An experimental rat model of local bone cancer invasion and its responsiveness to ethane-1-hydroxy-1,1-bis(phosphonate).

Line A Walker carcinoma differs from line B in that it does not elicit hypercalcemia and hypercalciuria when implanted in rats at various sites (s.c, i.m., intraaortically). However, Walker 256/A, unlike line B, may invade the tibia when implanted i.m. in the adjacent gastrocnemius muscle. This invasion was evaluated by measuring the increased weight of the bone and decreased calcium concentration per unit weight of the tibia, by reduced opacity to X-ray, and by the presence of tumor cells in the compact bone cortex. Ethane-1-hydroxy-1,1-bis(phosphonate), a diphosphonate derivative, at a dose of 10 to 30 mg/kg/day s.c., prevented cancer cell invasion of the tibia as judged by the above criteria. This inhibition was obtained with no apparent effect on the growth of Walker 256/A carcinoma.

Secretion of lipoproteins, apolipoprotein A-I and apolipoprotein E by isolated and perfused liver of rat with experimental nephrotic syndrome.

Nephrotic syndrome induced in the rat by the administration of puromycin aminonucleoside is accompanied by a hyperlipoproteinemia characterized by an elevation of all plasma lipoproteins, particularly of VLDL (1.006 g/ml) and HDL1 (1.050-1.090 g/ml). The increase of HDL1 is due to the accumulation of a lipoprotein species floating mainly in the density interval 1.050-1.090 g/ml, in which apolipoprotein A-I replaces apolipoprotein E as the major constituent peptide. This lipoprotein has been designated nephrotic HDL. The present study was conducted to establish whether nephrotic liver secreted more lipoproteins than the control liver and, in addition, produced a lipoprotein similar to nephrotic HDL found in plasma. Isolated livers from control and nephrotic rats were perfused with a lipoprotein-free medium for 3 h in a recirculating system. Lipoproteins were isolated by ultracentrifugation; apolipoprotein A-I and apolipoprotein E were measured in the whole perfusate at various time intervals. Nephrotic liver secreted twice as much VLDL and HDL2 and 30% more LDL and HDL1 than the control liver. This was accompanied by an increased secretion of both apolipoprotein A-I and apolipoprotein E, the levels of which were 6.5- and 2-fold, respectively, of those found in the control perfusates at the end of the perfusion. In view of the increased secretion of apolipoprotein A-I, the apolipoprotein A-I to apolipoprotein E ratio was much higher in the perfusates of nephrotic livers than in those of the controls. The concentration of apolipoproteins A-I and E in plasma of nephrotic rats was 7- and 2-fold, respectively, of that found in the plasma of the controls. In the perfusates of the nephrotic livers, we could not find a HDL1 (1.050-1.090 g/ml) rich in apolipoprotein A-I similar to that isolated from plasma (nephrotic HDL). We suggest that the latter is formed in the circulation from the intravascular modification of HDL2 secreted in excess by the liver.

Doxorubicin toxicity and pharmacokinetics in old and young rats.

Doxorubicin (Dx) toxicity was compared in old (24 months) and young (6 weeks) Crl:CD(SD) BR male rats, and a clear age-related increase was found. The mortality of all animals receiving a single i.v. Dx dose was followed for 270 days. Old rats died after doses of 2.5 mg/kg, while young animals died after doses two times higher, 5 mg/kg. In old rats body weight loss started 10 to 15 days after Dx, compared to 50 to 80 days for young animals. In young and old rats pharmacokinetic and metabolic studies of Dx were conducted in vivo and in the liver perfusion model. Peak levels of Dx and areas under the time/concentration curves (AUC) in serum and in several tissues of old rats were 1.5 to 2 times higher than in young rats. Concentrations of Dx metabolites in serum and tissues (doxorubicinol, Dxol, and doxorubicinone, Dxone) in young and old rats were not noteworthy. However, higher percentages of Dxone than Dxol were found in both groups in vivo and in vitro. Old livers appeared to produce more Dxone as a percentage, particularly in the bile, which was higher. Urinary elimination of Dx markedly slowed with age; only small amounts of the metabolites were eliminated in urine. In vivo and in vitro availability of Dx and its metabolites is discussed in view of their possible role in the greater toxicity observed in 24-month-old rats.

Pharmacokinetics of fotemustine and BCNU in plasma, liver and tumor tissue of rats bearing two lines of Walker 256 carcinoma.

