Dose-response relationship of phenobarbital promotion of diethylnitrosamine initiated tumors in rat liver

The dose-response relationship was determined in rats for the enhancement by phenobarbital of diethylnitrosamine (DENA)-initiated neoplastic nodules and hepatocellular carcinomas. Male Sprague-Dawley rats received a single oral dose of either 80 mg/kg DENA or water. Seven days later, the animals were divided into groups that started to receive 0, 62.5, 125, 250, 500 or 1000 ppm sodium phenobarbital in the drinking water. Animals from each group were killed at 48 and 70 weeks after the DENA. No significant difference was observed in the low response of neoplastic nodules among the DENA-initiated groups. The incidence of DENA-initiated hepatocellular carcinoma was enhanced at 70 weeks by 250, 500 and 1000 ppm sodium phenobarbital but not by 62.5 or 125 ppm sodium phenobarbital. Equal enhancement of the incidence of hepatocellular carcinomas was obtained with 250, 500 and 1000 ppm sodium phenobarbital. In non-DENA-initiated rats, phenobarbital did not induce neoplastic nodules or hepatocellular carcinomas. Our results suggest that a daily dose of at least 250 ppm sodium phenobarbital is required in order for it to exert tumor promoting activity.     

Effects of strain,sex, route of administration and partial hepatectomy on the induction by chemical carcinogens of gamma-glutamyltranspeptidase foci in rat liver

The incidence of gamma-glutamyltranspeptidase (GGT)-positive foci induced by 0.3 mmol/kg diethylnitrosamine (DENA) followed by promotion with 500 ppm sodium phenobarbital in drinking water and was the same in Fischer 344, Sprague-Dawley and Wistar-Lewis rats. There was no difference in the level of GGT-foci initiated by DENA, 7,12-dimethylbenz[a]anthracene (DMBA), or 1,2-dimethylhydrazine (DMH) followed by promotion with phenobarbital with respect to sex or route of administration including gavage and intraperitoneal injection. Maximal stimulation by partial hepatectomy of DENA initiation of GGT-foci occurred when the DENA was administered 18 h after the operation. Our results indicate that the optimal protocol for the rat liver foci assay consists of using partial hepatectomized rats of 1 of the 3 strains and of either sex. The test substance should be administered by either gavage or intraperitoneal injection so that maximal DNA binding coincides with the maximal rate of DNA replication resulting from partial hepatectomy.     

Effect of coadministration of phenobarbital sodium on N-nitrosodiethylamine-induced gamma-glutamyltransferase-positive foci and hepatocellular carcinoma in rats

The effect of concurrent administration of phenobarbital on the hepatocarcinogenicity of N-nitrosodiethylamine (diethylnitrosamine; DENA) in rats was investigated by determination of the incidence of gamma-glutamyltransferase (gamma-glutamyltranspeptidase) (GGT)-positive foci and liver tumors. Male outbred Sprague-Dawley rats received either a weekly oral dose of DENA (0.08 mol/kg), phenobarbital sodium (500 ppm) in their drinking water, or DENA and phenobarbital sodium concurrently. After 16 weeks, only the animals treated concurrently with DENA and phenobarbital sodium had GGT-positive foci (3.65 foci/cm2). At 30 weeks, the group treated with DENA and phenobarbital sodium exhibited more foci (23.6 foci/cm2) compared to the group that received only DENA (3.08 foci/cm2). The average size of foci in both of the DENA-treated groups was the same. The tumors in the group that received DENA plus phenobarbital sodium showed a greater incidence of GGT activity compared to the tumors in the DENA group. Under the conditions of this study the incidence of GGT-positive foci did not predict the incidence of hepatocellular carcinomas.     

