Absorption, tissue distribution, metabolism and elimination of taurine given orally to rats.

Three biodisposition studies with taurine were performed in male and female adult rats at dosages of 30 and 300 mg/kg. A single oral dose of (14)C-taurine was rapidly absorbed, distributed to tissues and excreted unchanged in urine. Elimination of radioactivity from intracellular pools was slow. Pre-treatment of animals for 14 days with unlabelled taurine did not significantly affect the fate of (14)C-taurine. At the higher dose there was more extensive excretion combined with a lower percentage of the dose in the carcass, indicating the possibility of saturation of the tubular reabsorption mechanism for taurine. Daily administration of unlabelled taurine for 14 days did not result in an increase in total taurine in the brain. The data indicate that exogenous taurine rapidly equilibrates with endogenous body pools and that any excess is rapidly eliminated by the kidneys.

Rabbit serum paraoxonase 3 (PON3) is a high density lipoprotein-associated lactonase and protects low density lipoprotein against oxidation.

The paraoxonase gene family contains at least three members: PON1, PON2, and PON3. The physiological roles of the corresponding gene products are still uncertain. Until recently, only the serum paraoxonase/arylesterase (PON1) had been purified and characterized. Here we report the purification, cloning, and characterization of rabbit serum PON3. PON3 is a 40-kDa protein associated with the high density lipoprotein fraction of serum. In contrast to PON1, PON3 has very limited arylesterase and no paraoxonase activities but rapidly hydrolyzes lactones such as statin prodrugs (e.g. lovastatin). These differences facilitated the complete separation of PON3 from PON1 during purification. PON3 hydrolyzes aromatic lactones and 5- or 6-member ring lactones with aliphatic substituents but not simple lactones or those with polar substituents. We cloned PON3 from total rabbit liver RNA and expressed it in mammalian 293T/17 cells. The recombinant PON3 has the same apparent molecular mass and substrate specificity as the enzyme purified from serum. Rabbit serum PON3 is more efficient than rabbit PON1 in protecting low density lipoprotein from copper-induced oxidation. This is the first report that identifies a second PON enzyme in mammalian serum and the first to describe an enzymatic activity for PON3.

Sensitization of 1,3-bis(2-chloroethyl)-1-nitrosourea and cisplatin cytotoxicity by 5-bromo-2'-deoxyuridine in human glioma.

5-Bromo-2'-deoxyuridine (BrdUrd) was found to increase the cytotoxicity induced by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and cisplatin in human glioma cells. At a fixed concentration of BrdUrd and BCNU, the greatest cell loss was observed in exponentially growing cells. As cells approached plateau growth, cytotoxicity was reduced as indicated by greater cell viability. Under varying growth conditions the percentage of thymine replacement by bromouracil in DNA, as determined by gas chromatography/mass spectrometry analysis, declined as cultures approached maximum density. These data indicate BrdUrd must be incorporated into DNA for the enhanced effect to be observed. In exponentially growing cells, sensitization was dependent upon both the concentration of BrdUrd and alkylating agent. Using regression analysis (at 95% CL), a relationship between the level of bromouracil in DNA and the extent of enhanced cytotoxicity was observed at two concentrations of BCNU (r2 = 0.99, 0.96). Although it is known that bifunctional alkylating agents exert cytotoxicity by forming cross-links between cDNA strands, increased cross-link formation was not observed in BrdUrd substituted DNA as determined by alkaline elution. The data suggest that DNA damage induced by halogenated pyrimidines may not involve interstrand cross-links and that these agents may be useful in the treatment of glioma in combination with alkylating agents.

Preferential cytotoxicity of cells transduced with cytosine deaminase compared to bystander cells after treatment with 5-flucytosine.