The plasma and tissue pharmacokinetics of fotemustine (diethyl-1-[3-(2-chlorethyl)-3-nitrosoureido]-ethylphosphonate+ ++) and BCNU (1,3-bis-[2-chlorethyl]-1-nitrosourea) were investigated in healthy control rats and in animals bearing either the nitrosourea-sensitive line A (W256/A) or the nitrosourea-resistant line B (W256/B) of Walker 256 carcinoma. The antitumor activities of these nitrosoureas were similar following i.v. doses ranging from 10 to 40 mg/kg. For both drugs, the survival of tumor-bearing rats was lower in the W256/B than in the sensitive W256/A line. Some sex differences were observed, female rats being more responsive than males to both drugs. Nitrosourea concentrations were assayed in plasma and tissues by differential pulse polarography so as to assess whether the pharmacokinetics could explain the differences in antitumor activity. The antineoplastic effects of fotemustine seemed to be influenced by its pharmacokinetics. The plasma AUC of the intact nitrosourea was higher in females than in males. Fotemustine was cleared 2-5 times more slowly than BCNU from tumor tissue, and its clearance was higher in W256/B- than in W256/A-bearing rats. This suggests that the antitumor activity in the responsive line might partly be due to longer exposure of the growing tumor to the drug. The distribution volume of both nitrosoureas in plasma was higher in tumor-bearing animals than in healthy controls, indicating that the tumor tissue probably constitutes an additional distribution space.

Hepatobiliary metabolism and urinary excretion of 4-demethoxydaunorubicin as compared to daunorubicin in rats.

The hepatic metabolism and biliary excretion of 4-demethoxydaunorubicin (4DDM) was studied in Crl: CD(SD) BR rats by the liver perfusion technique. In the same strains of rats urinary excretion was investigated in vivo. Daunorubicin (DM) was always included for comparison. The drugs and their metabolites were determined in the perfusion medium, in the bile and liver and in the urine by high-performance liquid chromatography with fluorimetric detection. Compared to its analogue DM, 4DDM markedly differed in the metabolic and excretory profile. The cumulative biliary and urinary excretion of 4DDM and the metabolites was quantitatively lower than that of DM (18% vs 36% of the dose) and was consistent with prolonged persistence of 4DDM in plasma in vivo. The extensive carbonyl reduction of 4DDM and DM observed in previous in vivo pharmacokinetic studies was also evident in this study. 13-hydroxy metabolites, daunorubicinol (DMol) and 4-demethoxydaunorubicinol (4DDMol), either as such or after glycosidic cleavage, i.e. 4DDMol aglycone, were present in appreciable amounts in the perfusion medium, bile, liver and urine. In the hepatobiliary system, however, the 13-hydroxy derivative of DM amounted to a much lower fraction than the DM aglycone (17% vs 50% of the total dose), 80% of the total 4DDM dose was accounted for by 4DDMol aglycone. In urine uncleaved DMol or 4DDMol represented more than 75% of the total amount excreted for both drugs. Conjugation, a major step in the excretion of aglycones, seems to play a minor role in the biliary and urinary excretion of 4DDM and 4DDMol.

Antitumor activity of the novel nitrosourea S10036 in rodent tumors.

The activity of the novel anticancer agent diethyl 1-3-(chloroethyl)-3-nitrosoureido ethyl phosphonate (S10036) was investigated on several rodent tumors. S10036 showed a good efficacy, comparable to that of the anticancer agent BCNU, against i.p transplanted P388 and L1210 leukemias. S10036 was very effective against the primary tumor and metastases of i.m transplanted M5076 reticular cell sarcoma of the mouse and against subline A of the Walker carcinoma of the rat. It was inactive against rodent tumors resistant to BCNU such as L1210/BCNU, ICIG-Ci4 murine fibrosarcoma and the Walker carcinoma subline B in the rat.

Caffeine does not bind covalently to liver microsomes from different animal species and to proteins and DNA from perfused rat liver.

Caffeine was found not to bind covalently to liver microsomal proteins from mice, rats and rabbits. Microsomes metabolized caffeine only to a limited extent, the highest rate (about 2% of the substrate concentration) being obtained with rabbit microsomal preparations. The rat liver perfusion technique represents a good model for in vitro caffeine biotransformation studies and therefore for covalent binding experiments. After 2 h perfusion caffeine was extensively metabolized mainly to dimethyl and monomethyl xanthines, a minor pathway to 1,3,7-trimethyluric acid was also seen. However, covalent binding studies using the liver perfusion technique did not reveal any appreciable amount of caffeine metabolites irreversibly bound to either microsomal and total proteins and to DNA.

L-asparaginase effects on inhibition of protein synthesis and lowering of the glutamine content in cultured rat hepatocytes.

Primary cultures of rat hepatocytes were exposed to various concentrations of L-asparaginase derived from Escherichia Coli. Protein synthesis was inhibited by about 33% and cellular glutamine was reduced proportionally to the enzyme concentration. However, protein synthesis was inhibited only by amounts of enzyme able to reduce glutamine to critical levels below 10 nmol/mg cell protein. These data suggest that the glutaminase activity which probably contaminates E. coli asparaginase may be responsible for reduced liver protein synthesis.

