Fluorescence quenching method for the determination of bleomycins A5 and A2 with halofluorescein dyes.

In weak acidic medium, the anticancer antibiotics bleomycin A5 (BLMA5) and bleomycin A2 (BLMA2) bind with halofluorescein dyes, such as erythrosin (Ery), eosin Y (EY) and eosin B (EB), to form ion-association complexes, which causes fluorescence quenching of halofluorescein dyes. The quenching values (DeltaF) are directly in proportional to the concentrations of bleomycins over the range 0.09-2.5 microg/mL. Based on this, a fluorescence quenching method for the determination of BLMA5 and BLMA2 has been developed. The dynamic range is 0.12-2.5 microg/mL for the determination of BLMA5 and 0.09-2.0 microg/mL for BLMA2, with detection limits (3sigma) of 0.04 microg/mL for BLMA5, 0.03 microg/mL for BLMA2, respectively. It has been applied to determine the two antibiotics in human serum, urine and rabbit serum samples. The recovery is in the range 90-102%. In this work, the optimum reaction conditions and the spectral characteristics of the fluorescence are investigated. The reasons for fluorescence quenching are discussed, based on the fluorescence theory.

Application of radioimmunoassay to monitor treatment of human cerebral gliomas with bleomycin entrapped within liposomes.

A radioimmunoassay was assessed for its suitability for monitoring the blood and urinary concentrations of bleomycin entrapped within large unilamellar liposomes in patients receiving therapeutic doses of the drug. Bleomycin entrapped within liposomes was administered by direct intracerebral injection to three patients with grade III to IV cerebral gliomas, twice weekly for up to six weeks. No clinically important toxic effects were observed which could have been attributed to these preparations, although all three patients showed progressive deterioration in their clinical condition. This type of treatment may be more beneficial to patients at an earlier stage of their disease.

Intravesical bleomycin in bladder tumour prophylaxis.

The authors have evaluated the efficacy, tolerability and systemic absorption of bleomycin administered by intravesical route in the prophylaxis of recurrences of superficial vesical tumours after endoscopic resection (TUR). Thirty mg of bleomycin dissolved in 30 ml of saline solution were instilled in the bladder once a week for the first month and then once a month for 12 months. The instilled fluid was held in the bladder for 1 hour. The systemic absorption of the drug was evaluated by a microbiological assay of bleomycin in plasma and in the perfusion liquid recovered from the bladder. Fifteen patients were treated; most had a long history of multiple recurrent tumours despite previous intravesical treatments. Thirteen patients presented tumour recurrences during the treatment; 4 of them were given a second course of instillations following a second TUR and 3 of these presented further recurrences. The authors conclude that the drug, administered intravesically to high-risk patients with the modalities employed in the present study, was not effective in preventing recurrences. It was well tolerated locally and was free from systemic toxicity. The amount of bleomycin that was not recovered from the bladder after a contact time of one hour was relatively high but the drug did not reach measurable levels in the peripheral blood.

A radioimmunoassay procedure for tallysomycin S10b in human plasma and urine.

A simple and sensitive radioimmunoassay (RIA) procedure was developed and validated for the analysis of tallysomycin S10b in human plasma and urine. The assay utilized antisera developed in rabbits, 125I-tallysomycin as the radioligand, and dextran-coated charcoal to separate free and bound antigen. The antibody was specific in that it did not cross-react with either tallysomycin A or bleomycin. The lower limit of quantification was 5 ng per ml plasma or urine, and the linear range of the assay was 5-320 ng tallysomycin S10b base per ml plasma or urine. The within-day assay variability (%RSD) for plasma and urine was 11% at a concentration of 50 ng per ml, and 3% and 7% for plasma and urine, respectively at 200 ng per ml. Within-day accuracy ranged from 100% to 108% of the theoretical value. Tallysomycin S10b was stable in human plasma and urine at concentrations of 20 and 160 ng per ml for at least 7 months when stored at -20 degrees C. The method was applied to the analysis of plasma and urine samples from a patient given tallysomycin S10b as part of a phase I study.

Endolymphatic application of bleomycin oil suspension in dog model.