In vitro experiments from our laboratory and others have suggested that herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) gene therapy depends on gap junctional intercellular communication (GJIC) to produce a strong bystander effect. Furthermore, we have shown that cells transduced with HSV-TK can be protected from GCV-mediated toxicity by GJIC with bystander cells. We wished to determine whether GJIC affected either the bystander or protective effect of the cytosine deaminase (CD)/5-flucytosine (5-FC) gene therapy approach, in which CD converts 5-FC to 5-fluorouracil (5-FU). To test this, we designed a coculture system using communication-competent WB rat hepatocytes and a noncommunicating subclone (aB1), which were transduced with CD and with antibiotic resistance genes so that we could independently determine the survival of the CD-containing or bystander cells. We found that, compared to the HSV-TK/GCV strategy, bystander killing resulting from treatment with CD/5-FC does not depend on GJIC. However, our most striking finding was that both communication-competent and -incompetent CD-transduced cells were preferentially killed, by a factor of up to 500, compared to bystander cells. The lesser dependence of the CD/5-FC system on GJIC, combined with the finding that most cancer cells lack the capacity for GJIC, suggest that the CD/5-FC system may be superior to the HSV-TK/GCV approach for gene therapy. However, the premature death of the CD-transduced 5-FU "factory" suggests that other strategies may be necessary to produce a sufficient quantity of 5-FU for a duration long enough to produce permanent tumor regression.

Regional pharmacokinetics of 5-fluorouracil in dogs: role of the liver, gastrointestinal tract, and lungs.

The purpose of this study was to determine the presence and extent of pulmonary elimination for 5-fluorouracil (FUra). A secondary aim was to characterize the relative importance of the liver, gastrointestinal tract, splanchnic region, and lungs toward the overall elimination of FUra. A total of 10 mixed-breed male and female dogs were used in these acute studies in which FUra was administered through a cephalic vein. Six dogs were studied at sequentially escalated dose rates of 0.125, 0.250, 0.500, 0.750, and 1.00 micromol/min/kg (8-fold range); four dogs were studied at sequentially escalated dose rates of 0.0625, 0.250, 0.750, 1.50, and 2.00 micromol/min/kg (32-fold range). Each infusion lasted 2 h, at which time steady-state plasma concentrations were obtained (i.e., portal vein, carotid artery, hepatic vein, and pulmonary artery), perfusion rates were measured (hepatic artery, portal vein, and cardiac output), and pharmacokinetic parameters were directly assessed. Pulmonary elimination of FUra was conclusively demonstrated. Although only 17% of the drug was extracted by the lungs at the lowest dose rate, pulmonary clearance (16.0 ml/min/kg) was on the order of splanchnic clearance (13.5 ml/min/kg), or larger. As the dose rate increased, pulmonary clearance was more easily saturated than splanchnic clearance. Thus, it appears that at increasing dose rates, the splanchnic region becomes a more significant pathway, whereas the lungs have a reduced role in the overall elimination of FUra.

Steady-state pharmacokinetics of delavirdine in HIV-positive patients: effect on erythromycin breath test.

OBJECTIVE: The steady-state kinetics of delavirdine and desisopropyldelavirdine were evaluated in human immunodeficiency virus-positive patients after escalating oral doses and after repeated oral administrations at the same dose level. STUDY DESIGN: Patients (n = 8 males) were given escalating oral doses of delavirdine mesylate, in a sequential fashion, over 14 days for phases 1 (200 mg every 8 hours), 2 (300 mg every 8 hours), and 3 (400 mg every 8 hours). Control patients (n = 4 males) were given 300 mg oral doses of drug every 8 hours for all three phases. Hepatic CYP3A activity was evaluated with the erythromycin breath test (ERMBT). RESULTS: In the escalating-dose group, delavirdine displayed nonlinear kinetics as indicated by the decreasing oral clearance, maximum steady-state plasma concentration/minimum steady-state plasma concentration ratio, and log-linear terminal rate constant, as well as by increasing half-life at higher doses; the ratio of desisopropyl-delavirdine formation clearance to elimination clearance was also reduced. In the control group, the kinetics of delavirdine and desisopropyl-delavirdine were unchanged. Plasma protein binding was linear for delavirdine in the escalating-dose and control groups; on average, the fraction unbound was about 2.3% and 2.0%, respectively. Hepatic CYP3A activity was markedly reduced after short- and long-term exposure to all doses of delavirdine mesylate. Delavirdine could maximally inhibit 70% to 75% of predose ERMBT values, with an IC50 of about 0.9 mumol/L. CONCLUSION: Delavirdine is a potent and reversible inhibitor of hepatic CYP3A; it is also a substrate for this CYP450 isoform. It is likely that delavirdine will exhibit drug-drug interactions when coadministered with other CYP3A substrates.

