Single subcutaneous dosing of cefovecin in rhesus monkeys (Macaca mulatta): a pharmacokinetic study.

Cefovecin is a third-generation cephalosporin approved for antibacterial treatment with a 14-day dosing interval in dogs and cats. This antibiotic may also be useful for zoo and wildlife veterinary medicine, because of its broad spectrum and long duration of activity. The aim of the study was to determine whether cefovecin is a suitable antibiotic to prevent skin wound infection in rhesus monkeys. Therefore, the pharmacokinetics (PK) of cefovecin after a single subcutaneous injection at 8 mg/kg bodyweight in four rhesus monkeys (Macaca mulatta) and sensitivity of bacterial isolates from fresh skin wounds were determined. After administration, blood, urine, and feces were collected, and concentrations of cefovecin were determined. Further, the minimum inhibitory concentrations (MIC) for bacteria isolated from fresh skin wounds of monkeys during a health control program were determined. The mean maximum plasma concentration (C(max) ) of cefovecin was 78 µg/mL and was achieved after 57 min. The mean apparent long elimination half-life (t½) was 6.6 h and excretion occurred mainly via urine. The MIC for the majority of the bacteria examined was >100 µg/mL. The PK of cefovecin in rhesus monkeys is substantially different than for dogs and cats. Cefovecin rapidly reached C(max) which however was lower than most of the MIC levels and with a very short t½. Therefore, cefovecin is not recommended for treating skin wounds in rhesus monkeys.

Selected pharmacokinetic parameters for Cefovecin in hens and green iguanas.

The third generation cephalosporin cefovecin has been shown to have an exceptionally long elimination half-life in dogs and cats, making it suitable for antibacterial treatment with a 14-day dosing interval in these species. Pharmacokinetic parameters for cefovecin were investigated in juvenile hens and green iguanas, following subcutaneous injections with 10 mg cefovecin/kg bodyweight. Preliminary studies in eight additional species of birds and reptiles were performed and results were compared with the parameters found in hens and green iguanas. The kinetics were characterized by rapid absorption with peak plasma concentration of 6 +/- 2 microg/mL in hens and 35 +/- 12 microg/mL in green iguanas. The mean plasma half-life for cefovecin was 0.9 +/- 0.3 h for hens and 3.9 h in green iguanas. Volume of distribution was 1.6 +/- 0.5 L/kg for hens and 0.3 L/kg for green iguanas and clearance was 1252 +/- 185 mL.h/kg for hens and 53 mL.h/kg for green iguanas. Results from preliminary studies did not differ notably from those seen in hens and green iguanas. Cefovecin is not suitable for the treatment of bacterial infections with a 14-day dosing interval in hens or green iguanas and seems not to be in a number of other bird and retile species either.

Putative biomarkers for evaluating antibiotic treatment: an experimental model of porcine Actinobacillus pleuropneumoniae infection.

Biomarkers of infection were screened for their possible role as evaluators of antibiotic treatment in an aerosol infection model of porcine pneumonia caused by Actinobacillus pleuropneumoniae (Ap). Following infection of 12 pigs, clinical signs of pneumonia developed within 20 h, whereafter the animals received a single dose of either danofloxacin (2.5mg/kg) or tiamulin (10 mg/kg). To test the discriminative properties of the biomarkers, the dosage regimens were designed with an expected difference in therapeutic efficacy in favour of danofloxacin. Accordingly, the danofloxacin-treated pigs recovered clinically within 24h after treatment, whereas tiamulin-treated animals remained clinically ill until the end of the study, 48 h after treatment. A similar picture was seen for the biomarkers of infection. During the infection period, plasma C-reactive protein (CRP), interleukin-6 and haptoglobin increased, whereas plasma zinc, ascorbic acid and alpha-tocopherol decreased. In the danofloxacin-treated animals, CRP, interleukin-6, zinc, ascorbic acid and alpha-tocopherol reverted significantly towards normalisation within 24h of treatment. In contrast, signs of normalisation were absent (CRP, zinc and ascorbic acid) or less marked (interleukin-6 and alpha-tocopherol) in the tiamulin-treated animals. Plasma haptoglobin remained elevated throughout the study in both groups. This indicates that CRP, zinc, ascorbic acid and to a lesser extent interleukin-6 and alpha-tocopherol might be used to evaluate antibiotic treatment of acute Ap-infection in pigs. The present model provides a valuable tool in the evaluation of antibiotic treatments, offering the advantage of clinical and pathological examinations combined with the use of biochemical infection markers.