Comparative kinetics of benz(a)anthracene, chrysene and triphenylene in rats after oral administration. I. Study with single compounds.

Distribution and elimination of benz(a)anthracene (B(a)A), chrysene (Ch), and triphenylene (Tr) were compared after oral administration of the single compounds to 7-8-week-old female rats. These four-ring isomers were chosen because of their different carcinogenicity. The compounds have high affinity for lipid-rich tissues such as brain, mammary and parametrial adipose tissue. The relative availability of these compounds in the tissues examined decreased in the order Tr greater than B(a)A greater than Ch, but fecal elimination diminished in the opposite order Ch greater than B(a)A greater than Tr. Availability and fecal elimination of single polycyclic aromatic hydrocarbons (PAH) were influenced by the dose and concentration of PAH in the vehicle.

Effect of fasting, induction, sex and age on clearance of benz(a)anthracene and chrysene by isolated perfused rat liver.

Hepatic clearances of benz(a)anthracene (B(a)A) and chrysene (Ch) by isolated perfused liver of female rats were similar when measured with hydrocarbons singly (B(a)A 4.3 ml/min, Ch 5.1 ml/min) or in a mixture (4.7 resp. 5.3 ml/min). Clearance of both isomers by livers from 24 h fasted donors was approximately halved. In vivo pretreatment with B(a)A (22.8 mg per kg for 2 days) significantly increased elimination of both hydrocarbons. Hepatic clearance of B(a)A was significantly higher in male rats than in females of the same age (8 weeks). Elimination of both hydrocarbons was significantly lower in 2 year-old males.

Comparative kinetics of oral benz(a)anthracene, chrysene and triphenylene in rats: study with hydrocarbon mixtures.

Distribution and elimination of benz(a)anthracene (B(a)A) was compared in blood, liver, brain, parametrial adipose and mammary tissue of young female rats after oral administration of the polycyclic aromatic hydrocarbons (PAH) singly or in equimolar mixtures with chrysene (Ch) or triphenylene (Tr). The relative availability of B(a)A in tissues was not influenced by Ch or Tr in the mixture. However, the presence of B(a)A halved the relative availability of Tr and raised that of Ch. The relative availability of the three isomers decreased in the order Tr greater than B(a)A greater than Ch; this may be correlated to their solubility in water.

Effects of chronic treatment with di-(2-ethylhexyl) phthalate on rat liver microsomal activities.

The effects of chronic di-(2-ethylhexyl)phthalate (DEHP) on liver microsomal activity were studied in rats. Daily doses of 50 and 500 mg/kg for 4 weeks did not affect O-demethylation, aromatic hydroxylation, N-demethylation, C3-hydroxylation, styrene monooxygenase, glutamic-oxalacetic and glutamic-pyruvic transaminases (GOT, GPT). Inhibition of glutathione-S-transferase A and C and induction of epoxide hydrase, glutathione-S-transferase B and nitroreductase activity were instead observed. Protein, cytochrome P-450 and reduced glutathione levels in liver did not appear to be affected by DEHP pretreatment.

Differential plus polarographic determination of submicrogram quantities of carmustine and related compounds in biological samples.

A polarographic method was developed to determine the antineoplastic agent carmustine and other nitrosoureas, such as N-methyl-N-nitrosourea and N-cyclohexyl-N-nitrosourea, in biological fluids at levels well below 1 microgram/ml or g. The stability of carmustine in different media was investigated to prevent losses during administration or assay. Examples of nitrosourea determination in biological samples are given.

Effects of roxithromycin, a new semisynthetic macrolide, and two erythromycins on drug metabolizing enzymes in rat liver.

The effects of a new semisynthetic macrolide, roxithromycin, on drug metabolizing enzymes of rat liver were compared with two erythromycins, the base (EB) and the estolate (EE), after 7 days' treatment with high oral doses (400 and 800 mg/kg daily). Dose-related higher concentrations of roxithromycin were reached in serum and liver than after EB or EE. The two reference erythromycins induced the synthesis of microsomal enzymes and formed inactive cytochrome P-450-metabolite complexes. N-Demethylation of erythromycin itself and aminopyrine was increased by the treatment. Liver microsomal enzyme activities were not induced and the inactive cytochrome P-450-metabolite complex was not formed after 400 mg/kg of roxithromycin and only to a very limited extent after 800 mg/kg (10% vs. 50% after EE). At the higher dose microsomal activities were not changed by roxithromycin and only aminopyrine N-demethylation was reduced.

