on the growth of MDA cells correlates with an increased proportion of 5-FUTP in the pyrimidine nucleotide pool and, consequently, with an enhanced incorporation of 5-FU into RNA, but not with inhibition of DNA synthesis.

The intratumoral content of 5-phosphoribosyl 1-pyrophosphate (PRPP) and the activity of the enzymes anabolizing and catabolizing the sugar phosphate were determined following i.p. administration of an LD10 dose of an L-glutamine antagonist or saline to tumor-bearing animals. Elevation of PRPP pool size following administration of L-[alpha S,5S]-alpha-amino-3-chloro-4,5-dihydro-5-isopazoleacetic acid (NSC-163501) (AT-125) was maximal at 8 hr and returned to pretreatment levels by 24 hr. In P388 leukemia, dose for dose, at 4 hr, 6-diazo-5-oxo-L-norleucine (NSC-7365) (DON) was the most potent of the L-glutamine antagonists in elevating basal PRPP pool size (50% above control) followed by AT-125 and azaserine, 300 and 100% above control respectively. Moreover, such augmentation in PRPP pool size preferentially affected P388 tumor rather than the small intestine. Following i.p. administration of LD10 doses of AT-125, DON and azaserine, the specific activities of PRPP anabolizing and catabolizing enzymes were determined. A significant inhibition of PRPP amidotransferase was demonstrated with DON and AT-125 (P less than 0.05), and no inhibition with azaserine. A similar modulation of PRPP pool size demonstrated in vivo following administration of 250 mg/kg of ART-125 in mice bearing colonic adenocarcinoma lines. It was suggested that a significant increase of PRPP pool size might cause the possible synergism of a selected L-glutamine antagonist and 5-fluorouracil as reported after the appropriately scheduled administration of methotrexate and 5-fluorouracil.

The pharmacologic effects and plasma concentrations produced by the ip administration of high doses of thymidine were determined in CDF1 mice. Within 15 min after the injection of thymidine at a dose of 4.075 g/kg (between LD0 and LD50), mean arterial blood pressure and heart rate fell precipitously and remained depressed for over 6 hr. During the experimental period, sedation and anuresis were also consistently observed in thymidine-treated mice. After a dose of 4.7 g/kg, millimolar plasma thymidine concentrations were maintained for 16 to 20 hr; by contrast, at a lower but nonlethal dose (3.8 g/kg), millimolar plasma thymidine concentrations were maintained for only 9 hr. It is suggested that sustained elevation of circulating thymidine in the range 1–10 m m for periods greater than 1 2 day may be associated with toxicity in mice.

Publisher Summary The biological activation of 5-fluorouracil (5-FU) is dependent on the operation of several enzymes. These can be aggregated into three principal groups: those that anabolize the drug, those that catabolize it, or its anabolites, and “target” enzymes whose function is finally interrupted following treatment. These families of enzymes are susceptible to two general types of regulation: physiologic control that is used to encompass genetic or biochemical modulation and pharmacologic control that is used to designate the synergistic or antagonistic actions of one drug on the activity of the other. As 5-FU continues to be used in the palliation of breast and gastrointestinal malignancies, more information is being gathered on these classes of enzymes, as well as on the manner in which they are regulated. This chapter discusses selected aspects of such regulation, with emphasis both on published and original findings in the field. Whenever possible, absolute specific activities of the enzymes involved are presented in order that reasonable intercomparisons can be made of their relative contributions to the metabolism of 5-FU.

An in vitro assay system measuring the production of phosphorylated nucleotide products of 5-FU by human tumor cytosols is described. Samples from 11 patients with breast cancer treated with 5-FU-containing chemotherapy regimens were assayed. Significant differences were seen in quantitative and qualitative patterns of phosphorylation between samples from chemotherapy responding and nonresponding patients. The rate of 5-fluorodeoxyuridylate production and the amount of nucleotide triphosphate produced were significantly P < 0.05 increased in chemotherapy-re- sponding patients. This in vitro assay system is a simple, reproducible procedure that requires further evaluation as a potential system for prospectively predicting clinical response to fluorinated pyrimidines.

