Biological disposition of intravenously administered 131I-labeled anti-EGF-receptor antibody (RG 83852) in the rat.

RG 83852 is a murine monoclonal antibody that preferentially inhibits the high-affinity binding of epidermal growth factor (EGF) to its receptor. Since overexpression of EGF receptor has been implicated in some human malignancies, the antibody is under investigation as a potential anticancer agent. The present work characterized the tissue distribution and elimination of 131I-labeled antibody in rats following i.v. administration. 131I-RG 83852 was given in a 2.22 mg/kg dose to rats, and 4, 24, 48, and 72 h afterwards 131I activity excreted in the urine and feces and that present in various tissues was determined. The plasma contained the highest concentration of radioactivity at all times. At 4 h the plasma contained about 12% of the injected dose (ID)/ml, and radioactivity in this compartment accounted for almost 70% ID. The plasma elimination of 131I-derived activity occurred linearily at a rate of about 0.48% ID/h. Except in the thyroid, the concentration of 131I activity in all tissues was much lower than in the plasma (tissue-to-plasma ratio < or = 0.1). In the thyroid, accumulation of radioactivity (4% ID at 24 h) was presumably due to trapping of 131I released from the antibody as a result of biodegradation. The urinary excretion occurred at a rate of about 0.5% ID/h; the fecal excretion was minimal. The biodistribution results are consistent with the protein structure of the antibody. Based on the available disposition data, it is proposed that elimination of the antibody involves degradation, a process that follows zero-order kinetics, followed by excretion of the labeled product(s) in the urine.

Dispositional characteristics of a tyrosine kinase inhibitor (RG 14620) in rats and rabbits following intravenous administration or dermal application.

Based on its epidermal growth factor receptor-mediated tyrosine kinase inhibitory activity, (Z)-alpha-[(3,5-dichlorophenyl)methylene]-3- pyridylacetonitrile (RG 14620) is undergoing evaluation as a topical drug for psoriasis. Disposition studies were conducted in rats and rabbits, using [14C]RG 14620, primarily to investigate percutaneous absorption. Plasma radioactivity and the unchanged drug along with material balance were determined following intravenous administration (2.5 mg/kg) of a solution in PEG 400 or dermal application (50 mg/kg) of a 5% petrolatum ointment. Following an intravenous dose, initial (5-min) plasma radioactivity concentrations were comparable (approximately 1700 ng-eq/ml) in both species, but the decline was slower in rabbits. After dermal application, maximum plasma concentrations were attained at 12 hr in rats and 24 hr in rabbits, and corresponded to approximately 160 and 90 ng-eq/ml, respectively. Overall, the unchanged drug represented < 22% of the total plasma radioactivity in both species, suggesting extensive metabolism. In the rat, fecal route was the major route of excretion, whereas in the rabbit, urinary and fecal excretion contributed to about similar extent. Based on dose-adjusted ratios of plasma radioactivity AUC0-24 hr values, extent of percutaneous absorption appeared to be 1.9% in rats and 0.6% in rabbits. The estimates were 0.6% and 1.2%, respectively, when AUCs were determined for the unchanged drug in plasma. However, based on cumulative excretion of radioactivity up to 96 hr, percutaneous absorption was 12.0% in the rat and 2.0% in the rabbit. The differences in estimates of absorption were attributed to slow but continued absorption of the drug from skin, even after removal of the dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Method for separation and determination of lactone and hydroxy acid forms of a new HMG CoA reductase inhibitor (RG 12561) in plasma.

The new drug RG 12561 (I) is a lactone that is undergoing clinical evaluation for its cholesterol lowering effect based on potent HMG CoA reductase inhibitory activity displayed by its open hydroxy acid form. To determine the dispositional characteristics of the drug, a method was developed for determination of the two forms in plasma. A 0.25-ml aliquot of plasma was deproteinized with 0.5 ml of methanol, and the lactone was extracted with hexane-ethyl acetate (75:25, v/v). The methanolic plasma was then acidified followed by extraction of the hydroxy acid with hexane-ethyl acetate. The extracts were dried, reconstituted and analyzed by isocratic, reversed-phase high-performance liquid chromatography using ultraviolet absorbance at 254 nm. The separations were performed utilizing a C18 column with mobile phase consisting of acetonitrile, 2-propanol and 0.1 M acetate buffer (pH 5), the proportions of which differed depending on the form of drug analyzed. The method was found to be selective and a quantitation limit of 50 ng/ml was established. Validation studies demonstrated that the method was sufficiently accurate and precise for determining disposition of the drug in the dog.