Chemopreventive effects of the aromatase inhibitor vorozole (R 83842) in the methylnitrosourea-induced mammary cancer model

The chemopreventive activity of the highly specific nonsteroidal aromatase inhibitor, vorozole, was examined in the methylnitrosourea (MNU)-induced rat model of mammary carcinogenesis. Various doses of vorozole (0.08-1.25 mg/kg body wt/day) were administered daily (by gavage) to female Sprague-Dawley rats starting at 43 days of age. Seven days later, the rats were given a single i.v. dose of MNU (50 mg/kg body wt). Rats were continually treated with vorozole until the end of the experiment (120 days post-MNU). Vorozole caused a dose dependent inhibition of mammary cancer multiplicity. The highest dose of vorozole (1.25 mg/kg body wt/day) decreased cancer multiplicity by approximately 90%, and simultaneously decreased cancer incidence from 100 to 44%. The next two highest doses of vorozole (0.63 and 0.31 mg/kg body wt/day) inhibited MNU-induced mammary cancer multiplicity by 70-80%. Even the two lowest doses of vorozole (0.16 and 0.08 mg/kg body wt/ day) decreased cancer multiplicity -50%. Serum level determinations were performed on a variety of endpoints at either 4 or 24 h following the last dose of vorozole. Insulin-like growth factor (IGF)-1 levels were slightly, but significantly, increased by vorozole treatment. Vorozole induced striking increases in serum testosterone levels at 4 h at all the dose levels employed. Testosterone levels were significantly elevated over controls at 24 h in rats given the lower doses of vorozole (0.08-0.31 mg/kg body wt/day), but were significantly lower than in rats administered the higher doses of vorozole (0.63 or 1.25 mg/kg body wt/ day). This result presumably reflects the limited half- life of vorozole in rats. In a second series of experiments, the effects of limited duration of dosing with vorozole (2.5 mg/kg body wt/day) or intermittent dosing with vorozole were determined. Treatment of rats with vorozole for limited time periods, from 3 days post-MNU administration until 30 or 60 days post-MNU treatment, resulted in significant delays in the time to appearance of palpable cancers. However, these limited treatments did not greatly affect the overall incidence or multiplicity of mammary cancers when compared with the MNU controls at the end of the study (150 days post-MNU). Finally, the effects of intermittent dosing with vorozole (2.5 mg/kg body wt/day) were examined. Rats were administered cycles of vorozole daily for a period of 3 weeks followed by treatment with the vorozole vehicle for the next 3 weeks (total of four cycles). Although this intermittent treatment did inhibit the appearance of new tumors during each of the periods that vorozole was administered, it did not cause regression of palpable cancers.      

A subnecrogenic dose of diethylnitrosamine is able to initiate hepatocarcinogenesis in the rat when coupled with fasting/refeeding

Caloric restriction causes a generalized decrease in growthrate and has been repeatedly associated with an inhibitory effecton cancer development in several systems. In contrast, exposureto complete fasting followed by refeeding is as metabolic conditionassociated with increased cell turnover in different organs,including the liver. The present study examines whether suchcondition is able to sustain the induction of initiated hepatocytesfollowing a subnecrogenic dose of diethylnitrosamine (DENA).Male Fisher-344 rats were fasted for 4 days and 1 day afterrefeeding they were given a single dose of DENA (20 or 200 mg/kgbody wt, i.p.). Negative and positive control groups were fedad libitum and injected with 20 and 200k mg/kg of DENA, respectively.One week later all animals were subjected to the resistant hepatocytemodel for the selection of hepato-cyte nodules and they werekilled 2 weeks thereafter. Results indicated the presence ofgamma-glutamyltransfer-ase (GGT) positive foci and nodules (38± 7/cm²) in rats regularly fed and given 200 mg/kg ofDENA, while virtually no focal lesions (<1/cm²) were foundin the group receiving 20 mg/kg of DENA and fed throughtoutthe experiment. However, a significant number of GGT positivefoci/nodules (14 ± 7) also developed in rats exposedto fasting and given 20 mg/kg of DENA 24 h after refeeding.No evidence of helpatocellular necrosis was found in the lattergroup follwing DENA administration. No effect of fasting wasobserved when rats received 200 mg/kg of DENA. It is concludedthat fasting/refeeding provides conditions which are able tosustain initiation in rat liver by a subnecrogenic dose of acarcinogen. These findings are in contrast with the commonlyreported inhibitory effect of chronic food restriction on variousstages of carcinogenesis, including initiation.     