3-Deazaguanine (3-DG), a purine analogue, has unusual antitumor activity against experimental mammary tumor models and a number of other solid tumors. Others have shown that mutant CHO cells deficient in hypoxanthine guanine phosphoribosyl transferase (HGPRTase) or adenine phosphoribosyl transferase (APRTase) are resistant to 3-DG. We developed a L1210 cell line resistant to 3-DG, L1210/3-DG, by subculturing the parent L1210/0 cells in the presence of increasing concentrations of 3-DG. The IC50 was 3.5 microM and 620 microM for L1210/0 and L1210/3-DG, respectively. Cytotoxicity studies proved the resistance to be stable. Examination of the baseline-specific activity of HGPRTase and APRTase showed that the former was 118-fold lower in L1210/3-DG than in L1210/0, and the latter demonstrated no difference. A 4-h treatment of the cell lines at IC50 doses showed 48% and 23% reductions in IMP dehydrogenase in L1210/0 and L1210/3-DG, respectively. The rate of de novo purine biosynthesis was studied by using [14C]formic acid. Formate flux increased 2-fold in L1210/3DG in concert with the observed deficiency of HGPRTase in the cell line. 3-DG uptake was studied with [14C]-labelled compound. The total radioactivity was 9-fold higher in L1210/0 than in L1210/3-DG at 2 h. Subsequent chromatographic separation of radioactivity showed the 3-DG and 3-deazaguanosine pools of the drug to be equal in both lines. However, 3-DG nucleotide pools at 1 min and 2 h were 2.5-fold and 16-fold lower, respectively, in L1210/3-DG than in L1210/0. 3-DG incorporation studies with radiolabelled drug demonstrated that 3-deazaguanine is incorporated in the acid-insoluble fraction of the cell. These studies conclude that HGPRTase, and not APRTase, is required for the activation of drug. Inhibition of IMP dehydrogenase is partially responsible for antitumor activity of the drug. The incorporation of drug into nucleic acids may be a major mechanism for its antitumor activity. Further studies using a cloned cDNA probe for hypoxanthine guanine phosphoribosyltransferase (HGPRT) demonstrated no change in the DNA arrangements of the L1210/3-DG cell line, and Northern blot analysis showed approximately equal expression of mRNA in both cell lines.

The antitumor activity of 5-fluoruoracil (5-FU) for two murine colonie adenocarcinomas was correlated with the concentration and the clearance of the active antimetabolite, 5-fluorodeoxyuridylate (FdUMP). Mice inoculated with a cell suspension of murine colonic adenocarcinomas 38 and 51 were treated with 5-FU (100 mg/kg i.p.) on 3 day post-transplantation. For mice bearing adenocarconoma 38, treatment with 5-FU was associated with a 97 per cent reduction in mean tumor weight a day 30 and a 77 per cent reduction at day 37 of tumor growth. In contrast, mice bearing colonic adenocarcinoma 51, treated with the same dose schedule of 5-FU did not demonstrate a reduction in the rate of tumor growth in vivo. Two hr after i.p. injection of 5-FU (100 mg/kg) the intracellular concentration of free FdUMP in the sensitive tumor 38 was 560 fmoles/μg of DNA. The active antimetabolite was maintained at a concentration in excess of 100 fmoles/μg of DNA for 72 hr. In contrast, the 2-hr free FdUMP concentration in the resistant tumor line 51 was 240 fmoles μg of DNA(P < 0.005), and a concentration in excess of 100 fmoles/μg of DNA was maintained for only 24 hr. There was no difference in the rate of progressive accumulation of the competitive metabolite, deoxyuridine monophosphate (dUMP), during the first 24 hr of the study. Two hr after i.p. injection of 5-FU (100 mh/kg), [3H] deoxyuridine ([3H]Udr) incorporation into DNA was reduced in both tumor lines to below 3 per cent of control. However, in the sensitive tumor, adeno-carcinoma 38, DNA synthesis was maximally inhibited for 72 hr, compared to 24 hr in the resistant adenocarcinoma 51. The reinitiation of DNA synthesis corresponded to the reduction of free FdUMP concentration to less than 100 fmoles/μg of DNA. There was no linear relationship between the FdUMP/ dUMP ratio and [3H]UdR incorporation into DNA in either tumor line. These data demonstrate that the peak tumor FdUMP concentration and the kinetics of its clearance correlated with the responsiveness of the two specific murine tumors to 5-FU. The measure of peak FdUMP level should be tested for its potential clinical application as a means of selecting patients with gastrointestinal and breast cancer to be treated with this agent.

