The effect of narasin on apparent nitrogen digestibility and large intestine volatile fatty acid concentrations in finishing swine

In the mouse, both the mdr1a and the mdr1b gene encode drug-transporting P-glycoproteins. The mdr1a P-glycoprotein is expressed in epithelial cells of, among others, the liver and the intestine. Furthermore, the mdr1b gene product is found in the liver but is not detectable in the intestine. To establish the potential involvement of P-glycoprotein in the elimination of cationic amphiphilic drugs from the body, we investigated biliary, intestinal, and urinary excretion in mice with a homozygous disruption of the mdr1a gene (mdr1a(-/-) mice). These mice are fully viable under laboratory conditions and have normal bile flow. Cumulative biliary excretion (expressed as percent of the intravenously administered dose excreted over a 1-hour period) of several cationic compounds was decreased as follows in mdr1a(-/-) mice compared with the wild-type animals: tri-n-butylmethylammonium (TBuMA), 0.7% versus 2.1%; azidoprocainamide methoiodide (APM), 3.8% versus 7.6%; and vecuronium, 22.7% versus 41.3%. The luminal secretion of both TBuMA and APM in the small intestine was profoundly decreased, respectively 4.6-fold (1.8% vs. 8.2% in the wild-type) and 7.9-fold (1.6% vs. 10.3% in the wild-type) in mdr1a(-/-) mice. Thus mdr1a P-glycoprotein contributes substantially to the removal of a wide variety of cationic agents from the body through intestinal and hepatobiliary secretion, but it evidently acts in concert with other transport system(s). These processes probably provide a protective mechanism limiting the overall rate of absorption as well as the bioavailability of potentially toxic organic amines.     

Full blockade of intestinal P-glycoprotein and extensive inhibition of blood-brain barrier P-glycoprotein by oral treatment of mice with PSC833

Mice lacking mdr1-type P-glycoproteins (mdr1a/1b [-/-] mice) display large changes in the pharmacokinetics of digoxin and other drugs. Using the kinetics of digoxin in mdr1a/1b (-/-) mice as a model representing a complete block of P-glycoprotein activity, we investigated the activity and specificity of the reversal agent SDZ PSC833 in inhibiting mdr1-type P-glycoproteins in vivo. Oral PSC833 was coadministered with intravenous [3H]digoxin to wild-type and mdr1a/1b (-/-) mice. The direct excretion of [3H]digoxin mediated by P-glycoprotein in the intestinal mucosa of wild-type mice was abolished by administration of PSC833. Hepatobiliary excretion of [3H]digoxin was markedly decreased in both wild-type and mdr1a/1b (-/-) mice by PSC833, the latter effect indicating that in vivo, PSC833 inhibits not only mdr1-type P-glycoproteins, but also other drug transporters. Upon coadministration of PSC833, brain levels of [3H]digoxin in wild-type mice showed a large increase, approaching (but not equaling) the levels found in brains of PSC833-treated mdr1a/1b (-/-) mice. Thus, orally administered PSC833 can inhibit blood-brain barrier P-glycoprotein extensively, and intestinal P-glycoprotein completely. These profound pharmacokinetic effects of PSC833 treatment imply potential risks, but also promising pharmacological applications of the use of effective reversal agents.     

Enhanced oral absorption and decreased elimination of paclitaxel in mice cotreated with cyclosporin A

Recent experiments in mice have demonstrated that the systemic exposure to p.o. administered paclitaxel is significantly enhanced with coadministration of the P-glycoprotein blocker SDZ PSC 833 (J. van Asperen et al, Br. J. Cancer, 76: 1181-1183, 1997). To facilitate further research on the feasibility of a clinically effective oral formulation of paclitaxel, it is important to know whether cotreatment with a commonly applied and commercially available P-glycoprotein blocker, e.g., cyclosporin A, has a similar effect. Here, we present a detailed study about the effects of cyclosporin A on the pharmacokinetics of p.o. and i.v. administered paclitaxel. Female FVB mice received a combined treatment of 5 or 10 mg/kg paclitaxel (either i.v. or p.o.) plus 0, 10, or 50 mg/kg cyclosporin A (p.o.). The plasma concentrations of paclitaxel were determined at several time points after drug administration using high-performance liquid chromatography. Calculated relative to the area under the plasma concentration-time curve of i.v. administered paclitaxel in mice treated without cyclosporin A, the oral bioavailability of paclitaxel increased from 9.3% up to 67% with coadministration of cyclosporin A. The bioavailability in mice cotreated with 10 or 50 mg/kg cyclosporin A appeared to be similar. The effect of cyclosporin A on the systemic exposure to p.o. administered paclitaxel was the result of both a significantly decreased clearance and an increased uptake. A histological examination revealed that the enhanced absorption was not caused by gastrointestinal toxicity. We conclude that cyclosporin A and SDZ PSC 833 are equally effective in increasing the systemic exposure to p.o. administered paclitaxel. These data are promising for the development of a clinically useful oral formulation of this cytostatic drug and indicate that cyclosporin A is a suitable agent for further research of this concept.     

