A high performance liquid chromatographic method of analysis of 4'-O-tetrahydropyranyladriamycin and their metabolites in biological samples.

A method for measuring 4'-O-tetrahydropyranyladriamycin (THP) and its metabolites in biological samples are described. By reversed-phase high performance liquid chromatography using fluorescence detection, THP and its metabolites were all separated on a single chromatogram within 18 minutes. A linear calibration curve was obtained up to 2,000 ng/ml of THP in plasma. The recovery of THP in the analysis was more than 95% above 5 ng/ml and 87.1% even at 1.25 ng/ml. Thus the lower limit was 1.25 ng/ml in biological samples. Blood levels and urinary excretion in mice and dogs were satisfactory measured by this analytical method.

[Effect of (2"R)-4'-O-tetrahydropyranyladriamycin, a new antitumor antibiotic, on the bone marrow function of rabbits. (1) Intravenous administration by a single bolus injection].

New Zealand White rabbits were treated with (2"R)-4'-O-tetrahydropyranyladriamycin-HCl (THP), a new antitumor antibiotic, by an intravenous bolus injection at a dose of 1, 2 or 4 mg/kg. The peripheral leucocyte counts decreased markedly at doses of 2 and 4 mg/kg 1 to 7 days after injection, and the lymphocytes and neutrophils were affected. The nucleated cell count decreased in the bone marrow. Especially 3 days after injection, remarkable reductions of erythroids and immatured myelocytes were observed, with a subsequent rise of the matured myelocytes ratio in bone marrow cell constituents. These changes resulted in a marked increase of M/E ratio. Doxorubicin also showed an inhibitory effect on the bone marrow function of rabbits but the effect was slightly lower than THP. These changes of bone marrow cells reverted 7 days after injection and the recovery of the reduced peripheral leucocyte was also observed 14 days after injection. Therefore, it can be concluded that THP showed suppressive but reversible effects on the bone marrow function of rabbits.

[Effect of (2"R)-4'-O-tetrahydropyranyladriamycin, a new antitumor antibiotic, on the bone marrow function of rabbits. (2) Repeated intravenous injections].

(2''R)-4'-O-Tetrahydropyranyladriamycin-HCl (THP), a new antitumor antibiotic, was intravenously injected to New Zealand White rabbits at a dose of 2.5 mg/kg every 2 weeks for 6 weeks (3 courses) or at a dose of 0.5 mg/kg/day daily for the first 5 days of a 2-week course for 6 weeks (3 courses). The total dose was 7.5 mg/kg in both dosing schedules. The peripheral leucocyte and erythrocyte counts decreased. The leucocyte count decreased to 57% of the initial count on Day 3 in the first course and then increased gradually. The decrease was smaller in the divided dosing schedule than the single dosing. The nucleated cells, especially immatured myelocytes and erythroids reduced remarkably. Subsequently the matured myelocyte ratio in bone marrow cell constituents increased and the M/E ratio increased. These changes were observed on Day 3 and reverted by Day 9 in each course. The divided dosing schedule resulted in a higher nadir. All the changes in the peripheral blood and the bone marrow reverted even after the 3 course-treatment.

[Effect of aclacinomycin A on bone marrow (author's transl)].

Japan White rabbits were treated with aclacinomycin A, a new anthracycline antitumor antibiotic, at a dose of 6.25 or 25.0 mg/kg by single intravenous, or 12.5 or 50.0 mg/kg by single oral administration, respectively. Beagle dogs were treated at a dose of 3.0 or 6.0 mg/kg by single intravenous injection. In rabbits in higher dose groups, RBC and WBC counts as well as lymphocyte ratio in peripheral blood decreased on day 1. Nucleated cell counts and erythroid elements in bone marrow decreased to raise M/E ratio (Myeloid/Erythroid ratio) on day 3. In a dog given at 6.0 mg/kg, WBC and platelet counts, lymphocyte and neutrocyte per cents in peripheral blood and also nucleated cells, particularly erythroid elements in bone marrow remarkably decreased on day 3 accompanied with an increase in M/E ratio. These changes were almost completely recovered by day 14 in both animals. No abnormalities were found in lower dose groups. Male Wistar rats, treated with the drug at a dose of 1.5 mg/kg by daily intraperitoneal injection for 30 days, showed slight decreases in peripheral WBC and RBC counts and M/E ratio in bone marrow. No change was observed in rats treated at 0.75 mg/kg and less for 30 days.

[Irritative effect of aclacinomycin A on the eye mucous membrane, skin and muscle (author's transl)].

Aclacinomycin A, a new anthracycline antitumor antibiotic, was given to rabbits by single instillation or single intracutaneous injection and to guinea pigs by single subcutaneous or intramuscular injection to examine the irritative effect. Slight dilatation of blood vessel and swelling in the conjunctiva and nictitating membrane were observed in the eyes of rabbits given 1% solution. Edema in the bulbar conjunctiva, iris and cornea was histologically noted. Washing of the eyes after drug instillation prevented these damages. Subcutaneous and intracutaneous injections of 1% solution (0.2 ml) gave hyperemia, swelling and necrosis at injection site. Cellular infiltration, fibrosis and necrosis were histologically detected. Intramuscular injection of 1% solution (0.2 ml) also provided induration and swelling at injection site. Histologically cellular infiltration and necrosis were observed. These irritative effects were much slighter in administration of 0.1% solution. No change was observed with 0.01% solution.