Regional pharmacokinetics of 5-bromo-2'-deoxyuridine and 5-fluorouracil in dogs: hepatic arterial versus portal venous infusions.

The purpose of this study was to determine the effect of route of hepatic administration of drug on the regional pharmacokinetics and systemic exposure of 5-fluorouracil (FUra) and 5-bromo-2-deoxyuridine (BrdUrd). A total of 13 mixed-breed male and female dogs were used in these acute studies. Each dog was administered hepatic arterial and portal venous infusions of a single drug, in a cross-over fashion, at two dose rates for a total of four sequential infusions. BrdUrd was studied at 0.250 and 0.500 micromol/min/kg, and FUra was studied at 0.125 and 0.500 micromol/min/ kg. Each infusion lasted 2 h, at which time steady-state plasma concentrations were obtained (ie., gastroduodenal artery, portal vein, hepatic vein, and femoral artery), perfusion rates in hepatic artery and portal vein were measured, and hepatic extraction (as opposed to extraction across the splanchnic region) was directly assessed. BrdUrd and FUra were found to be highly extracted across the liver (E(H) > or = 0.65) at the regional dose rates studied, resulting in low values for the fraction of drug escaping presystemic hepatic elimination (F(H) < or = 0.35). In addition, the regional kinetics (ie., hepatic extraction, fraction escaping first-pass elimination in the liver, and hepatic clearance) and systemic exposure (i.e., CFA) of FUra and BrdUrd were not significantly different following hepatic arterial versus portal venous infusions of drug. Thus, it appears that regional chemotherapy may be applied to halogenated pyrimidines following hepatic arterial, portal venous, and alternating regional dosing routes with no additional risk of systemic toxicity.

Effect of irradiation on bromodeoxyuridine incorporation in human colon cancer xenografts.

PURPOSE: Although we have characterized the incorporation of the thymidine analog bromodeoxyuridine (BrdUrd) into human colon cancer xenografts under a wide variety of conditions, little is known about the effect of radiation on subsequent incorporation. Because clinical protocols include, as one component, BrdUrd administration after radiation, it was important to confirm that irradiation did not prevent subsequent BrdUrd incorporation. Therefore, we studied the effect of irradiation on BrdUrd incorporation into HT29 human colon cancer xenografts. METHODS AND MATERIALS: Two types of experiments were performed. In the first, the effect of radiation on subsequent incorporation was measured. Tumors received doses of 0, 2, 8, and 12 Gy, animals were infused with BrdUrd for 4 days, and incorporation was assessed at the end of the infusion. In the second, the effect of radiation on the elimination of BrdUrd from tumors was determined. Animals were infused with BrdUrd, tumors were irradiated with either 0 or 12 Gy, and tumor incorporation of BrdUrd was measured 1 and 3 days later. RESULTS: Radiation affected neither the incorporation into nor the elimination of BrdUrd from human tumor xenografts. CONCLUSIONS: These findings support the feasibility of clinical trials interdigitating BrdUrd infusion and radiation.

Incorporation of 5-bromo-2'-deoxyuridine into colorectal liver metastases and liver in patients receiving a 7-day hepatic arterial infusion.

Preclinical and clinical data suggest that the combination of hepatic arterial bromodeoxyuridine (BrdUrd), a thymidine analogue radiation sensitizer, and high-dose three-dimensional conformal radiation therapy offer a high potential for improving the local control of intrahepatic cancers. A key step in the design of a successful protocol is to determine in patients the conditions for BrdUrd administration that would be expected to produce selective radiosensitization of the tumor. Therefore, we designed a clinical trial to assess BrdUrd incorporation into the DNA of hepatic colorectal metastases and normal liver after a 7-day continuous BrdUrd infusion at a dose rate of 25 mg/kg/day (the maximal tolerated dose for a 14-day infusion) for patients undergoing laparotomy for either resection of liver metastases or hepatic arterial catheter and pump placement. Thirteen patients were entered into this study. We found that the average replacement of thymidine by BrdUrd in the tumor and normal liver were 11.6 +/- 1.2% and 1.1 +/- 0.2%, respectively. This extent of incorporation would be expected to produce a single fraction radiation enhancement of 1.5 in the tumor without detectable sensitization of the normal liver. Immunohistochemical staining for BrdUrd revealed heterogeneity of incorporation with a range of approximately 60-80% of the cells labeled in different regions of the specimens. These findings suggest that hepatic arterial BrdUrd given at this dose and schedule has a high likelihood of producing clinically significant radiosensitization for patients with hepatic metastases from colorectal cancer. Furthermore, the demonstrated selectivity of tumor perfusion that can be obtained with hepatic arterial infusion combined with the high proliferative rate of colorectal metastases (versus normal liver) suggests that these patients may be good candidates for tumor-directed gene transfer therapy by using regionally delivered retroviral vectors.