Interplant alkaloid variation and Senecio vernalis toxicity in cattle.

Senecio vernalis and other plants containing pyrrolizidine alkaloids (PA) are implicated in the poisoning of cattle. The liver is a known target organ. In this study the content of the alkaloids senecionine (SCO), senkirkin (SKK) and seneciphyllin (SCP) and their toxic effects in cattle were studied. The content of these 3 compounds only varied by a factor of 2 within 10 plant collections at different locations in western Denmark (Jutland). However, individual alkaloids varied 3-fold, and the interplant variation for some of the PA up to 8-fold. SCO and SKK had very short half lives, 20 min and 70 min respectively. In cattle fed dried plant material corresponding to 200 and 400 g of fresh material for 10 d alanine aminotransferase, alkaline phosphatase and g-glutamyl transferase activities remained unchanged. Cattle subsequently fed fresh plant material up to 1 kg/d for 8 d also had no change in liver enzyme activities. Cattle did not show any clinical signs of poisoning, and no morphological liver changes were observed.

Expression changes of CYP2A and CYP3A in microsomes from pig liver and cultured hepatocytes.

The P450 enzymes of the liver are responsible for the metabolism of a wide range of chemical compounds, and hepatocytes are used in pharmacological and toxicological in vitro tests. Thus, it is important to know how stable these enzymes are in culture. We measured the activity of CYP2A and CYP3A in microsomes isolated from both pig liver and primary pig hepatocyte cultures, together with the apoprotein concentration using Western blotting. The CYP2A activity and apoprotein concentration decreased rapidly; only about 5 percent remained after 48 hr incubation, whereas the CYP3A activity and apoprotein concentration was constant. CYP3A was induced 3 times after exposure to rifampicin, whereas neither rifampicin nor pyrazole could induce CYP2A. The hepatocytes were also incubated with varying concentration of FCS and autologous serum, however without effect on the stability of CYP2A, nor did different concentrations of growth hormone and testosterone added to the cultures have any effect.

Cytochrome P450 sex differences in minipigs and conventional pigs.

Minipigs have become a popular alternative to the traditional non-rodent species although little information is available on their P450 system. The total P450, the enzyme activity and immunochemical levels of some of the most important drug metabolizing isoenzymes CYP1A2, CYP2A6, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 were measured in liver microsomes from 8 minipigs and 12 conventional pigs of both sexes and castrate conventional pigs. The mRNA expression was analyzed for 3 isoenzymes: CYP1A2, CYP2A6 and CYP2E1. The total P450 activity was slightly higher in minipigs compared to conventional pigs but no sex differences were detected. CYP1A2 activity was 4 times higher in female than in male minipigs. The activity of the male minipigs possessed the same activity as were identical to the conventional females, males and castrates. The activity of CYP2E1 was 4 times higher in female than in male minipigs and 2 times higher in female than in male pigs. No activity of CYP2D6 or CYP2C19 could be detected. The CYP3A4 activity detected in minipigs was higher than the activity in conventional pigs. A slight sex difference was seen in both strains. Correlations between enzyme activity and immunochemical levels were found for CYP1A2, CYP2A6 and CYP3A4 but not for CYP2E1. The mRNA concentration of CYP1A2, CYP2A6 and CYP2E1 was determined because the activity of these enzymes showed marked sex differences. A Spearman ranking correlation analysis between mRNA expression and enzyme activity showed a weak correlation for CYP2A6, but not for CYP1A2 and CYP2E1. These results seem to indicate that CYP2A6 could be transcriptionally regulated, whereas CYP1A2 might be post-transcriptionally regulated.

Characterization of the P450 system in Göttingen minipigs.

