Comparison of pharmacokinetics of clodronate after single and repeated doses.

OBJECTIVE: Pharmacokinetics of orally given clodronate disodium, a drug for the treatment of hypercalcemia and bone resorption, were studied after a single dose of 400, 800 and 1600 mg given randomly to 11 healthy volunteers in a crossover manner, in 7-14 hospitalized cancer patients given 400, 800 and 1600 mg twice daily, each dosage for one week, and during the customary therapy in 15 additional cancer patients treated in hospital with 400 mg thrice daily for > or = 2 weeks. METHODS: Clodronate concentrations in serum and urine were measured by capillary gaschromatography with mass-selective detection. Pharmacokinetic parameters were calculated with a three-compartmental model. RESULTS: After a single oral dose to healthy volunteers the absolute clodronate concentrations increased almost dose-dependently. The mean cumulative excretion in urine was 1.72-2.77% of the dose, an interindividual range being from 0.92% to 5.52%. With 800 and 1600 mg twice daily for one week to cancer patients the serum drug concentrations increased almost progressively with increasing the dose. In cancer patients serum drug concentrations were clearly higher and renal drug clearances (mean 25-62 ml/min) lower than in healthy volunteers (mean 123-149 ml/min). The mean urinary excretions were 2.24-3.14% of the dose and interindividual ranges from 0.18% to 19.0%. During the routine cancer therapy with 400 mg thrice daily, the clodronate excretions in urine on two successive days were on an average 3.26% (range 0.0-10.5%). CONCLUSIONS: Absolute concentrations in serum and excretions in urine of orally given clodronate increase dose-dependently, but during the maintenance therapy in hospitalized cancer patients the renal drug clearances seem to be lower than in healthy volunteers. This and the large interindividual variation in kinetics propose therapeutic monitoring of clodronate for optimizing the oral dose of the drug.

Inhibition of human and rabbit platelet aggregation by chlorophenoxyacid herbicides.

Inhibition of human platelet aggregation by eight chlorophenoxyacid herbicides was studied in vitro. Thrombocyte aggregation in the platelet-rich plasma was induced by 1.0-32.0 microM adenosine diphosphate (ADP), 0.32-32.0 microM adrenaline or 7.5-30.0 micrograms/ml collagen with and without chlorophenoxyacid (0.05-2.0 mg/ml). Platelet aggregation by each inducer was inhibited dose dependently by all the eight chlorophenoxyacids at concentrations between 0.1 and 2.0 mg/ml. Increasing the concentrations of ADP and collagen but not of adrenaline inhibited the antiaggregatory action of chlorophenoxy-acids. No essential differences in inhibitory effect were found between different chlorophenoxyacids varying in respect of their ring substituents and the length of the carboxylic side chain. In the platelet-rich plasma prepared from rabbits 2.5 h after subcutaneous injection of 2.4-dichlorophenoxyacetic acid or 4-chloro-2-methylphenoxyacetic acid (100-150 mg/kg), platelet aggregation by ADP was inhibited 20-30%, compared to plasma taken from the rabbits before the chlorophenoxyacid treatment. The inhibition had disappeared by 20-23 h after administration. The results indicate that chlorophenoxyacid herbicides inhibit human platelet aggregation. Furthermore, the inhibition is probably involved in haemorrhages known to occur in various tissues of animals intoxicated by chlorophenoxyacid herbicides.

Effect of central muscle relaxants on single-dose pharmacokinetics of peroral paracetamol in man.

Paracetamol (acetaminophen) at a single, 160-450 mg dose was given perorally in combination with central muscle relaxants (CMRs) carisoprodol (200 mg), chlormezanone (100 mg) or orphenadrine (35 mg) in a double-blind, randomized, cross-over study in 10 healthy volunteers. The pharmacokinetic parameters of paracetamol remained unaltered in the presence of the CMRs as compared with those observed after paracetamol without additives, in spite of nearly twenty-fold differences in the dissolution rate between the products. Paracetamol is absorbed mostly in the duodenum, and therefore there is enough time for even the slowly dissolving tablets to release the active principle before the gastric contents are transferred to the area of paracetamol absorption. Some CMRs are anticholinergic compounds and may affect intestinal motility. Our results show, however, that CMRs do not significantly alter the pharmacokinetics of paracetamol, and presumably the antipyretic or analgetic efficacy of paracetamol is not impaired when combination formulations of paracetamol and CMRs are used.

Distribution of three common chlorophenoxyacetic acid herbicides into the rat brain.