Expression, purification, and characterization of a recombinant 5-lipoxygenase from potato tuber

The involvement of mdr1a P-glycoprotein (P-gP) on the tissue distribution of the multidrug resistance-reversing agent SDZ PSC 833 was assessed by use of mdr1a (-/-) mice. The mdr1a (-/-) and wild-type mdr1a (+/+) mice received a 4-h constantrate i.v. infusion (2 micrograms/min) of [14C]SDZ PSC 833. Mice were sacrificed at 0, 0.5, 1, 2 and 4 h during infusion and at 0.5, 1, 3, 8 and 24 h after stopping the infusion. Blood and tissues were analyzed on total (14C) and parental SDZ PSC 833 concentrations. Mdr1a (-/-) mice exhibited increased SDZ PSC 833 accumulation in cerebrum, cerebellum and somewhat in testes and small intestine compared with the wild-type mice. The difference between mdr1a (-/-) and (+/+) brain (cerebrum and cerebellum) penetration depended on SDZ PSC 833 blood concentrations, because this cyclosporin analog apparently governs its own brain penetration by inhibiting the P-glycoprotein pump in mdr1a (+/+) mice. Thus the mdr1a (-/-)/(+/+) ratio of brain concentrations tended to decrease and increase at high and low blood concentrations, respectively. These findings clearly demonstrate the interaction of SDZ PSC 833 with the P-glycoprotein present at the blood-brain barrier. The SDZ PSC 833 distribution in other mdr1a P-glycoprotein-expressed tissues, as well as its metabolism and elimination, was not affected by the mdr1a gene disruption. This suggests that factors other than mdr1a P-gP are involved in the disposition of this multidrug resistance-reversing agent.     

Brain bilirubin content is increased in P-glycoprotein-deficient transgenic null mutant mice

P-glycoprotein (P-gp), encoded by the mdr1a gene, is an ATP-dependent plasma membrane protein that is expressed in abundance on the blood-brain barrier (BBB). P-gp limits the CNS influx and retention of a variety of lipophilic compounds. We hypothesized that brain bilirubin content after an i.v. bilirubin infusion would be increased in P-gp-deficient mdr1a null mutant transgenic mice (mdr1a(-/-)) compared with controls. Eighteen mdr1a(-/-) null mutant and 18 P-gp-sufficient wild type mice (+/+) were anesthetized and 50 mg/kg bilirubin infused through the tail vein. Brain bilirubin content (mean +/- SEM) 10 min after infusion was significantly higher in mdr1a(-/-) (18.1 +/- 2.4 nmol/g) compared with (+/+) mice (10.4 +/- 1.0 nmol/g). Brain bilirubin content declined 60 min after infusion but remained higher in mdr1a(-/-) (10.3 +/- 1.4 nmol/g) compared with (+/+) mice (5.3 +/- 0.9 nmol/g). Brain bilirubin clearance did not differ between groups (t 1/2 approximately 55 min). We conclude that P-gp-deficient mdr1a(-/-) mice have significantly higher brain bilirubin content compared with controls after an i.v. bilirubin load. These data suggest that 1) bilirubin is a substrate for P-gp and 2) the increased brain bilirubin content in mdr1a(-/-) mice is due to enhanced brain bilirubin influx. We speculate that BBB P-gp provides a protective effect against bilirubin neurotoxicity by reducing brain bilirubin influx.     

