Enhanced efficacy of a liposomal pentamidine preparation in Pneumocystis carinii in an ex vivo/in vitro culture model without feederlayer cells.

The effects of pentamidine isethionate, phosphatidylcholine-liposomes (PPC-lip) and combinations of both on the survival of Pneumocystis carinii trophozoites in a culture system without feederlayer cells were quantified. Pentamidine or PPC-lip alone reduced significantly the number of cells, with a clear dose dependence. Combinations of both drugs showed overadditive effects on cell counts reduction. Additional EM-investigations showed a rapid destruction of cell structures especially with the pentamidine/phospholipids-combinations. Our results could justify clinical tests with these combinations in AIDS patients in order to reduce the pentamidine dose, reduce local side effects with inhaled pentamidine while at least maintaining efficacy.

The use of short-term cultures of Pneumocystis for drug testing.

A method for short-term drug testing for Pneumocystis is described. Up to now there was no successful cultivation method without feederlayers. Parasites for inocula were obtained from nude rats, kept under conventional conditions. Pneumocystis separation was done by two centrifugation steps from lung homogenates. Cultivation in a modified Tegoshi medium resulted in preservation of parasites for five days, however there was no growth of P. carinii. Quantification was done microscopically by counting trophozoites in triplicate cultures. The percentage of cysts with intracystic bodies did not exceed 2% of parasites. Tests with pentamidine isethionate as a reference drug demonstrated the suitability of this system for drug evaluation.

Pentamidine aerosol for prophylaxis of Pneumocystis carinii pneumonia after BMT.

Following BMT there is a 5-15% risk of interstitial pneumonia caused by Pneumocystis carinii (PcP). Cotrimoxazole is therefore administered prophylactically, but may cause myelodepression, allergic reactions and nephrotoxicity. As PcP prophylaxis with pentamidine aerosol is effective in patients with AIDS, we conducted a prospective trial with regular inhalations of pentamidine. The aim of this study was to evaluate toxicity, safety, practicability and possible resorption of aerosolized pentamidine. We treated 31 allogeneic and 12 autologous BMT patients with 60 mg pentamidine 3 days before and 14 days after BMT. Starting 4 weeks after BMT, 300 mg pentamidine was given every 4 weeks for 6 months. There was no pneumonia caused by Pneumocystis carinii. The only noteworthy side-effects were cough (19.8%), salivation (9.6%), and sore throat (5.7%), of similar frequency after allogeneic or autologous BMT. Using high pressure liquid chromatography, pentamidine could only be detected in the serum of 33-54% of patients tested. In these patients the median serum levels were 7.5-9 ng/ml. We conclude that pentamidine aerosol has only minor side-effects, is well tolerated and safe, and is therefore an attractive alternative for PcP prophylaxis after BMT.

[Risk factors and prevention of pneumocystis carinii pneumonia after bone marrow transplantation]. / Risikofaktoren und Prophylaxe der Pneumocystis-carinii-Pneumonie nach Knochenmarktransplantation.

For six months after bone marrow transplantation (BMT) there is a risk of 5 to 15% to suffer from interstitial pneumonia due to pneumocystis carinii (PcP). Prophylaxis with trimethoprim/sulfamethoxazol is therefore routinely and successfully applied. However myelotoxicity, allergic reactions, augmentation of the risk of nephrotoxicity with cyclosporine A and noncompliance may be serious problems. Since the prophylaxis of PcP with pentamidine-aerosol proved to be effective in patients with AIDS, we conducted a prospective trial with regular inhalations of pentamidine. The aim of this study was to evaluate the toxicity, safety, practicability and possible resorption of pentamidine when applied as aerosol. The first of 43 patients were treated with 60 mg pentamidine on two days before, at the day of BMT and 14 days after BMT. Starting four weeks after BMT, 300 mg pentamidine were given every four weeks up to six months. After the study, the four 60 mg inhalations were replaced by two 300 mg inhalations before BMT, because this proved to be more convenient for the patients. There was no pneumonia due to pneumocystis carinii. The only noteworthy side effects observed were cough (19.8%), salivation (9.6%) and sore throat (5.7%). In general pentamidine was well tolerated and well accepted by the patients. Pentamidine could only be detected in the serum of 40 to 60% of all patients. In those patients the serum levels were 7.5 to 9 ng/ml and similar to concentrations found in comparable patients with AIDS. We conclude, that pentamidine-aerosol has only minor side effects, is well tolerated and safe and is therefore an attractive alternative for PcP-prophylaxis after BMT.