The distribution of three common 14C-labelled chlorophenoxyacetic acid herbicides (2,4-dichlorophenoxyacetic acid or 2,4-D, 2-methyl-4-chlorophenoxyacetic acid or MCPA, 2,4,5-trichlorophenoxyacetic acid or 2,4,5-T) into the different brain areas was studied in rats pretreated with toxic doses of the herbicides (238-475 mg/kg). Also, their binding to proteins in rat plasma was determined in vitro by increasing the concentrations of chlorophenoxyacetic acids in the incubate from 0 to 1 mg/ml. Both 2,4-D and MCPA pretreatments increased brain concentrations of 14C-labelled herbicides more markedly than 2,4,5-T pretreatments did. No essential differences were found in the distribution between the different brain areas. Protein-unbound fractions of 2,4-D and MCPA in the plasma were clearly higher than those of 2,4,5-T but the highest herbicide concentration increased the protein-unbound fraction of 2,4,5-T more (7-13-fold) than of 2,4-D and MCPA (5-fold). The results suggest that the greater increase in the penetration into the brain of 2,4-D and MCPA than of 2,4,5-T during their intoxication is due to some factors other than the changes in their binding to plasma proteins and mere enhanced diffusion through the blood-brain barrier.

Increase in the acute toxicity and brain concentrations of chlorophenoxyacetic acids by probenecid in rats.

1. Probenecid increased the acute toxicity of chlorophenoxyacetic acids (2,4-D, 2,4,5-T and MCPA) in rats. 2. Probenecid increased the brain to plasma ratios of all the three 14C-labelled chlorophenoxyacetic acids. The increase was due only partly to the displacement of chlorophenoxyacids from their binding sites in rat plasma proteins by probenecid. 3. Probenecid did not change significantly the intracerebral distribution pattern of 14C-labelled chlorophenoxyacetic acids.

The uptake of clodronate (dichloromethylene bisphosphonate) by macrophages in vivo and in vitro.

Clodronate (dichloromethylene bisphosphonate) accumualtes extensively in the bone by binding to apatite crystals. We found recently that the drug also accumulates in the spleen and, to a lesser extent, in the liver of mice and rats. In the present study, the role of macrophages in soft tissue accumulation was studied in mice by autoradiography and macrophage-depleting techniques, and also by isolated rat peritoneal macrophages. The localization of [14C]clodronate in mouse spleen showed that the drug concentrates in the marginal zone between the white and red pulp, which is known to be rich in macrophages. Pretreatment of mice with pure clodronate did not change the accumulation of [14C]clodronate in the spleen. However, when splenic and hepatic macrophages were eliminated by the liposome-encapsulated clodronate, only a weak [14C]clodronate accumulation occurred in these organs. Isolated macrophages did not take up free [14C]clodronate, but the addition of ferrous iron to the incubate resulted in the uptake of 14C activity by macrophages. They were probably stimulated by the insoluble clodronate-iron complex. The results suggest that 1) macrophages are involved in the accumulation of clodronate in the spleen and liver, and 2) combination of clodronate with extracellular iron is a prerequisite for the activation of macrophages to take up the drug complex. Since the spleen and, to a lesser extent, the liver are rich in iron released from destroyed red cells, accumulation of clodronate takes place in these organs.

Effects of 2,4-dichlorophenoxyacetic acid (2,4-D) on biogenic amines and their acidic metabolites in brain and cerebrospinal fluid of rats.

Effects of single subcutaneous doses of sodium 2,4-dichlorophenoxyacetate (2,4-D-Na) on biogenic amines and their acidic metabolites in rat brain and cerebrospinal fluid (CSF) were analyzed by high pressure liquid chromatography. After 200 mg/kg 2,4-D-Na, the cerebral concentration of 5-hydroxytryptamine (5-HT) was increased slightly and that of 5-hydroxyindoleacetic acid (5-HIAA) roughly 3-fold between 1 and 8 h after the administration. There was also a tendency towards slightly lowered dopamine (DA) levels. No statistically significant changes in brain concentrations of noradrenaline (NA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) or tryptophan (TRY) were found. At the same time, however, the maximal increase in DOPAC, HVA and 5-HIAA concentrations in the CSF was 2.3-5.8-fold. The dependency of biogenic amines and metabolites on 2,4-D-Na dose was studied by injecting s.c. 0, 10, 30 and 100 mg/kg and sacrificing the rats at 2 h. In the brain, there was a dose-dependent increase in concentrations of 5-HIAA (at the two highest doses) and HVA (at the highest dose) while in the CSF those of all three acidic metabolites increased at the two highest doses. The 10 mg/kg dose had no effect. The results agree with the hypothesis that 2,4-D inhibits the organic acid transport out of the brain, which should then result in increased cerebral levels of acidic metabolites of biogenic amines, but it may also have effects on the activity of serotoninergic and dopaminergic neurones.

