Mutual kinetic interaction between 5-fluorouracil and bromodeoxyuridine or iododeoxyuridine in dogs.

The pharmacokinetics of 5-fluorouracil (FUra) were studied alone and in the presence of either bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd) in five mixed breed hounds. FUra was administered intravenously over 1 min as a 5 mg/kg dose in all three treatments. BrdUrd or IdUrd was infused intravenously at 2.5 mg/hr.kg x 6 hr and FUra was given 3 hr into the 6-hr infusion of thymidine analog. Serial blood samples were obtained for 3 hr prior to and after FUra dosing. Plasma concentrations of FUra, BrdUrd and bromouracil (BrUra), and IdUrd and iodouracil (IUra) were measured by reversed-phase HPLC. Concomitant administration of BrdUrd or IdUrd resulted in substantial changes in the kinetics of FUra. In particular, the total plasma clearance of FUra was decreased and area under the plasma concentration-time curve was increased. There were also significant changes in the volume of distribution steady-state, and in the mean residence time and half-life of FUra. The data show that FUra had little or no effect on the disposition of BrdUrd or IdUrd. In contrast, FUra had a significant effect on the disposition of BrUra and IUra going to form catabolic products. The observed pharmacokinetic interaction in dogs is probably due to a competition between BrUra (or IUra) and FUra for the rate-limiting catabolic enzyme, dihydropyrimidine dehydrogenase. This kinetic interaction was mutual and of a smaller magnitude for FUra + BrdUrd than for FUra + IdUrd.

Biochemical modulation of 5-bromo-2'-deoxyuridine and 5-iodo-2'-deoxyuridine incorporation into DNA in VX2 tumor-bearing rabbits.

The thymidine analogues 5-bromo-2'-deoxyuridine (Brd-Urd) and 5-iodo-2'-deoxyuridine (IdUrd) compete with thymidine for incorporation into the DNA of replicating cells. This incorporation results in radiosensitizing effects which are directly related to the degree of analogue substitution. In vitro and in vivo evidence suggests that preadministration or coadministration of the thymidylate synthetase inhibitors fluorouracil and 5-fluoro-2'-deoxyuridine (FdUrd) can modulate analogue incorporation into DNA. We have evaluated in the rabbit VX2 tumor model the effects of thymidylate synthetase inhibitor (fluorouracil or FdUrd) coadministration (as 24-hour, intravenous infusions) on the incorporation of BrdUrd or IdUrd into the DNA of relevant normal tissues (bone marrow, gut mucosa) and intrahepatic VX2 tumor. Tissues were harvested and processed for gas chromatography-mass spectrometry analysis of the thymine, 5-bromouracil, and 5-iodouracil contents in hydrolyzed DNA. Coadministration of FdUrd resulted in statistically significant (P less than .01) enhancement of IdUrd incorporation into the DNA of intrahepatic VX2 tumor and normal (bone marrow and duodenal mucosa) rabbit tissues. Coadministered fluorouracil, on the other hand, significantly enhanced IdUrd incorporation only into DNA of intrahepatic VX2 tumor. Statistically significant enhancement of BrdUrd incorporation was achieved only with FdUrd coadministration and then only into the DNA of intrahepatic VX2 tumor. The percent of thymine replaced by analogue (I) is related to the steady-state arterial plasma drug concentration (C) by the Michaelis-Menten equation: I = I(MAX.) C/(C50 + C). The primary effect of FdUrd coadministration on BrdUrd incorporation into VX2 tumor DNA was a reduction of the C50 parameter (plasma BrdUrd concentration eliciting I = I(MAX)/2) from 8.17 microM to 1.78 microM. On the other hand, the I(MAX) parameter (I as C approaches infinity) was only slightly affected (29.7% to 25.2%). Thus, the degree to which the modulator enhanced analogue incorporation varied inversely with the analogue's steady-state plasma concentration. These results, which describe potential tissue specificity of modulator efficacy and characterize the effects of thymidylate synthetase inhibitor modulation on thymidine analogue incorporation pharmacodynamics, should provide guidance as to dose scheduling of BrdUrd and IdUrd in clinical trials for improved tumor specificity of uptake.

Microscopic analysis of arterial microsphere distribution in rabbit liver and hepatic VX2 tumor.