To explore the potential usefulness of a cytostatic agent deposited directly in lymph nodes, 4ml Bleomycin oil suspension (Oil-Bleo) was injected over one hour into hind leg lymphatics of seven dogs. Five of these dogs received a second, identical dose one week later into lymphatics of the contralateral hind leg. Peak serum concentration of Oil-Bleo after the first injection (7 dogs) was 12.3 micrograms/ml but after the second injection (5 dogs) was slightly lower (10.8 micrograms/ml). Maximum level of Oil-Bleo in blood was 12% of the endolymphatic dose and represented the "spillover" from lymph transport. In a control experiment, in which 60mg of aqueous Bleomycin was injected, the serum spillover was one-third higher. Large amounts of Oil-Bleo were stored in popliteal and retroperitoneal lymph nodes for several weeks. Twenty-four hours after injection the weight of "treated" lymph nodes was 73% greater than "untreated" nodes and one month later treated lymph nodes were still 37% heavier. After 24 hours, 4.7% of Oil-Bleo instilled was distributed within extracted lymph nodes, and one month later 0.12% was still detectable. By contrast, after aqueous Bleomycin infusion, only 0.05% was detected in these lymph nodes after only six hours. In general, lymph nodal architecture was preserved after Oil-Bleo. Together the findings suggest that Bleomycin oil suspension may be a useful agent for treatment of lymph nodal metastases by endolymphatic infusion.

[Studies on organ distribution, absorption and excretion of pepleomycin sulfate (NK631) (author's transl)].

1. Organ distribution of pepleomycin (NK631) in mice and rats was studied. NK631 was found at higher levels than bleomycin (BLM) in skin, lung, stomach, solid tumor, etc. in mice and rats. Furthermore NK631 was detected in the mesenteric and lumbar lymph node, esophagus and prostate in rats and also distributed at about twice as high levels as BLM in the AH109A hepatoma cell-metastasized lymph nodes. 2. For the elucidation of reason on low pulmonary toxicity of NK631 which is in spite of 1.5 times highly distribution in lung compared with BLM, inactivation of various BLMs by high molecular fraction of lung of mice and rats was determined. The order of inactivation rate of various BLMs in lung was as follows: BLM-M5196 greater than NK631 greater than BLM greater than BLM-HPE. There is an encouraging coincidence between index of pulmonary fibrosis in mice and inactivating rate in lung. 3. A comparative study on the serum level and urinary excretion of NK631 and BLM was performed in dogs. The blood level and urinary excretion rate of both drugs were almost similar. 4. The blood levels of NK631 were comparable to those of BLM in cancer patients.

[Absorption, excretion, distribution and metabolism of 3H-labelled pepleomycin sulfate (author's transl)].

Absorption, excretion, distribution and metabolism of pepleomycin sulfate (NK631) were investigated in rats after intravenous administration of 3H-NK631 by whole-body autoradiography, CM-Sephadex column chromatography and high performance liquid chromatography. The highest radioactivity was found in kidney, followed by cartilage, blood, lung, esophagus, adrenal gland and skin at 0.5 hour after the administration and little radioactivity was observed in central nerve system. The NK631 and deamide NK631 were identified as major (90%) and minor metabolites in the 24-hour urine.

Radioimmunoassay of bleomycin in plasma and urine.

Antibodies to bleomycin were raised by immunization of sheep and rabbits with bleomycin-albumin conjugates. The combination of a high-titre, high-avidity sheep antiserum and iodinated bleomycin produced a radioimmunoassay sensitive to 8 ng of bleomycin per ml of plasma or urine. Untreated specimens (100 microliter) of plasma or urine could be added directly to the assay tubes. The antiseerum was specific for bleomycin and showed no cross-reaction with other anticancer agents used in combination chemotherapy. Over a concentration range of 20-100 ng/ml, recovery of bleomycin from plasma was 110% and from urine, 93%. Repeated assay of plasma samples showed a decrease in bleomycin levels unless the samples were kept at 4 degrees C or below. Assay of bleomycin levels in plasma and urine from patients under treatment with bleomycin showed similarities with results reported using a microbiological assay. The radioimmunoassay offers a more reliable, rapid and sensitive method for the measurement of bleomycin.

Bleomycin, and antitumor antibiotic: improved microbiological assay and tissue distribution studies in normal mice.

A microbiological assay was developed for bleomycin, an antitumor antibiotic reported to be active in human trials. The assay bacterium was a strain of Escherichia coli which is resistant to ethionine. Studies revealed relatively high concentrations of bleomycin in the blood and urine of mice after a single dose, < 0.33 ld(10), injected intraperitoneally.