Autoradiographic examination of the organ distribution of 14C-labelled pentamidine in rats after intravenous and inhaled administration.

The organ distribution of 14C pentamidine was studied in Sprague Dawley rats by means of whole-body autoradiography following intravenous and inhaled application of pentamidine (Pentacarinat, CAS 140-64-7). The distribution time after intravenous administration of 5 mg pentamidine per kg rat was 30 min, 6 h, 24 h, and 7 days, respectively. The corresponding times after administration of aerosolized pentamidine were 30 min and 24 h. The distribution of radioactivity was also determined by measuring radioactivity in punch specimens of 100 microns sections. Besides renal excretion, the excretion of pentamidine in the bile and via the salivary glands was assessed by autoradiography. Further target organs included the spleen and the bone marrow. As early as after 30 min no radioactivity was detectable in the blood vessels. A lack of radioactivity in the brain tissue with accumulation of pentamidine in the meninges suggests that pentamidine does not cross the blood/brain barrier. Following intravenous administration the lung uptake of pentamidine was relatively low. However, increased drug concentrations were recorded in the bronchial system. The elimination time of pentamidine from the target organs was long. Lung pentamidine concentrations remained almost unchanged for a period of 7 days. After inhaled administration high levels of pentamidine were measured in the lung. 14C pentamidine was also located in the oropharyngeal and gastrointestinal tract, the drug stemming from pentamidine ingested or licked off the skin by the animals. No further target organs were verifiable. From the pharmacological point of view, these studies prove the advantages of pentamidine aerosol in the treatment of Pneumocystis carinii pneumonia (PCP) and provide information on extrarenal excretion mechanisms and deep compartments.

Single dose pefloxacin compared with multiple dose co-trimoxazole in cystitis.

In a double-blind multicentre study the efficacy and safety of a single-dose treatment with pefloxacin (800 mg) was compared with a five-day treatment regimen of 960 mg co-trimoxazole twice daily in the therapy of acute uncomplicated cystitis in women. In order to maintain blindness, patients in the pefloxacin group received placebo to complement the full number of tablets. Nine centres were involved; 155 patients received pefloxacin and 161 patients received co-trimoxazole. Of these, 140 patients treated with pefloxacin and 145 with co-trimoxazole were considered valid for efficacy and safety analysis. At the first follow-up, after seven to ten days, 97.1% of the pefloxacin group and 95.2% of the co-trimoxazole group were bacteriologically cured. At the second follow-up visit, after 28 to 42 days, the urine culture was negative in 95.0% of the pefloxacin group and 90.3% of the co-trimoxazole group. A single dose of 800 mg pefloxacin was demonstrated to be as safe and at least as effective as a five-day regimen of co-trimoxazole in the treatment of uncomplicated cystitis.

Therapeutic efficacy of two different cytostatic-linked phosphonates in combination with razoxane in the transplantable osteosarcoma of the rat.

Two new compounds, 4-[4-[bis-(2-chloroethyl)-amino-]phenyl]-1- hydroxybutane-1,1-bisphosphonic acid (BAD) and aminotris-(methylenephosphonato)-diamminoplatinum(II) (ADP) that both have cytostatic and osteotropic properties, have shown good therapeutic efficacy against an osteosarcoma which metastasizes and kills by lung metastases. We therefore combined each of these drugs with the antimetastic agent razoxane. Razoxane (20 mg/kg i.p., 5 days/week for 6 weeks) was administered in combination with either BAD (30 mg/kg i.p.) or ADP (37.5 mg/kg i.v.) twice weekly for 3 weeks. Tumour volumes, body weight, survival time and occurrence of metastases were recorded, in addition to the measurement of the metastasis area compared to the total lung area in serial histological lung samples. In both experiments, razoxane effected a significant increase in life span while being ineffective in tumour inhibition. Razoxane in combination with BAD displayed an enhanced anticancer activity which was not significant. ADP had a good antineoplastic activity and a large increase in survival time (144 per cent ILS). Razoxane used in combination with ADP did not influence antitumour efficacy. Median survivals of both ADP-treated groups were significantly longer than the razoxane-treated group. Analysis of the lung metastasis area showed a maximum of 57 per cent for the controls while all treated groups occupied a lesser area. The lowest metastases area was found with the combination treatment BAD + RAZ (18 per cent). This was considered an antimetastatic effect, while ADP treatment effected a time delay only. No change in metastatic pattern was observed in any of the treatment groups. Histological examination showed no effect on the capillaries in the proliferating region of the tumours that could account for the lower occurrence of metastases.