Microspheres conjugated to radioisotopes and chemotherapeutic agents are playing an important investigative and clinical role in the management of metastatic neoplasms. The purpose of our investigation was to histologically assess the basis for regional intra-arterial microsphere therapy, by comparing the spatial distribution of microspheres in the tumor and liver of experimental models of hepatic metastases. Three New Zealand white rabbits with hepatic VX2 tumor implants were arterially injected with hepatic doses of either 15 or 30 million blue-dyed, polystyrene microspheres (27 microns-diameter). Microscopic examination of random liver and tumor samples revealed that 6-12 times as many microspheres were embolized within tumor than in normal liver (p less than 0.002). The majority of microspheres aggregated into clusters of various size within liver and tumor vasculature, though analysis of cluster sizes illustrated an exponentially skewed distribution toward isolated microspheres. Approximately eight times as many clusters were observed in tumor than in liver (p less than 0.008). Finally, a morphometric analysis was used to quantitate the minimal distances separating microsphere clusters, the intercluster distance (ICD). Analysis of over three thousand intercluster measurements revealed a median ICD approximately five times lower in tumor than in liver (p less than 1 x 10(-8)). This microquantitative analysis provides a fundamental description of how regional intra-arterial microsphere therapy allows the targeted delivery of microspheres to neoplastic tissue, to potentially improve the therapeutic index in the treatment of hepatic metastases.

5-Bromo-2'-deoxyuridine incorporation into DNA in hepatic VX2 tumor-bearing rabbits.

The halogenated pyrimidine 5-bromo-2'-deoxyuridine (BrdUrd) possesses both radiosensitizing and antimetabolite effects through its incorporation as a thymidine analog in replicating DNA. To evaluate the regional advantage for treatment of hepatic malignancy by hepatic arterial infusion, BrdUrd was infused into either the hepatic artery (HA) or a central vein (iv) in 26 rabbits with intrahepatic VX2 tumor. After a 24-hr constant rate infusion of 10, 20, or 40 mg/kg/24 hr, the percentage BrdUrd incorporation into the DNA of bone marrow, duodenal mucosa, liver, and hepatic VX2 tumor was measured by gas chromatography/mass spectrometry methods. VX2 tumor BrdUrd incorporation was greater by HA than by iv routes (P less than 0.001). At doses of 10 and 20 mg/kg/day, HA to iv BrdUrd incorporation ratios for VX2 tumor significantly exceeded those for bone marrow and duodenum (P less than 0.05). At appropriate dose rates, hepatic arterial administration of BrdUrd provides a regional advantage for DNA BrdUrd incorporation in the rabbit intrahepatic VX2 tumor.

Steady-state arterial and hepatic venous plasma concentrations of 5-bromo-2'-deoxyuridine and 5-iodo-2'-deoxyuridine in animals--drugs which are subject to both splanchnic and extra-splanchnic elimination.

We have previously shown that 5-fluorouracil (FUra) in humans obeys Michaelis-Menten elimination kinetics. In this article we show that the related bromine and iodine-containing analogs in animals obey similar kinetics. Steady-state arterial (CssA) and hepatic venous plasma (CssV) concentrations of 5-bromo-2'-deoxyuridine (BrdUrd) are reported for 7 rabbits given 5 different infusion rates of BrdUrd and 5 dogs given 4 or 5 different infusion rates of BrdUrd. Steady-state arterial and hepatic venous plasma concentrations of 5-iodo-2'-deoxyuridine (IdUrd) are reported for 5 rabbits and 2 dogs given 5 different infusion rates of IdUrd. Each set of data could be fitted by a nonlinear least squares method to the equation: (equation; see text) where Vm/Q is the maximum difference, (CssA) - (CssV), and Km is the Michaelis constant. The estimated parameter Vm/Q and Km are compared for the two drugs and different species and also with the same parameters derived in the same manner from previously published data on fluorouracil in 8 cancer patients. The infusion rate needed to saturate the splanchnic elimination system (Rs in mumol/kg/min) was also estimated. For BrdUrd the mean value of Rs in the rabbit, namely 1.23, and in the dog, namely 1.25 mumol/kg/min are essentially the same.

Effects of hepatic arterial yttrium-90 microsphere administration alone and combined with regional bromodeoxyuridine infusion in dogs.