Promotion by polychlorinated biphenyls of enzyme-altered foci in rat liver

Aroclor 1254 is a complex mixture of polychlorinated biphenyls (PCB) that upon prolonged administration has been reported to produce hepatic tumors in mice and rats. The ability of Aroclor 1254 to promote enzyme-altered foci was determined in an initiation/promotion bioassay in rat liver. Initiation was accomplished in rats that received a 2/3 partial hepatectomy followed in 24 h by diethylnitrosamine (DENA). Aroclor 1254 was administered to each rat 7, 28 and 49 days after the DENA and some of the rats were killed 21 days after each dose of Aroclor. The liver of rats that received Aroclor 1254 on either day 7 or on day 7 and 28 contained an increased incidence of gamma-glutamyltranspeptidase (GGTase)-positive foci compared to partial hepatectomized and DENA treated rats given tricaprylin (the solvent for Aroclor 1254). Therefore, Aroclor 1254 was demonstrated to enhance the appearance of enzyme-altered foci after only a single oral dose.     

Study for tumor-initiating effect of acetaminophen in two-stage liver carcinogenesis of male F344 rats

The potential liver-tumor-initiating activity of acetaminophen(paracetamol, APAP) was investigated in male F344 rats. APAPwas administered by intragastric intubation either as 10 dosesof 1 g/kg body weight over 5 weeks or as a single dose of 0.5g/kg body weight 24 h after two-thirds partial hepatectomy.These initiating treatments were followed by administrationof 0.1% phenobarbital in the drinking water for 12 weeks asthe promoting regimen. Quantitative examination of placentalglutathione S-transferase-positive foci revealed no enhancingeffect of APAP on the induction of the foci consisting of morethan two positive cells with either initiating treatment. Ifsolitary positive hepatocytes were included in the effectivenumber of foci, 10 repeated doses of 1 g/kg APAP increased thenumber of foci while the validity of the single positive cellsis uncertain. This dose of APAP caused centrilobular necrosis.By ³²P-postlabeling, although the active metabolite of APAPformed DNA adducts when incubated with isolated DNA, no DNAadduct formation was detected in the liver of rats either fed0.1–1.5% APAP for 1 week or given 1 g/kg by gastric intubation.These results indicate that APAP possesses no tumor-initiatingactivity in the rat liver.     

Promotion mechanism of phenobarbital and partial hepatectomy in DENA hepatocarcinogenesis cell kinetics effect

Diethylnitrosamine (DENA, 10 mg kg-1 per day) was fed to rats for 2, 4 and 6 weeks. One week after the cessation of DENA, animals were submitted either to partial hepatectomy or to phenobarbital administration. Partial hepatectomy did not promote neoplastic transformation, except after a 6-week DENA treatment. A minimum of phenobarbital was required to reach a significant promoting effect in DENA carcinogenesis. A too-limited treatment was ineffectual but could be compensated for by prolonged DENA administration. The phenobarbital treatment became unnecessary when neoplastic nodules were present. Phenobarbital continuously given after the carcinogen administration promoted neoplastic transformation even after a subcarcinogenic DENA treatment (2 weeks). It accelerated the pathological evolution and increased the tumour incidence. In these conditions, phenobarbital increased the proliferation advantage of preneoplastic cells over normal cells. In the different experimental modalities, the promoting effect was associated with the induction of chronic cell proliferation, the inhibition of the rapid response to the 2/3 partial hepatectomy and the mitotic circadian rhythm normally present during liver regeneration. It is concluded that the promotion mechanism could consist in disturbing the mitotic control in order to maintain, for a long time, a chronic low level of cell proliferation permitting the selective growth of preneoplastic cells and their subsequent transformation.     