BBB transport and P-glycoprotein functionality using MDR1A (-/-) and wild-type mice. Total brain versus microdialysis concentration profiles of rhodamine-123

PURPOSE: The effect of P-glycoprotein (Pgp) on brain distribution using mdr1a (-/-) mice was investigated. METHODS: Fluorescein (Flu) and FD-4 were used to check whether blood-brain barrier (BBB) integrity was maintained in mdr1a (-/-) mice. The Pgp substrate rhodamine-123 (R123) was infused and total brain, blood and brain microdialysate concentrations in mdr1a (-/-) mice and wild-type mice were compared. RESULTS: Maintenance of BBB integrity was indicated by equal total brain/blood ratios of Flu and FD-4 in both mice types. R123 concentrations in brain after i.v. infusion were about 4-fold higher in mdr1a (-/-) than in wild-type mice (P < 0.05), without changes in blood levels. After microdialysis experiments the same results were found, excluding artifacts in the interpretation of Pgp functionality by the use of this technique. However the 4-fold ratio in brain was not reflected in corresponding microdialysates. No local differences of R123 in the brain were found. By the no-net-flux method in vivo recovery appeared to 4.6-fold lower in mdrla (-/-) mice compared with wild-type mice. CONCLUSIONS: Pgp plays an important role in R123 distribution into the brain. Using intracerebral microdialysis, changes in in vivo recovery by the absence or inhibition of Pgp (or active efflux in general) need to be considered carefully.     

The Preterm Prediction Study: recurrence risk of spontaneous preterm birth. National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network

Our study was designed to evaluate the pharmacokinetics, tissue distribution, toxicity and therapeutic efficacy of liposome-encapsulated paclitaxel (LET) in comparison to conventional paclitaxel. In normal mice, LET was much less toxic than the conventional drug. A dose of 32.5 mg/kg of conventional paclitaxel administered i.v. on three consecutive days produced 100% mortality by day three, while liposomal paclitaxel exhibited no mortality. The control group which received Diluent 12 (Chremophor EL and ethanol; 1:1 v/v), a vehicle used in conventional paclitaxel, 30% mortality was observed at this dosage level. In murine ascitic L1210 leukemia model, liposomal paclitaxel and conventional paclitaxel showed comparable antitumor activity. The pharmacokinetics of conventional paclitaxel and LET was studied in mice at dose levels of 5 mg/kg and 20 mg/kg. After intravenous administration of conventional paclitaxel at a dose of 5 mg/kg, the area under the plasma-concentration-time curve (AUC) was 2-fold lower and, the elimination half-life was 2-times shorter compared to LET. At a dose of 20 mg/kg, the terminal half-lives were comparable, however, conventional paclitaxel displayed non-linear pharmacokinetics with disproportionate increase in AUC. At the two dose levels studied, LET demonstrated linear kinetics. Tissue distribution of paclitaxel after administration of LET showed levels 10-fold higher in spleen and 3.5-fold higher in liver as compared to conventional paclitaxel. The significant decrease in toxicity shown by LET, coupled with an increase in plasma AUC and half-life indicates that LET may be a viable alternative to the therapeutic use of the conventional preparation of paclitaxel.     

Immediate implantation into the posterior maxilla after antroplasty: the Cranin-Russell Operation

KW-2149 is a new derivative of mitomycin C (MMC). The plasma concentrations, distribution, metabolism, and excretion of [3H]-KW-2149 in normal and tumor-bearing mice after i.v. administration of 16.6 mg/kg were investigated. The plasma radioactivity decreased biexponentially after i.v. administration in normal mice. However, the unchanged drug disappeared rapidly, showing a half-life (t1/2) of 9.7 min, which was shorter than MMC's (18 min). The radioactivity was excreted in mouse urine (33%) and feces (58%) within 144 h. High radioactivity was distributed in the gallbladder, liver, kidney, pancreas, and lung at 1 h after i.v. administration to normal mice. The tumor concentration was lower than the plasma or blood concentration. The lowest radioactivity was observed in the brain. The metabolic rate of KW-2149 was very rapid. The methyl sulfide form (M-16), the symmetrical disulfide dimer (M-18), and the albumin conjugate were detected in plasma, which possessed anticellular activity. The specific anticellular activity of these compounds against uterine carcinoma (HeLa S3) was 1/100, 1, and 1/20 respectively, as compared with that of KW-2149.     