Comparative study on cerebrovascular injuries by three chlorophenoxyacetic acids (2,4-D, 2,4,5-T and MCPA).

At toxic doses in rats, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA) caused injuries in blood vessels in the brain. 2. 2,4-D caused extravasations of the circulating Evans blue-albumin complex also in the spinal cord. 3. By contrast, potency in damaging cerebral vessels was almost non-existent with 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). 4. None of the three chlorophenoxyacetic acids caused such cerebrovascular injuries in mice, guinea pigs, Syrian hamsters, rabbits and chickens.

Distribution of [14C]clodronate (dichloromethylene bisphosphonate) disodium in mice.

The distribution of 14C-labeled clodronate (dichloromethylene bisphosphonate), a new bisphosphonate for the treatment of osteolytic bone metastases and hypercalcemia, was studied in mice by whole-body autoradiography and by measuring the 14C activities in various tissues [14C]Clodronate was administered into the tail vein, and its distribution was followed from 5 min to 90 days after the injection. The drug disappeared promptly from the plasma and accumulated intensively in the bone and moderately in the spleen. In both tissues, relatively high radioactivities were measured as late as 90 days after the [14C]clodronate administration. Small amounts of 14C activity were also detected in the liver for 90 days. The results agree well with the previous observations that bisphosphonates deposit rapidly in the bone. Our findings indicate further that clodronate accumulates in the bone and the spleen for several months.

Evidence for a dissimilarity of chicken oviducts differentiated by diethylstilboestrol and oestradiol-17 beta: a study of progesterone-induced egg-white protein (avidin) synthesis.

The growth and differentiation of chick oviducts were caused by daily diethylstilboestrol (DES) or oestradiol-17 beta (E2) injections, and the effects of these oestrogens on the progesterone-induced production of a biotin-binding egg-white protein (avidin) were studied. In the DES primed oviducts, but not in the E2 primed ones, both DES and E2 administered with progesterone potentiated avidin production 2 to 3-fold, even after 10-day oestrogen withdrawal. The results suggest that DES and E2 prime the avian reproductive target tissue differently.

The occurrence and production of avidin: a new conception of the high-affinity biotin-binding protein.

The production of avidin, a high-affinity biotin-binding egg-white protein, is not restricted to the avian, amphibian and reptilian oviducts. In the acute phase of inflammation, avidin is synthesized and secreted by various injured tissues in the domestic fowl, both male and female. Also in other avian species and a lizard, injured tissues produce an avidin-like biotin-binding factor. The non-oviductal production of avidin in domestic fowl has a great variety of inducers, for example acute inflammation caused by mechanical or thermal tissue injury, septic bacterial infection and (toxic) drugs, and even retrovirus-induced cell transformation. In culture, chicken embryo fibroblasts and yolk sac macrophages synthesize and secrete avidin. Besides the albumen, avidin may act as an antibacterial protein also in the tissues.

Correlation between brain tissue and cerebrospinal fluid concentrations of selected compounds in rats with intact and injured blood-brain barrier.

We examined how the concentrations of exogenous compounds (and their metabolites) in rat brain tissue correlate with their concentrations in the cerebrospinal fluid (CSF) (i) when the blood-brain barrier (B-BB) is intact and increasing doses of compounds are administered, and (ii) when the function of the B-BB is impaired and small constant doses are given. The impairment of B-BB function was caused by chlorophenoxyacetic acid herbicides. An excellent linear correlation between brain tissue and CSF concentrations, calculated from 14C activities, was obtained with 14C-p-aminobenzoic acid, 14C-antipyrine, and 14C-sucrose administered in increasing doses (to 320 mg/kg, i.v.) to B-BB-intact animals (r = 0.92 to 0.99, P less than 0.001). After the administration of small, constant, i.v. doses of 14C-methyl-4-chlorophenoxyacetic acid, 14C-2,4-dichlorophenoxyacetic acid, or 14C-p-aminobenzoic acid into B-BB-damaged rats, both brain tissue and CSF radioactivities increased about linearly 10-fold or more when the lesions progressed. A close correlation between brain tissue and CSF radioactivities was calculated with each compound (r = 0.97 to 0.99, P less than 0.001). In tests with small constant amounts of 14C-antipyrine, 14C-sucrose, and 125I-labeled human albumin, however, no marked linear correlation could be drawn. This was due to the fact that these compounds always caused roughly the same 14C activities both in the brain tissue and in CSF, i.e., their cerebral penetration was only slightly or negligibly affected by the B-BB impairment. The results suggest that significant changes in compound penetration into rat brain tissue can be monitored in the CSF.