Bromodeoxyuridine-mediated radiosensitization in human glioma: the effect of concentration, duration, and fluoropyrimidine modulation.

PURPOSE: To define the relative influence of duration of exposure, concentration, and modulation by fluorodeoxyuridines (FdUrd) on the incorporation of 5-bromo-2-deoxyuridine (BrdUrd) into DNA of a human malignant glioma line (D-54) in vitro and in vivo. IN VITRO STUDIES: an established human malignant glioma line (D-54) was exposed to a clinically achievable concentration of BrdUrd to model intravenous (1 microM BrdUrd) and intraarterial (4 microM BrdUrd) conditions. The influence of modulation was assessed using 1 nM FdUrd. Incorporation of BrdUrd, radiosensitization, and cytotoxicity were determined after 24, 72, and 120 h drug exposures. In Vivo studies: nude mice bearing D-54 xenografts were infused with BrdUrd at 100 mg/kg/day for 7 and 14 days or BrdUrd at 400 mg/kg/day for 5 days. The influence of modulation was assessed by combining 100 mg/kg/day of BrdUrd with 0.1, 0.3, and 1 mg/kg/day FdUrd for 7 days. Incorporation of BrdUrd into the DNA of tumor, gut, and marrow were determined. RESULTS: In Vitro: thymidine replacement and radiosensitization were a function of concentration, and incorporation began to plateau after 2 to 3 population doublings. Modulation with 1 nM FdUrd significantly increased incorporation. Radiosensitization was a linear function of thymidine replacement under all conditions tested. In Vivo: infusion with 400 mg/kg/day for 5 days resulted in greater tumor incorporation (10.3 +/- 0.4% thymidine replaced) than treatment with 100 mg/kg/day for 14 days (6.0 +/- 0.6% of thymidine replaced). Infusion of FdUrd with BrdUrd increased normal tissue incorporation of BrdUrd, but failed to increase BrdUrd incorporation in tumor cells. CONCLUSION: These results suggest that relatively short, high dose rate infusions may be preferable to long, low dose rate infusions. The potential benefit of FdUrd modulation demonstrated in vitro may be difficult to realize using continuous systemic infusions.

Kinetics of bromodeoxyuridine elimination from human colon cancer cells in vitro and in vivo.

We have previously shown that the thymidine analogue radiation sensitizer bromodeoxyuridine (BrdUrd) is incorporated into human tumors to a greater extent than into the livers of athymic mice bearing these tumors as xenografts. However, incorporation into the intestine and bone marrow exceeds that of the tumor (T. S. Lawrence, M. A. Davis, J. Maybaum, S. K. Mukhopadhyay, P. L. Stetson, D. P. Normolle, P. E. McKeever, and W. D. Ensminger, Cancer Res., 52: 3698-3704, 1992). We hypothesized that the ratio of tumor incorporation to intestinal or bone marrow incorporation might increase during a period of drug elimination following the termination of an infusion. To test this hypothesis, we infused athymic mice bearing HT29 human colon cancer xenografts with BrdUrd and measured incorporation in the tumor and normal tissues up to 7 days after the infusion was discontinued. In addition, we assessed the effect of exposure to BrdUrd on subsequent incorporation in vitro and in vivo through the use of a stable isotope of BrdUrd ("isotopic BrdUrd"), which could be differentiated from normotopic BrdUrd using the gas chromatographic-mass spectrometric assay. We found a significant increase in the ratio of BrdUrd in the tumor compared to bone marrow and intestine during the drug elimination period. We also found that BrdUrd incorporation slowed the kinetics of subsequent BrdUrd incorporation and elimination. These findings suggest that when the radiation dose-limiting organ is rapidly proliferative, such as the intestine or bone marrow, delivering radiation during a drug elimination period may improve the therapeutic index.