[Autoradiography study of organ distribution of 14C pentamidine following intravenous and inhalational administration in rats]. / Autoradiographische Untersuchung der Organverteilung von 14C-Pentamidin nach intravenöser und inhalativer Verabreichung an Ratten.

The organ distribution of 14C pentamidine was studied in Sprague Dawley rats by means of whole-body autoradiography following intravenous application and inhalation of aerosolized pentamidine. The distribution time after intravenous administration of 5 mg pentamidine per kg rat was 30 minutes, six hours, 24 hours, and seven days respectively. The corresponding times after administration of aerosolized pentamidine were 30 minutes and 24 hours. By measuring radioactivity in punch specimens of 100 microns sections, the distribution of radioactivity could also be determined semiquantitatively. Besides renal excretion, the excretion of pentamidine in the bile and via the salivary glands was assessed by autoradiography. Further target organs included the spleen and the bone marrow. As early as after 30 minutes no radioactivity was detectable in the blood vessels. A lack of radioactivity in the brain tissue, with concentration of pentamidine in the meninges, suggests that pentamidine does not pass the blood/brain barrier. Following intravenous administration, the lung uptake of pentamidine was relatively low. However, increased drug levels were recorded in the bronchial system. The elimination time of pentamidine from the target organs was long. The lung levels of pentamidine remained almost unchanged for a period of seven days. Following application by inhalation, high levels of pentamidine were measured in the lung. 14C pentamidine was also detectable in the oropharyngeal and gastrointestinal tract, the drug stemming from pentamidine ingested or licked off the skin by the animals. Other target organs could not be demonstrated. From the pharmacological point of view, these studies prove the advantages of pentamidine aerosol in the treatment of pneumocystis carinii pneumonia and provide information on extrarenal excretion mechanisms and deep compartments.

[Initial experiences with pentamidine aerosol in prevention of Pneumocystis carinii pneumonia following bone marrow transplantation]. / Erste Erfahrungen mit Pentamidin-Aerosol zur Prophylaxe der Pneumocystis-carinii-Pneumonie nach Knochenmarktransplantation.

After bone marrow transplantation there is a risk of 10% to acquire a pneumonia caused by pneumocystis carinii, if no prophylaxis is used. So far cotrimoxazol is the treatment of choice from day -14 before to day +180 after bone marrow transplantation. This substance, however, may cause allergic reactions, may augment the risk of nephrotoxicity of other drugs, and may be myelosuppressive. The prophylaxis with pentamidine-inhalation was used in 26 patients after bone marrow transplantation so far. It could be shown, that after salbutamol-inhalation 60 to 300 mg of pentamidine can be given safely in 14 to 28 days intervals. The main side effects were cough and dyspnea in some patients. Only minimal amounts of the drug could be detected in serum and urine after application. No toxic side effects and no pneumocystis carinii pneumonia were observed.

Anticancer-agent-linked phosphonates with antiosteolytic and antineoplastic properties: a promising perspective in the treatment of bone-related malignancies?