Effects of oestradiol-17 beta and diethylstilboestrol on progesterone-induced protein (avidin) production in chick oviduct: evidence for differences in the actions of steroidal and non-steroidal oestrogens.

The effects of diethylstilboestrol (DES) and oestradiol-17 beta on the production of a progesterone-induced protein (avidin) in the chick oviduct were studied. Chicks were pretreated with DES or oestradiol-17 beta daily for 3-28 days and progesterone (10 or 20 mg/kg) was administered 24 h after the last oestrogen injection. Diethylstilboestrol (2-20 mg/kg) administered with progesterone to chicks pretreated with DES increased avidin production after 16 h compared with that induced by progesterone alone. The potentiation of avidin production appeared even when DES was administered between 6 h before and 13 h after progesterone injection. The length of DES pretreatment did not affect the potentiation. The amount of avidin induced by progesterone in the chicks pretreated with oestradiol-17 beta was similar to that in the chicks pretreated with DES. Oestradiol-17 beta (2-20 mg/kg) administered with progesterone, however, did not affect avidin production. The results suggest that DES may have some non-oestrogenic effects on the production of progesterone-induced proteins.

Immunoelectron microscopic localization of a progesterone-inducible protein (avidin) in the chick oviduct mucosa.

Avidin, a specific progesterone inducible protein, was localized in the oviduct magnum mucosa of chicks treated with diethylstilbestrol (DES) or DES plus progesterone, using ultrastructural immunoperoxidase techniques. Diffusion technique and the double or triple layer peroxidase staining method were applied. The results obtained by immunoelectron microscopic peroxidase techniques with high resolution power indicated that progesterone stimulation in the DES-treated chicks resulted in avidin production in the goblet cells of the oviduct epithelium. The sensitive-antiperoxidase staining method revealed a slight avidin production in many goblet cells of chicks treated with only DES. This method also showed some avidin-positive ciliated epithelial cells in chicks treated with progesterone. This results suggest that some ciliated epithelial cells may have functional or metabolic properties characteristic of secretory goblet cells.

Increase in the penetration of tracer compounds into the rat brain during 2-methyl-4-chlorophenoxyacetic acid (MCPA) intoxication.

The penetration of different intravenous tracer molecules such as 14C-labelled 2-methyl-4-chlorophenoxyacetic acid (14C-MCPA), 14C-p-aminobenzoic acid (14C-PABA), 14C-sucrose, 14C-antipyrine and iodinated (125I) human albumin (125I-HA) into the brain and cerebrospinal fluid (CSF) was studied in MCPA-intoxicated and control rats. Toxic subcutaneous doses of sodium salt of MCPA (200-500 mg/kg) increased highly the brain/plasma and CSF/plasma ratios of 14C-MCPA and 14C-PABA, as compared to the muscle/plasma ratio. Probenecid (200 mg/kg) did not affect the cerebral MCPA concentration in the intoxicated animals. The tissue/plasma ratios of 14C-sucrose, 14C-antipyrine and 125I-HA were also increased in the brain and CSF of intoxicated animals, but the increases were less pronounced than those of 14C-MCPA or 14C-PABA. The results indicate that MCPA intoxication caused a selective damage of the blood-brain barrier in the brain areas studied.

Similarity of biotin-binding activity and immunoreactivity in chicken oviduct and non-oviduct avidin.

Biotin-binding and immunological methods were employed to demonstrate the similarity of oviduct and non-oviduct avidin in the chicken. Oviduct avidin was induced after oestrogen pretreatment by progesterone and non-oviduct avidin by intestinal tissue injury or by intraperitoneal actinomycin D administration. Avidin in the intestine, lung, bursa of Fabricius, plasma, pectoral muscle and liver after injury had biotin-binding activity similar to that of progesterone-induced oviduct avidin: (1) a temperature of 79-83 degree C was required for 50% of the maximum [14C]biotin uptake, (2) maximal exchange occurred only at 90 or 100 degree C and (3) denaturation of protein, i.e., loss of biotin-binding activity, was not yet observed at 100 degree C. Avidin in the intestine, lung, bursa of Fabricius, plasma and pectoral muscle also showed an identical cross-reaction with oviduct avidin. Furthermore, the increase in avidin-like biotin binding in the oviduct and most non-oviduct tissues was significantly correlated with the increase in avidin-like antigen in the tissue. This indicates that avidin induced in chicken non-oviduct tissues by injury or inflammation caused by actinomycin D administration is similar to progesterone-dependent oviduct avidin.