Clinical pharmacology of hepatic arterial infusions of 5-bromo-2'-deoxyuridine.

This investigation was undertaken to determine the pharmacokinetic parameters relevant to hepatic arterial (HA) infusion of 5-bromo-2'-deoxyuridine (BrdUrd) and to ascertain the maximum tolerated dose and related toxicities of BrdUrd administered as a 14-day HA infusion. In the pharmacokinetic study, 6 patients received a 2-h i.v. infusion (to steady-state) of BrdUrd at each of 5 escalating dose rates (10 to 160 mg/kg/day) with simultaneous blood sampling for BrdUrd levels from HA and hepatic venous catheters. Dose dependent HA and hepatic venous drug levels, total body clearance, hepatic extraction, and estimated regional exposure advantage were determined. The total body clearance of BrdUrd was high and dose rate dependent, falling from 3340 ml/min with a 10-mg/kg/day infusion to 2180 ml/min at a 160-mg/kg/day dose rate. Hepatic extraction was high and dose rate dependent as well, declining from 80% extraction at 10 mg/kg/day to 68% at 160 mg/kg/day. The calculated estimate for the exposure advantage achievable with HA as compared with i.v. infusion reflects the dose rate dependence of total body clearance and hepatic extraction and decreases from a 70-fold advantage at 10 mg/kg/day to a 30-fold advantage at 160 mg/kg/day. In the Phase I study aimed at determining the maximum tolerated dose, successive groups of 3 patients were administered continuous HA infusions for 14 days at escalated BrdUrd dose rates (5, 10, 15, 25, and 35 mg/kg/day) in order to ascertain dose-limiting toxicity. The maximum tolerated dose of BrdUrd for a 14-day continuous HA infusion was found to be 35 mg/kg/day with reversible thrombocytopenia as the sole dose-limiting toxicity. Skin and other toxicities were infrequent, minor, reversible, and non-dose dependent. No hepatic toxicity was detected despite direct drug infusion into the liver. The high total body clearance and hepatic extraction of BrdUrd substantiate its administration via the hepatic artery as a means to achieve higher exposure with intrahepatic tumors than can be obtained by systemic administration. Despite higher hepatic exposures, no hepatic toxicity was noted, and readily reversible systemic toxicity (thrombocytopenia) was dose limiting for the 14-day continuous HA infusion. Thus, HA infusion of the potent radiosensitizer BrdUrd is both pharmacokinetically rational and well tolerated.

Determination of plasma propofol levels using gas chromatography-mass spectrometry with selected-ion monitoring.

Propofol (2,6-diisopropylphenol, I.C.I. 35,868) is a rapid-acting, intravenous anesthetic agent recently introduced for the induction and maintenance of general anesthesia. This paper describes a gas chromatographic-mass spectrometric procedure using selected-ion monitoring for the determination of plasma propofol levels. The drug and the internal standard (thymol) were extracted from plasma into diethyl ether-pentane, and derivatized to their trimethylsilyl derivatives before analysis. The reproducibility of the daily standard curves had coefficients of variation ranging from 2.7% to 10.2%. The precision of the assay yielded a coefficient of variation ranging from 4.5% to 5.6%, and the concentration means for the seeded control samples were found to be within -1.6% to +0.6% of the theoretical values for propofol. No interfering peaks have been observed in application of this procedure to either normal volunteer or patient samples. The minimum detectable level under the conditions described was 0.20 ng propofol/ml plasma. This assay and a high-performance liquid chromatographic assay with fluorescence detection were both used to measure plasma propofol concentrations in 89 human plasma samples, and the correlation between the two methods was excellent.

Halogenated pyrimidine sensitization of low dose rate irradiation in human malignant glioma.