Bisphosphonates are compounds with a high affinity for bone and other calcified tissues. They inhibit tumor-induced bone destruction and the associated hypercalcemia by hindering the activity of the osteoclasts. Owing to a long biological half-life of bisphosphonates in the bone, a treatment using a prophylactic regimen seems possible. This paper summarizes preclinical studies with the bisphosphonate 3-amino-1-hydroxypropylidene-1,1-diphosphonic acid and two methyl derivatives; 3-N,N-dimethylamino-1-hydroxypropylidene-1,1-diphosphonic acid and 4-N,N-dimetyhlamino-1-hydroxybutylidene-1,1-diphosphonic acid with respect to their bone-protecting activity in therapy as well as in prophylaxis. To find substances that are useful for the treatment of primary tumor, as well as bone metastasis, we synthesized and tested cis-diammine[nitrilotris(methylphosphonato)(2-)-O1,N1]platin um(II) and cis-diammine[( bis-(phosphonatomethyl)amino]acetato(2-)-O1,N1)platinum(II), which contain both an osteotropic and an antineoplastic moiety. Experiments were carried out: (a) in the intratibial transplanted Walker carcinosarcoma 256B of the rat, which mimics osteolytic bone metastasis, and (b) in the transplantable osteosarcoma of the rat, which shows a histology and metastatic pattern similar to that found in man. These investigations indicate that it is possible to effect adjuvant therapy of bone metastases by combination of two compounds with different properties into one structure without losing the therapeutic characteristics of the parent compounds. They thus provide evidence that it may be possible to design compounds well suited for the therapeutic or prophylactic treatment of bone-related malignancies.

Assessment of the preclinical activity of budotitane in three different transplantable tumor systems, its lack of mutagenicity, and first results of clinical phase I studies.

The antitumor activity of budotitane was investigated in three different tumor systems--the transplantable murine ascitic-colon-adenocarcinoma MAC 15A, the TD-osteosarcoma of the rat, and the intramuscularly transplanted murine sarcoma 180. Marked inhibition of tumor growth was observed in the intramuscularly transplanted sarcoma 180, and cure rates of 50-80% were achieved in the colon adenocarcinoma MAC 15A. In contrast to these findings, bulotitane was inactive in the transplantable TD-osteosarcoma of the rat. Preliminary mutagenicity studies with the Salmonella typhimurium/mammalian microsome assay of Ames did not show any evidence of mutagenicity for the compound. The first results of the phase I clinical trials showed mild hepatotoxicity at a dose level of 15 mg/kg, dose-limiting nephrotoxicity at 21 mg/kg, and a reversible impairment of the sense of taste, beginning at a dose of 9 mg/kg.

Evaluation of spin labeled TEPA and CCNU analogs against osteosarcoma and MNU-induced mammary carcinoma of the SD-rat.

The effectiveness to reduce tumor growth by 1-(2-chloroethyl)-3-(1-oxyl-2,2,6,6-tetramethylpiperidinyl)- 1-nitrosourea (SLCNU) and N,N,N,'N'- bis(1,2-ethanediyl)-N"-(1-oxyl-2,2,6,6-tetramethyl- 2-piperidinylaminocarbonyl)-phosphoric triamide (SLDU) was studied in osteosarcoma and MNU-induced mammary carcinoma in the SD-rat. Both compounds elicited neither an inhibitory effect on these tumors nor an increase in the mean/median life span as compared to the control group.

Anticancer activity of bisphosphonic acids in methylnitrosourea-induced mammary carcinoma of the rat--benefit of combining bisphosphonates with cytostatic agents.

This study primarily describes the cytostatic activity of a bisphosphonate and of an alkylating agent linked bisphosphonate toward mammary carcinomas in vivo. Bisphosphonates had been shown to be therapeutically active in bone metastases. There is no animal tumor model available in which both primary mammary carcinomas and bone metastases can be studied simultaneously. Therefore, the Walker carcinosarcoma model, which was used as a model for bone metastasis in earlier studies, was combined with the M-methyl-N-nitrosourea (MNU) induced mammary carcinoma as a model for the primary tumor. Four-, or six-week treatment of MNU-induced mammary carcinomas in Sprague-Dawley rats with the new aromatic bisphosphonate 4[4-[bis(2-chloroethyl)-amino]-phenyl]-1-hydroxybutane-1, 1-bisphosphonate (BAD) showed higher antitumor activity than treatment with melphalan or with 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (APD) alone. BAD is the APD moiety covalently bound to a molecule derived from melphalan. A combination therapy with 11.75 mg/kg/day APD and 0.6 mg/kg/day melphalan showed the best therapeutic efficacy in this tumor model. In comparison to monotherapy with BAD, APD, or melphalan, a significantly higher rate of complete remissions was achieved. APD, itself, was not genotoxic in 3 employed short term assays. Since bisphosphonates had been shown to be therapeutically active in bone metastases, the antitumor potency of these compounds against experimental primary mammary carcinomas, coupled with the non-genotoxicity of APD and the inhibition of osteolytic bone metastases, might be an important advancement for adjuvant chemotherapy of human mammary carcinomas.