Greater antitumor efficacy of paclitaxel administered before epirubicin in a mouse mammary carcinoma

Combined treatment with paclitaxel and anthracyclines is increasingly being tested in clinical practice. Epirubicin is in general administered before paclitaxel. We have investigated, using a murine mammary adenocarcinoma, whether the efficacy and toxicity of this combination is influenced by treatment sequence, different time intervals and dose intensity. The tumor was transplanted into the right hind foot of C3D2F1 female mice. Paclitaxel was administered i.p. in doses ranging from 15 mg/kg to 75 mg/kg and epirubicin (i.p. or i.v.) in doses from 9 mg/kg to 30 mg/kg. The hepatic and peritoneal toxicity observed with epirubicin administration increased in combined treatments (stronger with i.p. than i.v. epirubicin administrations) and was dose-dependent. When paclitaxel and epirubicin were administered simultaneously or paclitaxel was given 24 h before epirubicin, the same tumor growth delays were obtained in all groups. A smaller effect was observed when paclitaxel was administered 24 h after epirubicin. Increasing the epirubicin or paclitaxel dose led to higher tumor growth delays but also an increased toxicity. In conclusion, in this experimental model, the administration of 45 mg/kg paclitaxel before 15 mg/kg epirubicin was very effective and the increased toxicity can be limited by introducing an interval of 24 h between drug administrations. These results should be considered when designing clinical trials.     

Availability of PSC833, a substrate and inhibitor of P-glycoproteins, in various concentrations of serum

BACKGROUND: P-glycoproteins are membrane-associated transporters that can render cells resistant to a variety of chemotherapeutic drugs. Reversal agents are (preferably nontoxic) drugs that can inhibit these P-glycoproteins and thereby overcome multidrug resistance. PSC833, a cyclosporin A analog, is a reversal agent that has shown potential in in vitro experiments and in clinical trials. We tested PSC833 to determine whether it is a transported substrate of human and murine P-glycoproteins associated with multidrug resistance (encoded by the human MDR1 gene and its murine homolog, mdr1a) and whether it can completely inhibit these P-glycoproteins under simulated in vivo conditions. METHODS: Monolayers of polarized LLC-PK1 pig kidney cells transfected with complementary DNA containing either MDR1 or mdr1a sequences were used to measure the directional transport of P-glycoprotein substrates under various serum conditions. RESULTS: In contrast to two previous studies, we found that PSC833 is transported by both the MDR1 and the mdr1a P-glycoproteins, albeit at a low rate. PSC833 has a very high affinity for the MDR1 P-glycoprotein, and its Michaelis constant (Km) for transport is 50 nM, fourfold lower than for cyclosporin A. Inhibition of drug transport by PSC833 is approximately eightfold less effective in 100% fetal bovine serum than in tissue culture medium containing 10% serum. The concentration of PSC833 necessary to fully inhibit transport of digoxin and paclitaxel (Taxol) under complete (i.e., 100%) serum conditions is higher than the plasma concentrations achieved in clinical trials. CONCLUSIONS: Although PSC833 binds efficiently to the MDR1 P-glycoprotein and is released only sluggishly, the high concentrations of PSC833 necessary to inhibit this P-glycoprotein under complete serum conditions in our in vitro system suggest that it may be difficult for PSC833 alone to produce total inhibition of P-glycoprotein activity in patients.     

In vitro and in vivo reversal of multidrug resistance by GF120918, an acridonecarboxamide derivative