Effects of ethinyl estradiol and tamoxifen on liver DNA turnover and new synthesis and appearance of gamma glutamyl transpeptidase-positive foci in female rats

We investigated possible mechanisms associated with promotionof hepatocarcinogenesis by the synthetic estrogens mestranol(M) and ethinyl estradiol (EE). Our first objective was to determinewhether chronic EE treatment was associated with hepatotoxicityaccompanied by regenerative hyperplasia. Female Sprague-Dawley(SD) rats were partially bepatectomized and their liver DNAprelabeled with [³H]thymidine. Two weeks later the rats weretreated with EE at three doses or M (using timed-release tabletsimplanted s.c.) or with 0.05% phenobarbital (PB) in the diet.The rats were killed 6 weeks later and the amount of [³H]thymidineremaining in liver DNA determined. The results showed that noneof the promoters caused hepatotoxkty as detected by the lossof prelabeled DNA. EE but not M or PB caused a dramatic dose-dependentenlargement of the pituitary. Subsequent experiments were carriedout to define the dose-time response of liver DNA synthesisto treatment with EE, M and PB. Female SD rats were treatedwith these agents and [³H]-thymidine incorporation into DNAwas determined at various times thereafter. The results showedthat EE at 2.5 µig/day and PB (0.05%) caused a rapid increasein liver DNA synthesis which peaked between 24 and 72 h andremained elevated for at least the next 7 days. Dose-responseexperiments with EE- and M-treated rats demonstrated that 24h after beginning treament, significant increased in liver DNAsynthesis could be detected at an EE dose as low as 0.1 µig/day;DNA synthesis was also significantly increased by M. The anti-estrogentamoxifen (T), did not cause increased liver DNA synthesis.However, T at 15 µig/day did inhibit the induction ofDNA synthesis caused by EE and M at 2.5 µig/day but notby PB (0.05%). The effect of T on promotion by EE of the appearanceof gamma glutamyl trans-peptidase (GGT)-positive foci was determined.Female SD rats were initiated with a single i.p. dose of diethylnitrosamineat 20 mg/kg given 24 h after partial hepatectomy. One week laterthe rats were treated with EE (5 µig/day), T (15 or 50µg/day) or EE plus T at both doses using timed-releasetablets. The rats were killed after 4 months and the incidence,number and size of the GGT foci determined. The results revealedthat, as expected, EE enhanced the appearance of GGT foci. Unexpectedly,T alone at both doses also enhanced the appearance of the foci.However, in rats treated with EE plus T at either dose, thenumber of foci was similar to that in the non-promoted placebo-treatedcontrols. T also prevented the pituitary hyperplasia and hypertrophyassociated with EE treatment.     

Threshold dose dependence in phenobarbital promotion of rat hepatocarcinogenesis initiated by diethylnitrosamine.

The dose-response of phenobarbital (PB) promotion of hepatocarcinogenesis in rats was investigated. Male F344 rats were given 1, 4, 16, 75, 300 or 1200 p.p.m. PB solutions given ad libitum as their drinking water for 39 weeks following initiation with a single i.p. injection of diethylnitrosamine (DEN) (100 mg/kg). At week 40, the incidence of hepatic tumors was increased clearly in the DEN + PB groups given 300 p.p.m. PB or above, as compared to that in the group given DEN only. Linear dose-response curves for numbers and sizes of enzyme-altered hepatic foci (gamma-glutamyl-transpeptidase or placental glutathione S-transferase positive foci) were obtained in the dose range 16-1200 p.p.m. PB. The minimum promoting dose level of PB for enzyme-altered foci, estimated from dose-response curves by the Logit model, was calculated to be 15-23 p.p.m. Thus while dose dependence was demonstrated over a large range, a threshold was evident at low doses.     