[General pharmacology of (2"R)-4'-O-tetrahydropyranyladriamycin, a new antitumor antibiotic].

General pharmacology of (2"R)-4'-O-tetrahydropyranyladriamycin hydrochloride (THP) was studied in experimental animals. Intravenous administration of THP showed no significant effect on the respiratory and cardiovascular systems, such as blood pressure, heart rate, ECG and respiration in anesthetized rabbits and dogs. But in rats and cats, THP produced a transient decrease in blood pressure resulted from vasodilation. The hypotension was not inhibited by antihistaminics. Contraction of isolated guinea-pig atria was stimulated by THP at high concentrations (10(-4) g/ml). THP inhibited the spontaneous movement of isolated rabbit ileum and rat uterus (virgin and pregnant) at high concentrations (10(-4) g/ml). In some isolated guinea-pig ileum preparations, THP partially (6 approximately 36%) antagonized the contraction inducing by acetylcholine, histamine, serotonin and barium chloride. Urine volume and urinary excretion of electrolytes were increased by intravenous injection of 5.0 mg/kg THP. Vascular permeability was progressed when administered intracutaneously. Hemolytic effect was shown at high concentrations (10(-4) g/ml) but no effect on the coagulation was found. No significant effect of THP was observed on the general behavior and central nervous system, autonomic nervous and peripheral nervous systems. Also, THP had no significant effect on gastrointestinal propulsion in mice, the mucous membranes of the stomach and duodenum of rats, or gastric acid and bile secretion in rats.

Acute local irritative effect of (2''R)-4'-O-tetrahydropyranyladriamycin, a new antitumor antibiotic.

(2"R)-4'-O-Tetrahydropyranyladriamycin hydrochloride (THP), a new antitumor antibiotic, was administered to rabbits at a concentration from 0.02 to 0.5% by instillation, or by intracutaneous, subcutaneous or intramuscular injection to study its local irritative effect. The irritative effect of THP increased with concentration. At a concentration of 0.5%, THP was irritant to the eye, skin and muscle but at a concentration of 0.1% practically no effect was observed. The effect was equal to or lower than that of doxorubicin. An instillation of 0.5% THP caused reversible irritation effect on the eye. Slight conjunctival responses (redness and chemoisis) were observed. Rinsing reduced the irritative effect. Intracutaneous injection of 0.1 ml of 0.5% THP caused well defined, moderate erythema, surface ulceration and dermal necrosis. Cutaneous muscle necrosis also occurred. At a concentration of 0.02%, dermal necrosis and inflammatory cell infiltration were observed. Erythema, as well as muscle necrosis and calcification with giant cell reaction and inflammatory cell infiltration were observed by an intramuscular injection at a concentration of 0.5%. Subcutaneous injection of 0.5% THP showed no irritative effect.

Pharmacokinetics and disposition of (2''R)-4'-O-tetrahydropyranyladriamycin in dogs.

The pharmacokinetics and physiological disposition of a novel anthracyclines, (2''R)-4'-O-tetrahydropyranyladriamycin (THP) were studied in dogs by an HPLC analysis. The THP administered intravenously (1.5 mg/kg) disappeared rapidly from the plasma immediately after an injection of the drug. The plasma level of THP was lowered in a triphasic pattern up to 24 hours and was simulated by a three-compartment open model in which the half-lives of alpha-, beta- and gamma-phase were calculated to be 0.0116 hour, 0.152 hour and 7.02 hours, respectively. The blood cell level of THP was about 10 times as high as the plasma level during the observation. In the study of tissue distribution of THP 2 and 8 hours after the administration, the highest concentration of THP was found in the spleen and lung and these concentrations were diminished quickly. However, in the lymph nodes and bone marrow concentrations of THP increased with a lapse of time. THP and its metabolites were excreted in the bile by 2.7% of dose during 8 hours in the bile-cannulated dogs. Urinary recovery of THP and its metabolites was about 1.3% of the dose up to 72 hours. In these experiment, THP was metabolized to THP-OH and ADM, and to aglycones which were excreted in conjugated forms. The results obtained from a similar study on ADM were compared and discussed.

[Studies on the absorption, excretion and distribution of aclacinomycin A: absorption, excretion and distribution of 14C- or 3H-aclacinomycin A in mice, rats and rabbits (author's transl)].