N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]- phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) has been selected from a chemical program aimed at identifying an optimized inhibitor of multidrug resistance (MDR). The potency of GF120918 is assessed by dose-dependent sensitization of CHRC5, OV1/DXR and MCF7/ADR cells to the cytotoxicity of doxorubicin and vincristine respectively: GF120918 fully reverses multidrug resistance at 0.05 to 0.1 microM and is half maximally active at 0.02 microM. The spectrum of drugs sensitized by GF120918 coincides with those having the classical MDR phenotype. In CHRC5 cells, 0.01-0.1 microM GF120918 enhances the uptake of [3H]daunorubicin and blocks the efflux from preloaded cells. It is also shown that GF120918 is still active several hours after being taken away from the culture medium showing that it is not, like verapamil, effluxed rapidly by P-glycoprotein. GF120918 effectively competes with [3H]azidopine for binding P-glycoprotein, pointing to this transport membrane protein as its likely site of action. After i.v. administration to mice, GF120918 penetrates thoroughly various organs that have a tissue level/blood level ratio above 10. It is eliminated from organs and blood with a half-time of approximately 2.7 h. It is well absorbed after p.o. administration. In mice implanted i.p. with the MDR P388/Dox tumor, a single i.v. or p.o. dose of GF120918 restores sensitivity of the tumor to a single i.p. dose (5 mg/kg) of doxorubicin administered 1 h later. A statistically significant effect is observed at 1 mg/kg GF120918 i.v. and maximal effect is reached at 5 mg/kg. Similarly, whereas neither drug alone is effective, GF120918 (10 mg/kg i.p.) associated with doxorubicin (5 mg/kg i.p.) inhibits the growth of the moderately MDR C26 tumor implanted s.c. as assessed by tumor size at day 19. GF120918 does not modify significantly the distribution or the elimination of doxorubicin in mice ruling out the possibility that the antitumor effects seen might be explained by pharmacokinetic interactions.     

K-ATP-blocking diuretic PNU-37883A reduces plasma renin activity in dogs

We determined the effects of the K-adenosine triphosphate (ATP)-blocking diuretic PNU-37883A on plasma renin activity (PRA) in conscious and anesthetized dogs. In conscious dogs, oral PNU-37883A (6-60 mg/kg) was less potent than hydrochlorothiazide (0.15-1.5 mg/kg) and furosemide (FURO; 0.3-3.0 mg/kg) but exhibited high natriuretic efficacy with little kaliuresis. Unlike the standard diuretics, PNU-37883A reduced PRA by 46-76%, and its high dose minimally affected 24-h urinary aldosterone excretion. PNU-37883A, 1 mg/kg i.v., also blunted the hyperreninemia induced by 1 mg/kg i.v. FURO. In cannulated dogs, 10 mg/kg i.v. PNU-37883A maximally increased fractional Na+ clearance 140% and reduced PRA 76%, but these effects were accompanied by a mean 13 mm Hg pressor effect. In anesthetized dogs, renal artery-infused PNU-37883A (3 mg/kg/h i.r.a.) increased Na+ excretion and reduced renal venous PRA independent of hemodynamics, whereas half this dosage selectively reduced renal venous PRA and renin release, independent of hemodynamics and natriuresis. These data demonstrate that the K-ATP blocker diuretic PNU-37883A reduces PRA in dogs after oral, i.v., and i.r.a. administration and could be a useful pharmacologic agent for exploring the role of K-ATP channels in regulating renin release.     

Complete regression of well-established tumors using a novel water-soluble poly(L-glutamic acid)-paclitaxel conjugate

Despite an intensive search, few water-soluble paclitaxel derivatives have been shown to have a therapeutic index superior to paclitaxel itself. We now report a water-soluble poly(L-glutamic acid)-paclitaxel conjugate (PG-TXL) that produces striking antitumor effects with diminished toxicity. A single i.v. injection of PG-TXL at its maximum tolerated dose (defined as that dose that produces a maximum 12-15% body weight loss within 2 weeks after a single i.v. injection) equivalent to 60 mg of paclitaxel/kg and at even a lower dose equivalent to 40 mg of paclitaxel/kg resulted in the disappearance of an established implanted 13762F mammary adenocarcinoma (mean size, 2000 mm3) in rats. (An equivalent dose of PG-TXL is the amount of conjugate that contains the stated amount of paclitaxel.) Similarly, mice bearing syngeneic OCA-1 ovarian carcinoma (mean size, 500 mm3) were tumor-free within 2 weeks after a single i.v. injection of the conjugate at a dose equivalent to 160 mg of paclitaxel/kg. The conjugate has little if any intrinsic tubulin polymerization activity in vitro and is >20 times less potent in supporting the growth of a paclitaxel-dependent CHO mutant cell line. PG-TXL has a prolonged half-life in plasma and greater uptake in tumor as compared with paclitaxel. Furthermore, only a small amount of total radioactivity from PG-[3H]TXL was recovered as free [3H]paclitaxel in either the plasma or the tumor tissue within 144 h after drug injection. Histological studies of tumor tissues obtained from mice treated with PG-TXL show fewer apoptotic cells but more extensive tumor necrosis as compared with paclitaxel treatment. These data suggest that in addition to its role as a carrier for selective delivery of paclitaxel to the tumor, PG-TXL exerts distinct pharmacological actions of its own that may contribute to its remarkable antitumor efficacy.     