Accelerated repopulation of mouse tongue epithelium during fractionated irradiations or following single doses

Mouse tongue mucosa was established as an animal model to study repopulation after large single doses or during continuous irradiation. A top-up irradiation technique was used employing priming doses or fractionated treatment to the whole snout (300 kV X-rays) followed by local test doses (25 kV X-rays) to elicit denudation in a confined field of the inferior tongue surface. Clearcut quantal dose-response curves of ulcer incidence were obtained to all protocols; animal morbidity, i.e. body weight loss was minimal. Repopulation following priming doses of 10 and 13 Gy started with a delay of at least 3 days and then progressed rapidly to nearly restore original tissue tolerance by day 11. During continuous fractionation over 1 to 3 weeks with 5 fractions/week and doses per fraction of 2.5, 3 and 3.5 Gy, repopulation was small in week one but subsequently increased to fully compensate the weekly dose at all dose levels. Additional measurements of cell density during a 4 weeks course of 5 x 3 Gy or 5 x 4 Gy per week showed only moderate depletion to 67% of the control figures. The fact that rapid repopulation is achieved at relatively moderate damage levels should be taken into account when the timing of a treatment split is considered.     

Effect of phenobarbital on the development of liver tumors in juvenile and adult mice

The effect of long-term exposure to phenobarbital (CAS: 50-06-6) subsequent to tumor initiation on the development of liver tumors in BALB/c and (C57BL/6 X C3H/Anf)F1 (B6C3F1) mice was determined. In male B6C3F1 mice that received either 15 or 45 ppm diethylnitrosamine [(DENA) CAS: 55-18-5] between 6 and 10 weeks of age, subsequent treatment with 500 ppm sodium phenobarbital in the drinking water resulted in the promotion of liver tumors. However, in male B6C3F1 mice initiated on day 15 of age with 25 mg DENA/kg, beginning long-term treatment of 500 ppm sodium phenobarbital at 4 weeks of age inhibited the development of liver tumors, whereas in male BALB/c mice initiated with 25 mg DENA/kg on day 15 of age, beginning the long-term treatment with 500 ppm sodium phenobarbital at 4 weeks of age promoted the development of liver tumors. Hence phenobarbital can either enhance or inhibit the formation of liver tumors, depending both on the mouse strain used and the animal's age at the start of exposure.     

Liver tumor promoting ability of corn oil gavage in B6C3F1 male mice

In chronic carcinogenic bioassays, chemicals being tested with low water solubility have been administered via corn oil gavage. The present study examined the effect of chronic corn oil gavage on hepatic tumor formation in the B6C3F1 male mouse. Mice were initiated with diethylnitrosamine (DENA) either at 15 days of age with a single i.p. injection (5 μg/gbw) (protocol 1) or at 4 weeks of age via the drinking water (15 mg/l) for a duration of 3 weeks (protocol 2). At weaning (protocol 1) or 8 weeks of age (protocol 2) initiated and untreated mice were administered either corn oil at a dose of 0.15 ml via gavage (once a day, 5 days/wk) or saline (0.15 ml via gavage, once a day 5 days/wk). All mice were killed at 28 weeks of age and hepatic lesions were quantitated. Only mice exposed to DENA demonstrated hepatic tumors. Mice treated with DENA (at 15 days of age) and corn oil gavage exhibited a significant decrease in the number of hepatic adenomas compared with DENA (at 15 days of age) only treated mice. No difference was noted in the number of hepatic adenomas between mice treated with DENA (at 4 wks of age) and corn oil gavage and mice exposed to DENA (at 4 wks of age) only.     

Validation in rats of two biomarkers of exposure to the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP): PhIP-DNA adducts and urinary PhIP

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adductsin white blood cells and tissues and unmetab-olized PhIP inurine were validated as biomarkers of exposure in male Fischer-344rats treated with daily PhIP doses ranging from 1 to 0.0001mg/kg. At the end of the 23 day treatment period all rats werekilled and their blood and 10 tissues were collected for isolationof DNA and analysis of PhIP-DNA adducts by ³²P-postlabelingand alkaline hydrolysis with GC/MS. PhIP-DNA adducts could bedetected only in animals receiving 1 or 0.1 mg/kg/day, withhighest adduct levels in the pancreas, heart and kidneys. Therewas a good correlation (r = 0.77, p < 0.005) between thetwo methods of analysis, with average adduct levels determinedby ³²P-postlabeling     

