Pharmacokinetic study of darbepoetin alfa: absorption, distribution, and excretion after a single intravenous and subcutaneous administration to rats.

KRN321 is a hyperglycosylated analogue of recombinant human erythropoietin (rHuEPO, epoetin alfa), and its absorption, distribution, and excretion have been studied after a single intravenous and subcutaneous administration of 125I-KRN321 at a dose of 0.5 microg kg-1 to male rats. The half-lives of immunoreactive radioactivity in the terminal phase after intravenous and subcutaneous administration were 14.05 and 14.36 h, respectively, and the bioavailability rate after subcutaneous administration was 47%. The total radioactivity in tissues was lower than that in the serum in all tissues excluding the thyroid gland and skin at the injection site (subcutaneous administration). The maximum concentrations were observed in the bone marrow or skin at the injection site followed by the thyroid gland, kidneys, adrenal glands, spleen, lungs, stomach and bladder. The radioactivity found in trichloroacetic acid-precipitated fractions suggested that a high-molecular weight compound, unchanged or mixed with endogenous protein, distributed to the tissues after administration. The whole-body autoradiographic findings in both groups were in agreement with the tissue distribution mentioned above. The blood cell uptake of KRN321 was low for both groups. The excretion ratios of radioactivity into urine and faeces up to 168 h were 71.4 and 14.1% after the intravenous administration and 74.9 and 12.0% after the subcutaneous administration. There was no difference in the excretion profile of radioactivity between the two groups.

Role of antennary structure of N-linked sugar chains in renal handling of recombinant human erythropoietin.

To elucidate the role of the branched structure of sugar chains of human erythropoietin (EPO) in the expression of in vivo activity, the pharmacokinetic profile of a less active recombinant human EPO sample (EPO-bi) enriched with biantennary sugar chains was compared with that of a highly active control EPO sample enriched with tetraantennary sugar chains. After an intravenous injection in rats, 125I-EPO-bi disappeared from the plasma with 3.2 times greater total body clearance (Cltot) than control 125I-EPO. Whole-body autoradiography after 20 minutes of administration indicated that the overall distribution of radioactivity is similar, but 125I-EPO-bi showed a higher level of radioactivity in the kidneys than control 125I-EPO. Quantitative determination of radioactivity in the tissues also indicated that radioactivity of 125I-EPO-bi in the kidneys was two times higher than that of control 125I-EPO. The difference in plasma disappearance between 125I-EPO-bi and control 125I-EPO was not observed in bilaterally nephrectomized rats. The distribution of 125I-EPO-bi to bone marrow and spleen was similarly inhibited by simultaneous injection of excess amounts of either the nonlabeled EPO-bi or control EPO. These results indicate that the low in vivo biologic activity of EPO-bi results from rapid clearance from the systemic circulation by renal handling. Thus, the well-branched structure of the N-linked sugar chain of EPO is suggested to play an important role in maintaining its higher plasma level, which guarantees an effective transfer to target organs and stimulation of erythroid progenitor cells.

Cardiotoxicity of combined administration of adriamycin and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats-with special reference to 125I-MIBG cardioautoradiography and histopathological findings.

We studied whether adriamycin(ADM)-induced myocardial disorder in rats is advanced when recombinant human granulocyte colony-stimulating factor (rhG-CSF) is administered. Rats were divided into three groups (15 rats/group), i.e. the ADM group, the ADM+rhG-CSF group, and the vehicle-treated control group. ADM (2 mg/kg, i.p.) was administered for the first 2 days in each cycle and 10 days of administration of rhG-CSF (50 micrograms/kg, s.c.) was started two days after the second dose of ADM was given in each cycle. The dosing cycle was repeated 3 times. One day after the last dose, the following parameters were analyzed: peripheral blood and bone marrow cells, electrocardiogram (ECG) and histopathological findings. Four hours after intravenous administration of 125I-metaiodobenzylguanidine (125I-MIBG), accumulation of 125I-MIBG in some organs and findings from autoradiography (ARG) of the heart were examined. ECG revealed an extended ventricular activation (VAT) time in the ADM and ADM+rhG-CSF groups. In histopathological analysis, vacuolar degeneration of the myocardium was observed in both the ADM and ADM+rhG-CSF groups. The degree of change was the same for both groups. The accumulation of 125I-MIBG in the heart was lower in both the ADM and ADM+rhG-CSF groups than in the control group. The same tendency was observed in ARG, but the difference between the ADM group and the ADM+rhG-CSF group was not significant. These results suggest that administration of rhG-CSF at the standard clinical dose does not aggravate ADM-induced myocardial disorder. However, because this disorder may be more clearly manifested by treatment with higher doses of ADM, it is necessary to conduct further studies on the methods of dosing and administration.