Increasing incidence of childhood class V lupus nephritis

1. Homozygously mdr1a gene disrupted mice (mdr1a(-/-) mice) and wild type mice (mdr1a(+/+) mice) were used to develop a method for P-glycoprotein (P-gp) function imaging non-invasively and to study the effect of a P-gp reversal agent on its function in vivo. 2. [11C]verapamil (0.1 mg/kg) was administered and the changes in tissue concentrations were determined ex vivo by organ extirpation and in vivo with PET. To block P-gp function, cyclosporin A was administered. 3. Biodistribution studies revealed 9.5-fold (P < 0.001) and 3.4-fold (P < 0.001) higher [11C]verapamil in the brain and testes of mdr1a(-/-) mice than in mdr1a(+/+) mice. Cyclosporin A (25 mg/kg) increased [11C]verapamil levels in the brain and testes of mdr1a(+/+) mice in both cases 3.3-fold (P < 0.01 (brain); P < 0.001 (testes)). Fifty mg/kg cyclosporin A increased [11C]verapamil in the brain 10.6-fold (P < 0.01) and in the testes 4.1-fold (P < 0.001). No increases were found in the mdr1a(-/-) mice. This indicates complete inhibition of P-gp mediated [11C]verapamil efflux. 4. Positron camera data showed lower [11C]verapamil levels in the brain of mdr1a(+/+) mice compared to those in mdr1a(-/-) mice. [11C]verapamil accumulation in the brain of mdr1a(+/+) mice was increased by cyclosporin A to levels comparable with those in mdr1a(-/-) mice, indicating that reversal of P-gp mediated efflux can be monitored by PET. 5. We conclude that cyclosporin A can fully block the P-gp function in the blood brain barrier and the testes and that PET enables the in vivo measurement of P-gp function and reversal of its function non-invasively.     

Preclinical antitumor activity of water-soluble paclitaxel derivatives

PURPOSE: Five water-soluble paclitaxel derivatives were extensively evaluated for their antitumor activities relative to the parent drug. METHODS: Both subcutaneous (s.c.) murine (M109 lung) and human (A2780 ovarian, L2987 lung) tumor models were used for this purpose. RESULTS: Consecutive daily intravenous (i.v.) paclitaxel therapy of mice bearing s.c. M109, beginning on day 4 or 5 posttumor implant and continuing for 5 days, resulted in a range of maximum gross log cell kill (LCK) values (reflective of delays in tumor growth) and maximum relative median survival time (% T/C) values (reflective of increases in lifespan) of 1.0-2.1 and 132-162% (and one outlying result of 235%), respectively. Against the same tumor model, using the same treatment schedule, each of the water-soluble derivatives was active, with maximum LCK of 1.3-2.5 and T/C of 124-254%. These LCK and %T/C values were always within 0.5 LCK and 15%, respectively, of the concomitantly obtained maximum effects of paclitaxel. When tested in several experiments against staged (50-100 mg) s.c. A2780 tumors, using various i.v. treatment schedules, the water-soluble derivatives achieved a maximum LCK of 1.4-3.8. Evaluated in parallel, paclitaxel achieved a maximum LCK of 2.1-4.5 following every other day x 5 i.v. therapy. When paclitaxel was assayed in several experiments using the staged (50-100 mg) s.c. L2987 tumor model, maximum LCK of 0.9->4.1 were produced following every other day x 5 i.v. therapy. Concomitant testing of the water-soluble derivatives, using the same i.v. treatment schedule, resulted in maximum LCK of 0.2->4.1. In each of the tumor models used, the consistently active, and usually the most active, water-soluble derivative was BMS-185660. The levels of activity observed were comparable (within 1 LCK) to those achieved concomitantly using paclitaxel, and its potency was only slightly inferior to the parent drug. CONCLUSIONS: Based on the evaluations performed in three distal site tumor models, we conclude that BMS-185660 is a water-soluble paclitaxel derivative with preclinical antitumor activity comparable to that of the parent drug.     