Multi-day fractionated administration schedule for paclitaxel

PURPOSE: To establish the feasibility of fractionating paclitaxel administrationby utilizing daily one-hour infusions for three, four or fivedays with dose escalating to determine the patterns of hematologicand non hematologic toxicities. PATIENTS AND METHODS: Forty patients received 87 courses of daily fractionated paclitaxelfor three, four or five days; cycles were repeated every 21days. Six patients received concomitant daily cisplatin. Themedian number of cycles delivered per patient was two with arange of one to six. RESULTS: Cumulative doses per cycle ranged from 120 mg/m² to 250 mg/m²with 25% of the cycles delivering 200 mg/m² or more. Ten cycles(11.5%) were associated with dose limiting neutropenia (grade3 [7 cycles]; grade 4 [3 cycles]). No hypersensitivity reactionswere observed and no patient required cytokine support. No patientrequired hos-pitalization. CONCLUSION: Administering paclitaxel on a daily fractioned schedule in anambulatory setting is logistically feasible; does not requirepremedication; is associated with a toxicity pattern similarto single day schedules (e.g. 24-hour or three-hour infusion);is capable of delivering a higher dose per cycle than published96- or 120-hour infusion schedules; and could possibly be escalatedto doses higher than 250 mg/m² in carefully selected patients.The optimal dose rate for five-day multifrac-tionated administrationof paclitaxel is 40 to 50 mg/m²/d or a cumulative cycle doseof 200 to 250 mg/m² and does not require cytokine usage. Addingcisplatin on a fractionated daily schedule may accentuate theneurotoxicity associated with both agents. A prospective comparisonof four-day fractionated vs. four-day continuous infusionalpaclitaxel has been proposed as a randomized study to determineclinical differences in response, dose intensity and toxicity. paclitaxel, schedule-dependent administration     

The Carcinogenic Agent Diethylnitrosamine Induces Early Oxidative Stress,Inflammation and Proliferation in Rat Liver,Stomach and Colon: Protective Effect of Ginger Extract

Background: Diethylnitrosamine (DENA), a well-known dietary carcinogen, related to cancer initiation of variousorgans. The present study investigated the deleterious mechanisms involved in the early destructive changes of DENAin different organs namely, liver, stomach and colon and the potential protective effect of GE against these mechanisms.Methods: Adult male albino rats were assigned into four groups. A normal control group received the vehicle, anothergroup was injected with a single necrogenic dose of DENA (200 mg/kg, i.p) on day 21. Two groups received oral GE(108 or 216 mg/kg) daily for 28 days. Sera, liver, stomach and colon were obtained 7 days after DENA injection. Serumaspartate transaminase and alanine transaminase were detected as well as reduced glutathione (GSH), malondialdehyde,nitric oxide metabolites, interleukin 1β, tumor necrosis factor (TNF-α), alpha-fetoprotein (AFP) and nuclear factorerythroid2-related factor2 (Nrf2) in liver, stomach and colon. Histopathological studies and immunohistochemicalexamination of cyclooxygenase-2 (COX2) were conducted. Results: DENA induced elevation in liver function enzymeswith significant increase in oxidation and inflammation biomarkers and AFP while decreased levels of Nrf2 in liver,stomach and colon were detected. Histologically, DENA showed degenerative changes in hepatocytes and inflammatoryfoci. Inflammatory foci displayed increased expression of COX2 in immunohistochemical staining. GE-pretreatmentimproved liver function and restored normal GSH with significant mitigation of oxidative stress and inflammatorybiomarkers compared to DENA-treated group. AFP was reduced by GE in both doses, while Nrf2 increased significantly.Histology and immunostaining of hepatic COX-2 were remarkably improved in GE-treated groups in a dose dependentmanner. Conclusion: GE exerted a potential anti-proliferative activity against DENA in liver, stomach and colon viaNrf2 activation, whilst suppression of oxidation and inflammation.     