PURPOSE: To determine the potential advantage of combining halogenated pyrimidine radiosensitization and continuous low dose rate irradiation in human malignant glioma. METHODS AND MATERIALS: An established glioma line (U-251) was incubated with 5-bromo-2-doxyuridine (BrdUrd) at clinically achievable concentrations at three dose rates of interest--100 cGy/min (typical of external beam therapy), 43 cGy/hr (typical of temporary afterloaded implants), and 12 cGy/hr (typical of permanent implants). RESULTS: After exposure to 1 microM BrdUrd, the greatest enhancement ratio was seen at the 12 cGy/hr dose rate, implying a BrdUrd induced inverse dose rate effect independent of a G2M block. Under these conditions, the mean inactivation dose after 1 microM BrdUrd exposure was equivalent for 100 cGy/min and 12 cGy/hr. CONCLUSION: These results support the use of halopyrimidines as sensitizers of temporary afterloaded and permanent implants.

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.

The potential superiority of bromodeoxyuridine to iododeoxyuridine as a radiation sensitizer in the treatment of colorectal cancer.

Although the thymidine analogues 5-bromo-2'-deoxyuridine (BrdUrd) and 5-iodo-2'-deoxyuridine (IdUrd) have been used successfully as radiation sensitizers in clinical trials, it is not clear which of these agents is the more promising to pursue. To begin to assess this question with regard to colorectal cancer metastatic to the liver, a study was carried out using HT29 human colon cancer cells in culture and implanted in nude mice as xenografts. Cells and animals were treated with BrdUrd +/- the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine (FdUrd), and the results compared to our previous studies with IdUrd +/- FdUrd (T. S. Lawrence, M. A. Davis, P. E. McKeever, J. Maybaum, P. L. Stetson, D. P. Normolle, and W. D. Ensminger. Cancer Res., 51: 3900-3905, 1991). Using cultured cells, it was found that FdUrd (at concentrations of greater than 10 nM) increased: (a) the incorporation of BrdUrd into the DNA of cultured tumor cells; (b) BrdUrd-mediated radiosensitization; (c) BrdUrd-mediated increase in radiation-induced DNA damage; and (d) BrdUrd-mediated decrease in the repair of radiation-induced damage. The incorporation of BrdUrd was greater than or equal to the incorporation of IdUrd previously determined under the same exposure conditions. Studies using nude mice bearing HT29 xenografts showed that FdUrd increased BrdUrd incorporation more into tumors than into the normal liver. Most tumor cells incorporated BrdUrd (labeling index after a 4-day infusion = 87 +/- 2%; SE); in the liver, labeling was confined chiefly to nonparenchymal cells. In both the presence and absence of FdUrd, the incorporation of BrdUrd into tumors was significantly and consistently greater than the incorporation of IdUrd measured under the same conditions of drug administration (by a factor of 1.2-3.6). Furthermore, the administration of BrdUrd +/- FdUrd tended to produce less weight loss and hematological toxicity than IdUrd +/- FdUrd. These findings suggest that BrdUrd may be superior to IdUrd as a radiation sensitizer in the treatment of colorectal cancer metastatic to the liver.

Cyclosporine metabolism by P450IIIA in rat enterocytes--another determinant of oral bioavailability?

Cyclosporine is converted to its major metabolites (M-17, M-1, and M-21) in human liver by enzymes belonging to the P450IIIA subfamily. These enzymes are also present in rat and human enterocytes; however, the possibility that CsA is metabolized in enterocytes has not been previously investigated. We therefore directly compared metabolism of 3H-CsA in microsomes prepared from liver and jejunal enterocytes. M-17, M-1, and M-21 were the major CsA metabolites produced by enterocyte microsomes. This metabolism appeared to be catalyzed by P450IIIA, because pretreatment of rats with the P450IIIA inducer dexamethasone significantly increased the rate of CsA metabolism in enterocyte microsomes and preincubation of enterocyte microsomes with anti-P450IIIA IgG inhibited the production of CsA metabolites by greater than 95%. To determine if enterocyte P450IIIA metabolizes CsA in vivo, rats were pretreated with the P450IIIA inducer dexamethasone, the P450IIIA inhibitor erythromycin, or vehicle alone. At laparotomy, 2 mg/kg of 3H-CsA was injected into a sealed loop of jejunum, and after collection of the mesenteric venous blood draining this segment for 45 min, the production of M-17 and M-1 was measured. In the control group, a mean of 3.9% of the recovered radioactivity was found as M-1 and M-17. In the rats pretreated with dexamethasone, a mean of 8.4% of the radioactivity was found as M-1 and M-17 (P less than 0.05 relative to control) and this decreased to 2.3% in the group pretreated with erythromycin (P = 0.08 relative to control). We conclude that P450IIIA in jejunal enterocytes readily metabolizes CsA. Furthermore, the metabolism of CsA by enterocytes in vivo is substantial and likely contributes to "first pass metabolism" of orally administered CsA. Our observations provide novel hypotheses to explain some important drug interactions and interpatient differences in CsA dosing requirements.