Diazinon toxicokinetics, tissue distribution and anticholinesterase activity in the rat

The toxicokinetics, tissue distribution, and anticholinesterase (antiChE) activity of diazinon were investigated in the rat. Plasma concentrations most adequately fitted a two-compartment open model after i.v. administration of 10 mg/kg and a one-compartment model after oral administration of 80 mg/kg. Diazinon elimination half-life following i.v. and oral dosing was 4.70 and 2.86 h, respectively. The oral bioavailability was found to be low (35.5%). Hepatic extraction ratios after i.v. administration of 5 or 10 mg/kg were 54.8% and 47.7%, respectively, suggesting that low systemic oral bioavailability can be explained by a first-pass effect in the liver. Diazinon was found to be approximately 89% protein-bound in plasma within the concentration range 0.4-30 ppm. The highest concentration of diazinon after i.v. administration was found in the kidneys, when comparing to liver, kidney, brain. Both red blood cell (RBC) acetylcholinesterase (AChE) and plasma ChE activities were inhibited rapidly (44% and 17% at 10 min, and 36% and 13% min for i.v. and oral administration, respectively), but inhibition of RBC AChE was greater than that of plasma ChE.     

Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins

The mdr1-type P-glycoproteins (P-gps) confer multidrug resistance to cancer cells by active extrusion of a wide range of drugs from the cell. To study their physiological roles, we have generated mice genetically deficient in the mdr1b gene [mdr1b (-/-) mice] and in both the mdr1a and mdr1b genes [mdr1a/1b (-/-) mice]. In spite of the host of functions speculatively attributed to the mdrl-type P-gps, we found no physiological abnormalities in either strain. Viability, fertility, and a range of histological, hematological, serum-chemical, and immunological parameters were not abnormal in mdr1a/1b (-/-) mice. The high level of mdrlb P-gp normally present in the pregnant uterus did not protect fetuses from a drug (digoxin) in the bloodstream of the mother, although the protein did reduce drug accumulation in the adrenal gland and ovaries. Pharmacologically, mdr1a/1b (-/-) mice behaved similarly to the previously analyzed mdr1a (-/-) mice, displaying, for instance, increased brain penetration and reduced elimination of digoxin. However, both mdr1a and mdr1b P-gps contributed to the extrusion of rhodamine from hematopoietic progenitor cells, suggesting a potential role for the endogenous mdr1-type P-gps in protection of bone marrow against cytotoxic anticancer drugs. This, and the normal viability of mdr1a/1b (-/-) mice, has implications for the use of P-gp-blocking agents in cancer and other chemotherapy. mdr1a/1b (-/-) mice should provide a useful model system to further test the pharmacological roles of the drug-transporting P-gps and to analyze the specificity and effectivity of P-gp-blocking drugs.     

Similar bioavailability of single-dose oral and intravenous mesna in the blood and urine of healthy human subjects