In vivo and in vitro investigations on biological effects of aromatic bis-(2-chloroethyl)amino-bisphosphonic acids, new agents proposed for chemotherapy of bone tumors: cytostatic activity in rat osteosarcoma; toxicity and genotoxicity in liver and bone marrow; mutagenicity in S. typhimurium.

Two new aromatic bis-(2-chloroethyl)-amino derivatives (BCMP and BAD) which are linked to osteotropic bisphosphonates were investigated for their therapeutical efficacy in rat osteosarcoma. Furthermore their genotoxic potential in vitro was determined in S. typhimurium and in mammalian cells. Finally, parameters for toxicity and genotoxicity were determined in liver and bone marrow cells following in vivo treatment. It was shown that BAD was of higher therapeutic effectiveness than BCMP. Both compounds induced approximately a two fold increase of his+ revertants in S. typhimurium TA1535 following metabolic activation by subcellular liver fractions. Both compounds also induced amplification of SV40 DNA in SV40 transformed cells (CO631). This endpoint may be of importance for acquired resistancy of cells during therapy. DNA-single strand breaks were induced by BCMP but not by BAD in liver cells and CO631 cell line. Following in vivo treatment BCMP was of higher genotoxic activity in liver cells than BAD. In comparison, genotoxicity of both compounds was much lower in bone marrow cells than in liver cells. BCMP was again more potent than BAD in inducing DNA single strand breaks, whereas BAD was more toxic. The higher therapeutic efficacy of BAD together with its lower genotoxic properties makes this compound superior to BCMP as a candidate for applied chemotherapy in humans.

Protective effects of a prophylactic treatment with the bisphosphonate 3-amino-1-hydroxypropane-1,1-bisphosphonic acid on the development of tumor osteopathies in the rat: experimental studies with the Walker carcinosarcoma 256.

The present report primarily describes protective effects of a long-term prophylactic treatment with 3-amino-1-hydroxypropane-1,1-bisphosphonic acid (APD) on the development of tumor-induced osteolytic bone destructions. Pretreatment with daily intravenous doses of APD 9.5 mg/kg for 1 week resulted in a significant reduction of Walker carcinosarcoma 256-induced bone destruction, when Walker cells were transplanted intraosseously (2 x 10(6) tumor cells/rat) 7 weeks later. Shorter pretreatment periods (4, 2 or 1 week prior to tumor inocculation) resulted in a nearly total inhibition of bone destruction as well as tumor-induced hypercalcemia. Tumor growth itself was not inhibited by APD pretreatment. Histological and microradiographical findings are reported. Consistent to our experiments and with regard to new immunocytological methods to detect single metastatic tumor cells in the bone marrow, potential risk groups may be defined which may profit from the prophylactic APD treatment to inhibit tumor-induced bone destructions.

Pharmacokinetics of the osteotropic diphosphonate 3-amino-1-hydroxypropane-1,1-diphosphonic acid in mammals.

3-Amino-1-hydroxypropane-1,1-diphosphonic acid (APD) is a potent drug in treatment of tumor induced osteolytic bone destruction and other bone diseases. The fate of 14C-labelled APD was studied in rats following oral and intravenous application. Intestinal absorption of APD was about 0.5% of the dose administered and radioactivity was mainly deposited in bone tissue. Liver, kidneys and blood contained very small amounts of radioactivity. After intravenous application, autoradiographic studies revealed high APD accumulation in sites of high bone turnover, in tracheal cartilage, and--only at high doses--in the liver. Biliary excretion of 14C-APD was very low. Body distribution of 14C-APD in mice was similar to that in rats. After intravenous application, APD was mainly excreted in the urine, in which no metabolites could be detected. Biological half-life of APD in rat bone was approximately 300 days. This seemed to be an important fact with regard to a possible use of APD in prophylactic treatment of cancer patients to prevent bone metastases.