A new anthracycline antitumor antibiotic, aclacinomycin A, was labeled with 3H uniformly or with 14C simultaneously at the anthracycline nucleus and L-rhodosamine. These labeled drugs were administered intravenously to normal dd mice, solid type Sarcoma 180 tumor-bearing ICR mice, normal or pregnant Wistar rats and normal rabbits, respectively. 14C-Aclacinomycin A given to rabbits (5 mg/kg) was rapidly cleared from the blood and transferred to tissues. But low level of radioactivity (equivalent to about 0.5 mcg/ml) was remained in the blood even 8 approximately 10 hours after administration. About 45% of the radioactivity were recovered from the urine and 20% from the feces by 72 hours after administration. Tissue levels of 3H-14C-aclacinomycin A given to normal and tumor-bearing mice were highest in the lungs and spleen. Higher distribution was observed also in the liver and kidneys 2 hours after administration. Bioassay revealed that the drug was present in the lungs and spleen in biologically active form and in the liver and kidneys in inactive form, respectively. In the tumor tissue the radioactivity was low but it persisted for 48 hours. Autoradiography with 14C-aclacinomycin A in rats demonstrated that radioactivity due to the drug distributed in the lungs, spleen, kidneys, thymus, intestine, lymph nodes, bone marrow, salivary gland, hypophysis and pineal body but it was rapidly cleared. About 0.2% of radioactivity given to a pregnant rat were transferred to a fetus when 14C-aclacinomycin A was administered intravenously on the 18 approximately 19th day of pregnancy.

[Studies on the absorption, excretion and distribution of aclacinomycin A: absorption, excretion and distribution of aclacinomycin A in mice, rabbits and dogs by photometric assay (author's transl)].

An anthracycline antitumor antibiotic, aclacinomycin A, was given to mice, rabbits or dogs intravenously to study the pharmacokinetics by photometric assay based on the absorption of anthracycline ring. The drug was rapidly eliminated from the blood in these animals. Drug levels were much higher in the blood cells than in the plasma. Tissue levels in dogs were 50 approximately 100 times higher than the blood levels, which showed the drug was rapidly transferred from the blood to tissues after administration. Higher levels were observed in the lungs, spleen and lymph nodes, where the drug was present as aclacinomycin A itself and the glycoside-type metabolites that were biologically active. The active form was also detected in the pancreas, heart, thymus, bone marrow and gastrointestinal tract. In the liver and kidneys, biologically inactive aglycone-type metabolites were observed. About 2 approximately 4% of the drug given to rabbits or dogs was recovered in the urine by 72 hours after administration, in which only 10% of the excreted drug was active form in rabbits but about 65% in dogs. The rest was inactive aglycone-type metabolites that were excreted almost in the conjugated form. Biliary excretion also contributed to the total clearance of the drug. Aclacinomycin A was absorbed even by oral administration in rabbits and dogs. Tissue distribution of the drug orally given to dogs was similar to that in intravenous administration, except that higher levels of active form were detected in the gastrointestinal tract and of inactive form in the liver.

Radioimmunoassay for aclacinomycin A.

An antibody against aclacinomycin A (ACM) was produced in a rabbit by immunization with ACM-bovine serum albumin conjugate. The radioimmunoassay (RIA) was based on the competition of unlabeled anthracycline with 3H-labeled ACM for binding sites on a specific antibody. Antibody-bound and free antigen were separated by selective adsorption on dextran-coated charcoal. The antibody reacted equally with ACM and its metabolites, MA144 M1, MA144 S1 and aklavin, and could precisely distinguish aklavinone-related aglycones, adriamycin and daunomycin. RIA was sensitive in the range of 1 approximately 10 pmol per assay. The quantities of ACM and its metabolites in human plasma were practically determined without any pretreatment of physiological samples.

Immunological cross-reactivities of various anthracycline antibiotics against aclacinomycin A antisera.

Aclacinomycin A (ACM) antisera were obtained from the rabbits immunized with 4"'-deoxo-4"'-(R)-amino-ACM- or N,N-didemethyl-ACM-bovine serum albumin conjugate, and their immunoreactivities were tested with ACM-related anthracyclines. It was found that the binding ability with the ACM antisera was markedly decreased by the following structural changes in ACM: N,N-didemethylation of the rhodosamine moiety; 6-O- or 4-O-methylation; removal of the methoxycarbonyl group at C-10; hydroxylation at C-1, C-2 or C-11. It was less affected by some alterations in a side chain at C-9 or by deglycosidation of the terminal mono or disaccharide. The binding of the aglycone (aklavinone) was very weak.

Molecular cloning, characterization, and expression of wheat cystatins.

We cloned four kinds of cDNAs of wheat cystatins (WCs), WC1, WC2, WC3, and WC4, from the seed. They had 47-68% amino acid sequence similarities to other plant cystatins. WC1, WC2, and WC4 had 63-67% similalities to one another while 93% of amino acids were identical between WC1 and WC3. This suggested that WCI, WC2, and WC4 should be regarded as the isoforms of wheat cystatins. The mRNAs for WC1, WC2, and WC4 were all expressed in seed at an early stage of maturation and, after that, their quantities decreased gradually. However, each of the mRNAs was again expressed one day after the start of germination and the expression continued for the following five days. WC1 seemed to be expressed at a higher level than WC2 and WC4. Immunostaining for looking at site-specific expression of each WC demonstrated that both WC1 and WC4 existed in the aleuron layer and embryo, but in the endosperm the only existing species was WC1. Differences in mRNA level and tissue localization found for the WCs may suggest their differential physiological roles.