Dose-response studies of MeIQx in rat liver and liver DNA at low doses

2-Amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx) is aheterocyclic amine mutagen found in cooked meats and is carcinogenicin mice and rats at high doses (mg/kg body wt). Humans, however,are exposed to low amounts (p.p.b.) in the diet, and the effectscaused by exposure to human equivalent doses of MeIQx have beendifficult to determine accurately. We report on the effect ofMeIQx exposure on liver bioavailability, hepatic DNA bindingand MeIQx persistence in both liver tissue and liver DNA afteracute (24 h), and subchronic (7 day and 42 day) exposures inmale Sprague-Dawley rats. Male Sprague-Dawley rats were administered[2-¹⁴C] MeIQx either by gavage or in the diet for 1, 7 or 42days (1x10^-6mg/kg day up to 3.4x10^-2 mg/kg day dose) and the[2-¹⁴C]MeIQx was measured by accelerator mass spectrometry (AMS).Assessment of the kinetics of hepatic MeIQx DNA adduct formationover 42 days (1.1x10^-4 mg [2-¹⁴C]MeIQx kg daily dose) showsthat steady-state [2-¹⁴C]MeIQx tissue concentrations of 138± 15 pg/g liver and DNA adduct levels of 113 ±10 ag adduct/µg DNA were reached at 14–28 days and28 days respectively. The relationship between administereddose and either hepatic MeIQx DNA adduct levels or MeIQx tissuelevels are linear for the 24 h, 7 day and 42 day exposures.Furthermore, MeIQx adducts persist for at least 14 days afterexposure ceases. These data suggest that bloavailability andDNA adduction by MeIQx increase linearly with increasing dosefor both acute and subchronic exposures. These data also showthat MeIQx DNA adducts are useful in predicting daily exposureand support a linear extrapolation in the risk assessment ofMeIQx. However, the quantitative relationship between DNA adductsand tumor formation will also depend on the specific tissueand the subsequent steps needed for tumor progression.     

Boron Neutron Capture Therapy: Effects of Split Dose and Overall Treatment Time

New clinical protocols are being developed that will entail the administration of considerably higher doses of the boron delivery agent boronophenylalanine (BPA) than those in current clinical use. Fractionation (2 or 4 fractions) of BPA mediated boron neutron capture therapy (BNCT) is also under consideration at some clinical centres. Given the considerably higher infusion volumes that will be entailed in the delivery of BPA in the new high dosage protocols, there will be a requirement to extend the gap between fractions to 2 or more days. In order to assess the effects of a 2 fraction protocol on the therapeutic efficacy of BPA mediated BNCT, a series of split dose irradiations (two equal fractions) were undertaken using the rat intracranially implanted 9L gliosarcoma model. A single dose exposure to BPA mediated BNCT of 3.0Gy resulted in long term survival levels of 50%. Survival levels increased to 71% and 77% with a 3 and 5 day gap between dose fractions (two equal fractions), respectively, using the same total dose. A further increase in the time interval between dose fractions to 7 days resulted in a reduction in survival to 36%. However, there was no significant difference between the single dose and the 3, 5 and 7 day survival data (P > 0.1) The difference between the 5 and 7 day split dose survival data was of border-line significance (P = 0.05). It is anticipated that mucositis, could become a potential problem in future BNCT clinical protocols involving higher doses, larger field sizes or multiple fields. The potential sparing of the oral mucosa, due to repopulation during the interval between the two fractions, was investigated using a series of split dose BPA mediated BNC irradiations. The ventral surface of the rat tongue was utilised as a model for oral mucosa. The ED₅₀ (50% incidence) values for the ulceration end point were 3.0±0.1,3.2±0.1,3.0±0.1 and 3.6±0.1Gy, for 3, 5, 7 and 9 day splits between doses, respectively. It is evident from this data that there were no significant changes in the ED₅₀ (p < 0.001) until the 9 day dose split, when the ED₅₀ value was 20% higher than the ED₅₀ value after a 7 day split. It was concluded that the two fraction BNCT protocol, with dose splits of up to 5 days, did not diminish the therapeutic response of the rat 9L gliosarcoma, when compared with a single dose BNCT protocol. Tolerance of the oral mucosa to BNC irradiation was not increased until there was a 9 day gap between fractions. However, the beneficial effects of dose sparing at this time interval between doses, would probably be counteracted by a reduction in the therapeutic effectiveness of the BNCT modality, due to repopulation of tumour clonogens between doses.     