Effects of in vivo treatment with a new fluorinated macrolide (P-0501A) and other erythromycins on drug clearance and hepatic functions in perfused rat liver.

The hepatic clearance and the effects of a new fluorinated macrolide (P-0501A) on the functions of the isolated, perfused rat liver were compared with two known erythromycins--the base and the estolate--after 7 days of treatment (1.36 mmol/kg po daily). The in vitro metabolism of the antibiotics was induced to different extent but only the base and P-0501A were cleared from the perfusate and the liver faster than in untreated animals. In untreated rats the therapeutically active form of P-0501A was excreted in the bile more than the base and the estolate; after pretreatment, biliary excretion of all erythromycins was nearly double. The content of inactive, complexed cytochrome P-450 was increased only by the base and estolate, with various effects on microsomal activities (some induced, e.g. aminopyrine demethylation, other reduced, e.g. pentobarbital clearance). The clearance and biliary excretion of sulphobromophthalein was not affected by treatment with P-0501A or the base, but was significantly reduced by estolate.

Effects of a new fluorinated macrolide (P-0501A) and other erythromycins on drug metabolizing enzymes in rat liver.

The effects of a new fluorinated macrolide (P-0501A) on drug metabolizing enzymes of rat liver were compared with three erythromycins--the base, the stearate and the estolate--after 7 days of dosing (1.36 mmol/kg po daily). The three erythromycins induced the synthesis of microsomal enzymes, but the products of their metabolism inactivated cytochrome P-450 in the order base less than or equal to stearate less than estolate. N-Demethylation of erythromycin and aminopyrine increased, while O-demethylation of 4-nitroanisole was reduced and hydroxylation of aniline was not changed after in vivo treatment. Pentobarbital sleeping time was prolonged and liver glutathione levels were lower in treated rats than in controls. In contrast to the three erythromycins, P-0501A did not induce the synthesis of microsomal enzymes, did not form an inactive complex with cytochrome P-450 and did not affect mono-oxygenase activities or pentobarbital narcosis.

Pharmacokinetics of nitrosoureas: levels of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) in organs of normal and Walker 256/B carcinoma bearing rats after i.v. bolus.

The disappearance of 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU) from plasma, liver, kidney, lung, brain, spleen, tumor tissue and epididymal adipose tissue of Walker 256/B carcinoma-bearing rats and healthy animals was measured by differential pulse polarography after i.v. bolus of the drug. Only BCNU, not its decomposition products, was detected by the polarographic assay. Levels of BCNU in liver of tumor-bearing animals were significantly lower (about 10 times) than those on healthy rats. A bi-exponential fit was used to calculate the kinetics of BCNU in plasma, kidney, lung and brain, but no difference could be found between healthy and Walker tumor-bearing rats. BCNU disappeared faster from adipose tissue of tumor-bearing animals than from normals.

Pharmacokinetics of nitrosoureas: comparison of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) after oral and intravenous administration to rats.

Differential pulse polarographic assay of intact nitrosoureas revealed the lower bioavailability of CCNU (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea) in stomach and liver after oral administration to rats in comparison to BCNU (1,3-bis-(2-chloroethyl)-1-nitrosourea); blood levels of CCNU were below the detection limit of the method (20 ng). After i.v. bolus the CCNU concentration in plasma fell faster than that of BCNU. The rate of CCNU decomposition during incubation with blood at 37 degrees C was 3 times lower than that of BCNU.

Two lines of Walker carcinoma 256: their peculiarities and different interactions with the host.

Two sublines of Walker 256 carcinoma have been characterized for their ability to metastasize and to induce cachexia. The invasive, metastasizing line A induced terminal anorexia in rats with a mean survival time of 27 +/- 1.5 days. The non-invasive line B induced early anorexia and cachexia with a mean survival time of only 15 +/- 1 days. At death, the line B tumor was still smaller than the line A one, and no metastases were detectable. These two sublines are discussed as a composite model for studying anorexia and cachexia together with invasion and metastasis.

Polarographic assay of submicrogram quantities of cis-dichlorodiamineplatinum (II) in biological samples.

Differential pulse polarographic assay of the antineoplastic agent cis-dichlorodiamineplatinum II and its analogues was performed after acid oxidative hydrolysis (HClO4, HNO3, HCl) of biological samples (plasma, tissue homogenates, urine) and reaction with ethylenediamine. Platinum levels and kinetics were determined in blood and urine of patients with non-oat-cell lung carcinoma. Detection limit of the polarographic assay was 0.5 ng platinum; analytical error was +/- 3%. Levels of free cis-dichlorodiamineplatinum (II) in plasma fell in samples stored at -20 degrees C; the half-life of free drug was 38 h.