Brachytherapy by embolization with radiotherapeutic microspheres following intraarterial infusion of a radiosensitizer represents an attempt to combine several selective modalities into a more potent, focused attack on regionally confined tumors. In pursuit of this goal, we examined the ability of foxhounds with surgically implanted hepatic arterial (HA) delivery systems to tolerate a clinically relevant dosage of HA yttrium-90 (Y-90) by microsphere administration either alone or preceded by a 28-day constant HA infusion of either 5-bromo-2'-deoxyuridine (BUDR) or a control solution. Five dogs received BUDR (10 mg/kg/day) and five a control buffer infusion for 28 days immediately prior to the administration of Y-90-coated 15 micron resin microspheres (equivalent of 5000 rads to the entire liver) to each dog on day 31. In all animals, blood counts, bilirubin, amylase, appetite, weight, and behavior remained unchanged. Dogs receiving the microspheres after buffer infusion alone exhibited no hepatic enzyme alanine aminotransferase or alkaline phosphatase elevation. Alanine aminotransferase and alkaline phosphatase levels both rose during the third week of BUDR infusion, and while subsequent microsphere administration further increased enzyme levels, these levels had largely normalized by necropsy on day 82. At necropsy, the type and degree of hepatic toxicity among the animals receiving radioactive microspheres was comparable to that previously described in patients receiving external beam hepatic irradiation at conventional doses (2000-3000 rads). Also noted was a radiation-induced cholecystitis (due in large part to the gallbladder's total reliance on the hepatic artery for blood supply). One resin microsphere dog exhibited a small quantity of microspheres in the lungs causing focal radiation-induced granulomas suggesting the need to assess shunting of microspheres through the liver in clinical studies. Thus, HA Y-90 microspheres with BUDR can produce acceptable, nonlethal, and tolerable toxicities in this dog model suggesting that clinical studies of this combination are not likely to be contraindicated by synergistic toxicity. Although HA BUDR did not contribute significantly to the toxicity of the Y-90 microspheres, HA BUDR by itself administered uninterrupted for 4 weeks may, like HA FUDR (clinically), cause chemical hepatitis/cholangitis. The unexpected fragmentation of the resin spheres (albeit without myelosuppression) has led us to begin studies with a recently developed nondisruptible glass microsphere (ThereSphere) in which the Y-90 is part of the glass matrix and cannot leach.(ABSTRACT TRUNCATED AT 400 WORDS)

A dog model using an implanted system for protracted hepatic arterial chemotherapy.

A model for hepatic arterial chemotherapy studies using large dogs and an implantable infusion pump has been developed. Using this technique near complete perfusion (greater than 90%) of the liver can be achieved in vivo as determined by hepatic arterial perfusion scintigraphy with technitium 99m macroaggregated albumin. The system is reliable and has been in use for a total of 1353 days (mean of 104 days, range 52-239) in 13 dogs. Pump implantation causes no apparent acute liver damage based on pre- and post-operative alkaline phosphatase and serum glutamic-pyruvic transaminase determinations and does not affect the general mobility or behavior of the animals. Careful placement of the catheter and attention to the physicochemical properties of the solutions loaded are factors contributing to the success of the model. The model permits comprehensive preclinical pharmacokinetic and toxicologic studies of new or preexistent chemotherapeutic agents in the same device that will be used for later administration in human subjects. By providing the means to examine and develop new treatment modalities, it enables the design of even more potent cytotoxic therapy directed into the tumor vascular bed.

Effects of intraventricular methotrexate on cerebrospinal fluid monoamine metabolites in rhesus monkeys.

Previous work suggested that methotrexate (MTX) may cause encephalopathy by inhibiting biosynthetic pathways for dopamine and serotonin in the brain. We examined this issue by measuring the neurotransmitter metabolites homovanillic acid and 5-hydroxyindoleacetic acid in the lumbar CSF of rhesus monkeys receiving continuous intracerebroventricular infusions of MTX or dichloromethotrexate. Infusion of the lowest dose (0.05 mg/day) produced a large (300%) rise in homovanillic acid levels and a modest elevation in 5-hydroxy-indoleacetic acid. During higher dose infusion, which was associated with clinical encephalopathy, the biogenic amine metabolites fell from their previous elevated levels. In one encephalopathic monkey, an injection of 1 mg of leucovorin produced a marked elevation in CSF monoamine metabolites within 1 hour and rapid clinical recovery. In contrast, leucovorin produced no change in monoamine metabolites in control animals. The data suggest that MTX may block egress of monoamine metabolites from CSF at the lower doses and suppress neurotransmitter turnover at toxic doses which cause encephalopathy. Serial measurements of CSF monoamine metabolites deserve further investigation as biochemical markers for toxic effects of MTX on neuronal metabolism in the CNS.

Zinc inhibition of calmodulin: a proposed molecular mechanism of zinc action on cellular functions.

Calcium stimulates, and zinc inhibits, a wide variety of cell types. In the erythrocyte, we have found calcium and zinc to have antagonist actions in a variety of systems. An important mechanism for calcium effects on cells is activation of calmodulin. Calmodulin is a small ubiquitous protein which, when activated by calcium, has a large array of cellular regulatory functions. We now report that calmodulin function is inhibited by low concentrations of zinc. Zinc inhibition of calmodulin provides a rational molecular mechanism for the diverse cellular inhibitory effects of zinc, as well as for zinc's antagonism of calcium effects.