Combination Phase I/II Study of Irinotecan Hydrochloride (CPT-11) and Carboplatin in Relapsed or Refractory Non-Hodgkin's Lymphoma

Irinotecan hydrochloride (CPT-11) is a new derivative of camptothecinwhich inhibits topoisomerase I. Phase II studies have demonstratedthat CPT-11 is active against a broad spectrum of neoplasmsincluding intractable non-Hodgkin's lymphoma. An early phaseII study in lymphoma suggested that a schedule of daily infusionsof 40 mg/m²/day for three or five consecutive days is more effectivethan a single infusion of 200 mg/m² every three to four weeks.Carboplatin is also an active agent against lymphoma, and preclinicalstudies have shown that CPT-11 and its active metabolite havea synergistic effect with platinum compounds. To evaluate themaximal tolerated dose (MTD) and the therapeutic efficacy ofCPT-11 in combination with carboplatin in relapsed or refractorynon-Hodgkin's lymphoma, we conducted a combination phase I/IIstudy. The starting dose of CPT-11 was 20 mg/m²/day (days 1through 3 and 8 through 10), and dose escalations of 5 mg/m²/dayincrements were planned, with a fixed dose of carboplatin (300mg/m², day 1). Six of the eight patients receiving both agentsat the starting dose level developed critical toxicities suchas grade 4 hematologic (neutropenia 6/8, thrombocytopenia 1/8)and grade 3 non-hematologic toxicities (diarrhea 2/8, transaminaseelevation 1/8). Further dose escalation of CPT-11 was halted,and the starting doses were judged to be the MTDs. The responserate (25%, 2/8) to the combination of the MTDs was not superiorto that of CPT-11 alone in a previous phase II study (38%, 26/69),and the MTD of CPT-11 in combination with carboplatin was lessthan half the single-agent dose. We conclude that carboplatinis not recommendable for combination with CPT-11 in lymphomapatients. Other suitable agents for such a combination shouldbe sought.     

Preclinical biochemical pharmacology and toxicology of piritrexim, a lipophilic inhibitor of dihydrofolate reductase

Piritrexim (PTX), 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidin e, formerly called BW 301U, is a potent small-molecule inhibitor of dihydrofolate reductase (DHFR) that enters cells rapidly by passive diffusion and thus does not depend upon the transport-mediated uptake that can limit cell entry of methotrexate (MTX). PTX is as active as MTX in inhibiting DHFR and mammalian cell growth. In vivo, PTX is active against Walker 256, L1210, P388, Sarcoma 180, and Ehrlich ascites tumors. After iv administration of [14C]PTX to rats, the elimination profile of intact drug from plasma was first order with a half-life (t1/2) of 38 minutes. PTX penetrates extensively into tissues and its tissue:plasma concentration ratios are generally 10-fold higher than those reported for MTX. When administered systemically, PTX inhibits the DHFR-dependent conversion of sepiapterin or 7,8-dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4), demonstrating that PTX enters brain at pharmacologically relevant concentrations. Pharmacokinetic studies in the dog indicated a mean plasma t1/2 (after iv dose) of 2.15 hours, total body clearance of 0.625 liters/hr/kg and steady-state volume of distribution of 1.82 liters/kg; the absolute bioavailability was 0.64. Toxicologic studies were conducted in rats and dogs that received daily doses for 1, 5, or 90 days. In dogs, oral doses of 480 (single dose), 25 (5 daily doses), and 2.5 mg/kg (90 daily doses) were lethal, whereas 240 (single dose), 2.5 (5 daily doses), and 0.5 mg/kg (90 daily doses) produced reversible alterations in clinical toxicity and histopathologic parameters. The lethal toxicity of PTX in dogs given 25 mg/kg/day for 5 days is prevented by oral calcium leucovorin rescue with either 0.75 or 3.0 mg/kg every hour for 4 hours on any of the 5 treatment days. The general pharmacologic profile indicates that PTX should be free of CNS, cardiovascular, and respiratory side effects at clinically useful doses.