Modulation of iododeoxyuridine-mediated radiosensitization by 5-fluorouracil in human colon cancer cells.

The incorporation of 5-iodo-2'-deoxyuridine (IdUrd), a thymidine analog radiosensitizer, can be increased by the use of modulators such as 5-fluorouracil (FUra). FUra is a particularly attractive potential modulator to use against colorectal cancer, as it is the most active single agent in the treatment of this disease. To begin to define the conditions for the optimal combination of IdUrd and FUra in the treatment of patients with colorectal cancer, a study was conducted of the effect of FUra on IdUrd-mediated radiosensitization in cultured HT29 human colon cancer cells. It was found that when cells were exposed to concentrations of IdUrd typical of those obtained through intravenous exposure (1-3 microM), FUra (1 microM) increased radiosensitization beyond that which would be predicted for the same extent of incorporation produced by incubation with IdUrd alone. This increase appeared to result from a combination of at least two effects: FUra-mediated cell cycle redistribution and increased IdUrd incorporation. When a higher concentration of IdUrd (10 microM) was used with FUra (1 microM), cell cycle distribution returned to nearly normal, and radiosensitization was equal to that predicted by the extent of incorporation of IdUrd. These data demonstrate that the combination of FUra and IdUrd can produce radiosensitization both through increased IdUrd incorporation and cell cycle redistribution. Furthermore, they suggest that, in the presence of a modulator, it may not be necessary to achieve high levels of IdUrd incorporation to produce significant tumor radiosensitization.

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.

Fluorodeoxyuridine-mediated modulation of iododeoxyuridine incorporation and radiosensitization in human colon cancer cells in vitro and in vivo.

A study was conducted to assess the potential of 5-fluoro-2'-deoxyuridine (FdUrd) to increase the incorporation and radiosensitizing properties of 5-iodo-2'-deoxyuridine (IdUrd) using HT29 human colon cancer cells both in vitro and in nude mice bearing these tumors as xenografts. The purpose of this study was to assess (a) whether FdUrd could increase IdUrd efficacy using clinically achievable concentrations of drugs; (b) the relationships among radiosensitization, DNA damage and repair, and analogue incorporation; and (c) whether FdUrd improved the selectivity of IdUrd incorporation into tumor cells compared to normal tissues. It was found that FdUrd, at clinically achievable concentrations (1-100 nM), significantly increased IdUrd incorporation under all conditions but particularly when the IdUrd concentration was less than or equal to 10 microM. FdUrd increased IdUrd-mediated radiosensitization in proportion to the increase in IdUrd incorporation. FdUrd potentiated the ability of IdUrd to increase radiation-induced DNA double-strand breaks and to slow their repair. When IdUrd alone (100 and 200 mg/kg/day) was infused into nude mice bearing tumors, the extent of thymidine replaced in the tumor was 1.6 +/- 0.4 (mean +/- SE) and 2.5 +/- 0.4%, respectively. The combination of FdUrd (0.1 mg/kg/day) and IdUrd (100 mg/kg/day) increased the incorporation in the tumor to 5.3 +/- 0.9% with less toxicity than resulted from the use of 200 mg/kg/day of IdUrd alone. These data show that FdUrd is an effective biomodulator, because, for the same extent of normal tissue incorporation, the combination of IdUrd and FdUrd produces significantly greater incorporation into the tumor compared to the use of IdUrd alone. Furthermore, they suggest that the regional application of FdUrd with IdUrd, either through the use of regional infusions or in combination with focused irradiation, could potentially improve the outcome of treatment of localized gastrointestinal cancer.