Improvement of anemia in W/WV mice by recombinant human erythropoietin (rHuEPO) mediated through EPO receptors with lowered affinity.

We studied the effects of recombinant human erythropoietin (rHuEPO) on anemic W/WV mice which manifested severe anemia accompanied by mutation of the W gene encoding tyrosine kinase type receptor (c-kit gene) of bone marrow hematopoietic cells. Nine-week-old male W/WV mice or normal littermates (+/+) were used. Since serum EPO concentration in W/WV mice increased in proportion to severity of anemia, EPO production in the kidneys of these animals was found to be regulated normally. Hematocrit in +/+ mice increased and a maximal response was also obtained with 2,000 IU/kg of rHuEPO. On the other hand, hematocrit in W/WV mice increased in a dose-responsive manner by administration with 2,000 and 10,000 IU/kg, showing different responses to rHuEPO in these two types of mice. The responsiveness of W/WV mice to rHuEPO was low in terms of increases in erythroblastic precursor cells (CFU-E), and immature cells in the bone marrow. Scatchard analysis of the specific binding of 125I-rHuEPO against bone marrow cells revealed that the different responsiveness to rHuEPO between W/WV and +/+ mice may be correlated with differences in affinity of EPO receptor of bone marrow cells in these mice. From these results, a high dose of rHuEPO is capable of improving the anemia in W/WV mice that had EPO receptors with lowered affinity, indicating the possible effectiveness of rHuEPO in anemic patients with EPO receptor abnormality.

Differential role in lipid peroxidation between rat P450 1A1 and P450 1A2.

The role of cytochrome P450 (P450) in lipid peroxidation induced by NADPH or peroxide was investigated in a reconstituted system. When cumene hydroperoxide, t-butyl hydroperoxide and hydrogen peroxide were used as initiators, the rates of malondialdehyde (MDA) formation were much higher in a reconstituted system containing P450 1A1 than those observed in a reconstituted system containing P450 1A2. In contrast to peroxide-induced lipid peroxidation, P450 1A2 catalysed NADPH-induced lipid peroxidation more effectively than did P450 1A1 regardless of the presence of ADP-Fe(NO3)3. Carbon monoxide inhibited NADPH-induced formation of MDA in a reconstituted system containing P450 1A2, but not P450 1A1. In addition, superoxide dismutase (SOD) was an effective inhibitor in a NADPH-induced lipid peroxidation system catalysed by P450 1A2 but not by P450 1A1. These results suggest that a peroxide-induced reaction might proceed readily with P450 1A1, whereas P450 1A2 mainly functions in NADPH-induced lipid peroxidation via generation of an active oxygen species. It is furthermore indicated that the difference in the effect of SOD in NADPH-induced lipid peroxidation depends on the P450 used.

Polyamine lowered the hepatic lipid peroxide level in rats.

This is the first report for the hepatic lipid peroxide lowering effect of spermine in vivo. The influence of administration of polyamines on hepatic lipid peroxide level has been investigated by using normal or carbon tetrachloride (CCl4)-treated rats. Spermine was found to lower the hepatic lipid peroxide level most efficiently among polyamines used in CCl4-treated rats. In addition, the extent of liver enlargement caused by CCl4 treatment was reduced by spermine administration. Lipid peroxide lowering effect of spermine was also observed in normal rats. Hepatic spermine content was significantly increased in both normal and CCl4-treated rats after administration of spermine. Clear inverse relationship between the content of lipid peroxide and the concentration of spermine was observed. In reconstituted system containing NADPH-cytochrome P-450 reductase and extracted hepatic microsomal lipid, spermine inhibited the NADPH-dependent lipid peroxidation effectively at the concentration of 0.1 mM. From these results, we concluded that spermine exerted an inhibitory effect of lipid peroxidation in vivo as well as in vitro.