An improved method for isolating Ca2+-resistant myocytes from the adult rat heart.

Maintaining viability in cardiac myocytes isolated from adult rats using collagenase is difficult in Ca-containing media due to cell damage that occurs on reintroduction of Ca after perfusing the heart with the Ca-free medium needed to isolate myocytes with collagenase. Recently it has been proposed that Ca-free perfusion of isolated rabbit interventricular septa leads to cellular Na overload which, on reintroducing Ca, produces influx of toxic concentrations of Ca due to the Na/Ca exchange mechanism in the sarcolemma. We have found that replacing a portion of the 118 mM NaCl in the Ca-free perfusion medium with 69 mM LiCl dramatically increased the proportion of Ca-tolerant cardiac myocytes isolated from adult male rats with collagenase. Myocyte viability was maintained over a four hour period of incubation at 37 degrees C in 1 mM Ca.

Comparative effects of adriamycin and DNA-non-binding analogues on DNA, RNA, and protein synthesis in vitro.

Drug-DNA binding is claimed to be the basis by which the antitumor antibiotic adriamycin (doxorubicin) inhibits DNA and RNA synthesis in vitro. However, in preliminary studies the DNA-non-binding adriamycin analogue N-trifluoroacetyladriamycin-14-valerate (AD 32) showed somewhat greater inhibition of DNA and RNA synthesis than adriamycin under identical conditions. The kinetics of macromolecule synthesis inhibition induced by adriamycin and AD 32, and the two principal DNA-non-binding metabolites of AD 32, N-trifluoroacetyladriamycin (AD 41) and N-trifluoroacetyladriamycinol (AD 92), have now been subjected to comparative study in cultured CEM (human leukemic lymphoblastic) cells. At equimolar concentrations (10 microM), or at concentrations related to their 50% growth-inhibitory values vs CEM cells, AD 32 was consistently found to be more inhibitory than adriamycin of DNA and RNA synthesis, as measured by the incorporation of tritiated thymidine and uridine, respectively, into acid-precipitable fractions relative to untreated controls. Marked inhibitory activity was apparent with 10 microM AD 32 even at the earliest sampling time (15 min); with adriamycin at the same concentration the maximal effect was not achieved until 3 h. AD 32 at 4.8 microM concentration continued to show strong inhibition of nucleic acid synthesis, whereas adriamycin at 1.0 microM was essentially inactive. Like AD 32, AD 41 and AD 92 showed greater inhibition than adriamycin of DNA and RNA synthesis at the early sampling times, although in all instances the effects of AD 32 were more profound. AD 32 at 10 microM concentration produced a moderate but significant inhibition of the incorporation of tritiated methionine into protein compared with adriamycin, which at this concentration was not active. Parallel HPLC analytical studies with similar drug-treated cultures indicated that, while small amounts of adriamycin were found in cells treated with 10 microM AD 32, the amount of adriamycin present at 15 min was only a small fraction (less than 5%) of the amount of adriamycin achieved at 3 h in cultures treated with 1.0 microM adriamycin, a concentration already shown to be only slightly inhibitory of nucleic acid synthesis under the culture conditions. The present study thus confirms the marked DNA and RNA synthesis-inhibitory effects of AD 32, and establishes that this inhibitory activity is not due to conversion of AD 32 into adriamycin. These findings accordingly call into question the validity of the drug-DNA binding mechanism as the explanation for the nucleic acid synthesis inhibitory effects seen with ADR.

Doxorubicin in pregnancy: possible transplacental passage.

Two pregnant women with lymphoproliferative disorders were treated with doxorubicin-containing regimens. Both patients delivered shortly after a dose of doxorubicin. One child was healthy and the other was stillborn. Measurements of anthracycline levels in placental, cord, and fetal tissues by high-performance liquid chromatography suggest that doxorubicin may be transported across the placenta.

Relationship of adriamycin concentrations to the DNA lesions induced in hypoxic and euoxic L1210 cells.