A study was made of the in vivo effects of equitoxic doses of AT-125 and 5-FU combination, being administered either simultaneously (% ILS 152) or with a 6-h pretreatment with AT-125 (% ILS 184). To examine the biochemical basis for the scheduled synergism, measurements were made of the concentration of PRPP, the specific activities of CPS II, cytidine, thymidine, uridine, deoxyuridine kinases, and fluorinated nucleotide formation in P388 tumors and the small intestine. Two hours after in vivo simultaneous treatment of mice bearing tumors the concentration of PRPP increased 9- and 6-fold above baseline in the tumor and the small intestine, respectively. In the AT-125 pretreatment arm the concentration of PRPP increased 18- and 7-fold above baseline in the tumor and the small intestine, respectively. CPS II activity was reduced to 28%-18% of control in the tumors in the simultaneous and pretreatment groups, respectively, whereas it remained unchanged in the small intestine. Specific activities of cytidine kinase (5.5 +/- 1), thymidine kinase (4.0 +/- 1.6), uridine kinase (35.6 +/- 6.5), and deoxyuridine kinase (2.4 +/- 1.1) nmol/mg protein/h remained unchanged with treatment. In concert with the increased intratumor concentration of PRPP, fluorinated nucleotide formation was proportionally increased in the treatment arms. These results indicate the importance of drug scheduling of the above two agents in treating P388 leukemia.

The antitumor activity of 5-fluorouracil (FUra) in four murine colonie adenocarcinomas was correlated with the basal pool sizes of phosphoribosyl pyrophosphate (PRPP). Mice inoculated with 20–30 mg fragments of murine colonic adenocarcinomas 26 and 38 (FUra-sensitive), and 51 and 11A (FUra-resistant), were treated approximately 2 weeks later with FUra (200 mg/kg, i.p.). In mice bearing adenocarcinomas 26 and 38, intravenous administrations on days 3, 10 and 17 of the highest nontoxic dose of 73 mg/kg produced a tumor growth delay of 14.8 and 11.8 days respectively. In contrast, mice bearing colonic adenocarcinomas 51 and 11A, treated with the same dose schedule of FUra, demonstrated a tumor growth delay of 5 and 5.4 days respectively. The basal levels of PRPP in FUra-sensitive tumors 26 and 38 were 8.7 and 4.0 μM, whereas those in FUra-resistant tumors 11A and 51 were 2.4 and 2.8 μM respectively. Two hours after i.p. injection of FUra (200 mg/kg), FUra-sensitive tumors (26 and 38) showed a substantial reduction in basal levels of PRPP to 1.30 and 2.80μM respectively; FUra-resistant tumors (51 and 11A) demonstrated a significantly smaller decrease in basal pool size levels. At 24 hr there was a full restitution of intratumoral PRPP to the previous basal levels. Four hours following i.p. injection of FUra at 200 mg/kg, the group of enzymes which could perturb PRPP pool size were assayed, namely PRPP synthetase (EC 2.7.6.1), hypoxanthine-guanine phosphoribosyl transferase (EC 2.4.2.8), adenine phosphoribosyl transferase (EC 2.4.2.7), and orotate phosphoribosyl transferase (EC 2.4.2.10). The specific activity of PRPP synthetase in the FUra-sensitive line (colon tumor 26) was 3-fold higher than in the FUra-resistant tumors examined (colon 51 and 11A); moreover, only in the sensitive line was the specific activity of orotate phosphoribosyl transferase increased significantly following treatment with FUra. Thus, these data suggest that measurement of the basal intratumoral PRPP levels, together with the determination of specific activities of PRPP synthetase and oratate phosphoribosyl transferase, should be tested for its potential clinical application as a means of selecting patients with gastrointestinal and breast carcinomas to be treated with FUra.

In order to determine whether a simultaneous infusion of N-(phospho-n-acetyl)-L-aspartic acid (PALA) was able to increase the rate of antitumor response to 5-fluorouracil (5-FU), a pilot study was conducted. Of 10 evaluable patients with previously drug-untreated colorectal carcinoma, there were only two partial responses, lasting 2.3 and 1.6 months. No partial responses were observed in three evaluable patients with soft tissue sarcomas. The dose-limiting toxicity was dermatitis. The simultaneous infusion of PALA and 5-FU is not likely to produce a high number of antitumor responses of long duration in patients with colorectal carcinoma.