Although mesna has been used for more than a decade to reduce the incidence of hemorrhagic cystitis induced by ifosfamide and cyclophosphamide, the disposition of i.v. and oral mesna has not been adequately described. To obtain accurate bioavailability data for the design of mesna regimens, we developed procedures to preserve and measure mesna and dimesna in the blood and urine and studied 25 volunteer subjects who received single doses of i.v. mesna and four different formulations of oral mesna in a five-way randomized crossover study. The dose-adjusted area under the blood concentration-time curve showed no difference in bioavailability for i.v. and oral mesna; however, the maximum mesna concentration after oral doses was 16% of that estimated for i.v. doses. The short initial half-life of i.v. mesna indicated that mesna was rapidly cleared; however, the blood concentrations of mesna uniformly exceeded those of dimesna after oral as well as i.v. doses, which suggested that reduced mesna and oxidized mesna disulfide are in equilibrium. The ratio of mesna:dimesna was higher in protein-free plasma than it was in the urine, which suggested that most urinary mesna is produced by glomerular filtration of mesna rather than by renal tubular reduction of dimesna. The sum of mesna and dimesna excretion after the i.v. doses (73% of the dose) and the four oral formulations (68-73%) showed no difference in urinary bioavailability, consistent with the blood data. However, the urinary bioavailability of the therapeutically active free-thiol mesna was greater after i.v. doses (40% of the dose) than it was after oral doses (31-33%). The ratio of oral:i.v. mesna excretion ranged from 0.52-1.23 (mean, 0.82) among the 24 subjects. Urinary mesna concentrations exceeded 50 microM in all subjects for up to 12 h after oral doses as compared to 4 h after i.v. doses. About 90% of this mesna was excreted by hour 2 after i.v. doses and by hour 9 after oral doses. The mean maximum concentration of mesna in blood and excretion into urine were both 2.6 h after dosing. The oral formulations thus showed sustained urinary excretion, and their urinary bioavailability approached that of i.v. mesna.     

P-glycoprotein expression in Ehrlich ascites tumour cells after in vitro and in vivo selection with daunorubicin

Fluctuation analysis experiments were performed to assess whether selection or induction determines expression of P-glycoprotein and resistance in the murine Ehrlich ascites tumour cell line (EHR2) after exposure to daunorubicin. Thirteen expanded populations of EHR2 cells were exposed to daunorubicin 7.5 x 10(-9) M or 10(-8) M for 2 weeks. Surviving clones were scored and propagated. Only clones exposed to daunorubicin 7.5 x 10(-9) M could be expanded for investigation. Drug resistance was assessed by the tetrazolium dye (MTT) cytotoxicity assay. Western blot was used for determination of P-glycoprotein. Compared with EHR2, the variant cells were 2.5- to 5.2-fold resistant to daunorubicin (mean 3.6-fold). P-glycoprotein was significantly increased in 11 of 25 clones (44%). Analysis of variance supported the hypothesis that spontaneous mutations conferred drug resistance in EHR2 cells exposed to daunorubicin 7.5 x 10(-9) M. At this level (5 log cell killing) of drug exposure, the mutation rate was estimated at 4.1 x 10(-6) per cell generation. In contrast, induction seemed to determine resistance in EHR2 cells in vitro exposed to daunorubicin 10(-8) M. The revertant EHR2/0.8/R was treated in vivo with daunorubicin for 24 h. After treatment, P-glycoprotein increased in EHR2/0.8/R (sevenfold) and the cell line developed resistance to daunorubicin (12-fold), suggesting that in EHR2/0.8/R the mdr1 gene was activated by induction. In conclusion, our study demonstrates that P-glycoprotein expression and daunorubicin resistance are primarily acquired by selection of spontaneously arising mutants. However, under certain conditions the mdr1 gene may be activated by induction.     

A comparative diuretic and tissue distribution study of bumetanide and furosemide in the dog

Intravenous dose-response data obtained from renal clearance studies in anesthetized dogs indicated that bumetanide was approximately 30-fold more potent than furosemide in enhancing sodium excretion. After the administration of 0.01 mg/kg of bumetanide or 1.0 mg/kg of furosemide, the relationship between i.v. diuretic activity and tissue distribution was evaluated. In dog renal clearance experiments, bumetanide and furosemide significantly enhanced urine flow, sodium and potassium excretion. Inulin clearance as an estimate of glomerular filtration rate was not altered by either drug, but sodium reabsorption was decreased with bumetanide (13%) and furosemide (12%). At these diuretic doses, both compounds were bound to dog plasma protein to about the same extent (86-91%), although total plasma levels were 100-fold higher for furosemide. Within 1/2 hour after the i.v. administration of 14C-bumetanide or 14C-furosemide, 86 to 99% of the 14C in urine, plasma, kidney, and liver appeared as unchanged drug. One minute after maximal diuresis bumetanide was found to have a higher affinity (3-fold) for kidney compared to furosemide. These data offer a possible explanation for the i.v. diuretic potency difference between these two compounds. Furthermore, the lack of significant difference in plasma protein binding and the absence of urinary metabolites of either drug suggest that other factors may also contribute to the marked differences in diuretic activity between bumetanide and furosemide.