It is essential to establish the activity and regulation of the cytochrome P450 system of species selected for toxicological and pharmacological studies. The minipig has become a popular substitute for the traditional non-rodent species although little information is available on its P450 system. The total P450 and the enzyme activity of the most important drug-metabolizing isoenzymes: CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 were measured in liver microsomes from 4 minipigs and 8 conventional pigs of both sexes. Immunochemical levels were determined for 4 of teh isoenzymes. The total P450 activity was slightly higher in minipigs compared to conventional pigs but no sex difference was detected. CYP1A2 activity (7-ethoxyresorufin) was 4 times higher in female minipigs than in male minipigs. The activity in male minipigs was almost identical to the activity in conventional pigs. The activity of CYP2E1 (chlorzoxazone) was 4 times higher in female than in male minipigs and 2 times higher in female than in male conventional pigs. No activity of CYP2D6 (debrisoquine) and CYP2C19 (mephenytoin) could be detected. The CYP3A4 activity (testosterone) detected in minipigs was higher than the activity in conventional pigs. A weak sex difference was seen in both strains. Western blotting using anti-human CYP2E1 and CYP3A4 confirmed the results obtained in the enzyme activity assays, while only CYP1A2 correlated with the activity in the conventional strain. The total P450 enzyme activity was close to the levels reported for human beings, as were the activities of CYP2E1 and CYP3A4.

p53 mutations in non-small cell lung carcinomas in Hong Kong.

Lung resections from 50 Chinese patients in Hong Kong diagnosed as having non-small cell lung carcinoma were examined for the presence of mutations in the p53 gene by polymerase chain reaction single-stranded conformation polymorphism methods and for aberrant protein expression by immunostaining techniques. Eight-point mutations in the evolutionarily conserved exon 5 through 8 regions were detected. Abnormal expression of p53 detectable by immunostaining techniques was seen in 23 specimens tested. There was no statistically significant correlation between the detection of p53 aberrations and age, sex, smoking history, histologic type, and tumor stage. Aberrant p53 protein levels detectable by immunostaining were significantly associated with the clinical and nodal staging of the tumors.

[Cloning and sequencing of CTP synthetase cDNA from Chinese hamster cells].

The CTP synthetase cDNA was cloned from Chinese hamster lung cells V79. By comparison of the amino acid sequence of CTP synthetase with the counterpart of other species published, it was show that the CTP synthetase was a highly conserved and stable gene among the mammalian cells, but more differences of the CTP synthetase sequence could be seen between procaryotic and eukaryotic cells. This phenomenon suggested that the catalysing model of the enzyme might have some small differences in the organisms.

Method for estimation of benzo[a]pyrene DNA adducts.

Polycyclic aromatic hydrocarbons, e.g., benzo[a]pyrene (B(a)P) are known carcinogens/mutagens. These compounds may be metabolized by the P450 mixed function monooxygenase to more nucleophilic compounds which may form adducts to the cellular macromolecules, e.g., DNA, RNA, and proteins. We have used synchronous fluorescence scanning for the assay of DNA adduct formation. In our earlier work with in vitro exposed human lymphocytes we estimated the adduct formation (femtomoles B(a)P per microgram DNA) to be higher than that estimated by other workers. We suggested that this difference may be related to the DNA isolation method used. In order to elucidate these differences we compared DNA adduct formation in human lymphocytes where DNA was isolated by the two different methods, i.e., using phenol extraction or the Gene Clean method. The data demonstrate that the phenol extraction procedure gives a yield of adducts per microgram DNA lower than that obtained by the Gene Clean method. The principle of the Gene Clean method for DNA isolation is protein denaturation by means of NaI followed by catching of DNA by absorption on silica particles. In contrast, the phenol extraction method is based upon phenol-mediated denaturation of proteins in the cell lysate leaving the hydrophilic nucleotides in the aqueous phase. However, during adduct formation more lipophilic adducts derived from DNA may redistribute between the aqueous phase and the phenol phase. In support of this theory we found higher adduct concentration per microgram DNA by the Gene Clean method 40 to 60 times than that found by the phenol method.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of in vitro acute cytotoxicity data obtained on human lymphocytes with known calculated human LD values of 20 selected drugs.

The acute in vitro cytotoxicity of 20 selected drugs at different concentrations was assayed biochemically by the leakage of DNA and lactate dehydrogenase from cells to medium. These two methods were supplemented with the MTT assay. The selected drugs had known estimated human LD values and had previously been tested in HeLa cells using morphological changes as an endpoint. The mitochondrial assay system usually gave smaller values than the other two methods. Spearman rank correlation analysis revealed correlation coefficients between the human estimated LD values and the LC(50) values found in the MTT, LDH and DNA assays of 0.68, 0.67 and 0.68, respectively.