Exponentially growing L1210 mouse leukemia cells were incubated with Adriamycin (ADR) under hypoxic (95% N2:5% CO2) or euoxic conditions (95% air:5% CO2) for 1 hr at 37 degrees at a drug concentration ranging from 2.8 X 10(-8) to 2.8 X 10(-4) M, i.e., from levels attained clinically by bolus delivery to the high levels used as an i.p. drug dwell or experimentally, in in vitro conditions. High-pressure liquid chromatography analyses showed diminishing efficiency in drug uptake by the cells as the dose was increased. There were no significant differences between hypoxic and euoxic cells in drug uptake and metabolism. The frequency of DNA protein-associated single-strand breaks and DNA-protein cross-links per 10(6) nucleotides, detected by the alkaline elution technique, increased with the dose in the range of 2.8 X 10(-8) to 2.8 X 10(-6) M in both euoxic and hypoxic cells and declined thereafter. However, the number of DNA lesions relative to a normalized drug level declined steadily, starting with the 2.8 X 10(-7) M concentration. Concentrations greater than 2.8 X 10(-6) M of ADR induced still another type of lesion, direct DNA strand breaks, only in euoxic cells. The results indicate that a common mechanism of interaction between drug and DNA is present in hypoxic and in euoxic cells at low ADR, while an O2-dependent mechanism becomes operational in euoxic cells at high ADR levels.

Protein-associated DNA breaks and DNA-protein cross-links caused by DNA nonbinding derivatives of adriamycin in L1210 cells.

The effects of Adriamycin derivatives on L1210 mouse leukemia cells were studied with the DNA alkaline elution assay. The exposure of exponentially growing cells to approximately equitoxic concentrations of N-trifluoroacetyladriamycin-14-valerate (13.8 microM) and its metabolites, N-trifluoroacetyladriamycin (9.0 microM) and N-trifluoroacetyladriamycinol (43.7 microM), for 1 hr in vitro resulted in a high frequency of protein-associated DNA breaks and DNA-protein cross-links. These effects were comparable to those observed with Adriamycin (2.8 microM) and with adriamycinol (26.9 microM). In contrast to Adriamycin and its metabolite adriamycinol, N-trifluoroacetyladriamycin-14-valerate and its two major metabolites do not bind to DNA. Despite the absence of this direct interaction, N-trifluoroacetyladriamycin-14-valerate and its metabolites produce alterations in DNA comparable with the effects of intercalating agents. No evidence for conversion of N-trifluoroacetyladriamycin-14-valerate to Adriamycin or adriamycinol was found in L1210 cells. The similar effects on DNA macromolecules, observed between intercalating and non-DNA-binding anthracyclines, are consistent with the concept that mechanisms other than direct interaction with DNA play a role in the toxic effects of these compounds.

Distribution of radioactivity and anthracycline-fluorescence in tissues of mice one hour after [14C]-labeled AD 32 administration. Evidence for tissue aglycone formation.

Levels of radioactivity and total anthracycline fluorescence in tissues of A/JAX mice were compared 1 h after IV administration of unlabeled or [14C]-labeled AD 32 (50 mg/kg). Highest levels of both fluorescence and radioactivity were found in the small intestine (including contents) and liver, a result consistent with the known hepatobiliary excretion of AD 32 and metabolites. Significant accumulations of radioactivity and fluorescence were found in kidney, spleen, large intestine (including contents), lung, and heart. Lesser levels were found in muscle and fat. Little radioactivity and fluorescence were found in brain. Liquid chromatographic analysis of extracts of small intestine and liver homogenates showed N-trifluoroacetyladriamycin (AD 41) as the major fluorescent species, and also revealed N-trifluoroacetyladriamycinol (AD 92) and occasional low levels of AD 32. In addition, there was a major peak of nonfluorescent radioactive material and two fluorescent nonradioactive signals (unknowns 1 and 2), indicative of cleavage of the radiolabel from the chromophore.

Comparative effects of (+)- and (-)-isomers of propranolol and INPEA on (+)-tubocurarine- and succinylcholine-induced neuromuscular blockade.

The effects of the (+)- and (-)-isomers of propranolol and INPEA on the neuromuscular blocking action of (+)-tubocurarine and succinylcholine were studied in the tibialis anterior-peroneal nerve preparation of the rabbit and the isolated phrenic nerve-diaphragm preparation of the rat. (+)-Tubocurarine-induced neuromuscular blockade was intensified by (+)- and (-)-isomers of INPEA whereas it was unaffected by isomers of propranolol in both preparations. In the rabbit tibialis anterior-peroneal nerve preparation, only the (-)-isomers of propranolol and INPEA increased the duration of succinylcholine-induced neuromuscular blockade without affecting its magnitude. Higher doses of both (+)- and (-)-isomers of propranolol and INPEA potentiated succinylcholine-induced blockade in the rat phrenic nerve-diaphragm preparation. The results obtained indicate that only the increase in duration of succinylcholine-induced blockade with beta-adrenoceptor blockers can be related to beta-blocking property of these compounds.