Changes in cellular lipid synthesis of normal and neoplastic cells during cytolysis induced by alkyl lysophospholipid analogues.

Susceptibility of eight different cell types of murine or human origins to alkyl lysophospholipid analogue (ALP)-induced cytolysis correlated well with a selective, dose-dependent inhibition of radiolabeled oleic acid incorporation into phosphatidylcholine (PC) and a concomitant stimulation of incorporation into neutral lipids (NL), mainly triacylglycerols. In resistant cells (murine macrophages, L929S, K562, and rMeth A) a counts per minute NL/counts per minute PC ratio of 0.8-1.0 was observed with 30 micrograms ALP/ml; in sensitive tumor targets (Meth A, HL60, YAC, and ABLS-8.1) values greater than 2.7 were found with 5-10 micrograms ALP/ml. Changes in lipid metabolism preceded cytolysis in Meth A fibrosarcoma cells. In degradation experiments the percentage of total lipid radioactivity in PC was reduced after 24 hours to 47% compared to that in controls in sensitive Meth A with 10 micrograms ALP/ml. The macrophage-PC was unaffected at the same concentration. Sensitivity to ALP was independent of cell proliferation. Resistance was not restricted to normal cells and was inducible in Meth A (and rMeth A).

Inhibition of human tumor colony formation by the new alkyl lysophospholipid ilmofosine.

The cytostatic-cytotoxic effect of the new thioether alkyl lysophospholipid analogue Ilmofosine (1-hexadecylthio-2-methoxymethyl-rac-glycero-3-phosphocholine, BM 41.440) on colony formation of 30 different spontaneous human tumors was investigated in vitro with the use of a methylcellulose monolayer assay. Twenty-five neoplasms were sensitive to Ilmofosine within the concentration range tested (0-16 micrograms/ml). The most susceptible tumors were 2 colon carcinomas, 2 squamous cell carcinomas and 1 small-cell carcinoma of the lungs, 2 myosarcomas, 2 ovarian carcinomas, 1 gallbladder carcinoma, and 1 pleural mesothelioma with 50% colony formation inhibition doses ranging from 1.5 to 4.0 micrograms/ml. Due to the sensitivity criteria of the U.S. National Cancer Institute (greater than or equal to 70% inhibition of colony formation at 10 micrograms/ml), 15 of 30 tumors tested have to be considered as susceptible to Ilmofosine.

Ether phospholipids as inhibitors of the arachidonoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase in macrophages.

1-Alkylglycerophosphatide analogs which are known to activate macrophages to enhanced tumor cytotoxicity are structurally closely related to 1-acyl-sn-glycero-3-phosphocholine. In this study we have examined the influence of some of these compounds and of platelet-activating factor (PAF-acether, 1-0-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine) on the arachidonoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase (EC 2.3.1.23) in homogenate of bone-marrow-derived murine macrophages. This enzyme is suggested to be involved in the control of the availability of the icosanoid precursor, arachidonic acid. Kinetic experiments revealed apparent Km and V values for 1-palmitoyl-sn-glycero-3-phosphocholine of 6.0 microM and 16.10 nmol/mg protein per min, respectively. When the 1-palmitoyl-sn-glycero-3-phosphocholine concentration was equal to Km, the enzyme was dose-dependently inhibited by 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine with a 50% inhibition at 30 microM. The kinetic parameters in the presence of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (K'm = 10.0 microM, V' = 11.40 nmol X mg-1 X min-1) suggest that this alkyl phospholipid is a mixed-type inhibitor. All other alkyl analogs tested (1-O-methyl-2-O-octadecyl-rac-glycerol-3-phosphocholine, racemic PAF-acether, L-PAF-acether, D-1-O-hexadecyl-sn-glycero-3-phosphocholine, 1-O-octadecyl-rac-glycero-3-phosphocholine) inhibited the enzyme to various degrees. Arachidonic acid transfer to the 1-alkylglycerophosphatide analogs themselves could be ruled out under the assay conditions used. Therefore, we conclude that the arachidonoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase can be inhibited by synthetic and naturally occurring ether phospholipids in homogenate of bone-marrow-derived murine macrophages.

In vivo antitumor activity of ilmofosine.

Ilmofosine is a cytostatic/cytotoxic thioether phospholipid derivative. The in vivo anti-tumour activity of this compound was investigated in a methylcholanthrene (MethA)-induced fibrosarcoma and in the 3Lewis-lung carcinoma systems, respectively. Ilmofosine showed antineoplastic and antimetastatic properties at oral doses ranging from 0.625 to 40 mg/kg/day. Combination of Ilmofosine (p.o.) together with either cyclophosphamide (p.o.) or cis-DDP (i.v.) resulted in synergistic effects in vivo. These results demonstrate the in vivo antitumour activity of Ilmofosine in two tumour systems. The data indicate that direct cytostatic/cytotoxic effects of Ilmofosine are mainly responsible for its antitumour activity in vivo and which are increased by other cytotoxics.

Single dose, dose-escalating trial with fozivudine tidoxil (BM 21.1290).

BACKGROUND AND OBJECTIVES: In vitro and in vivo experimental data for fozivudine tidoxil [BM21.1290 (FZD) an ether-lipid conjugate of zidovudine] have shown better efficacy, no myelotoxicity and better tolerability compared with zidovudine. Therefore, the objectives of our study were to evaluate the safety of FZD in patients with human immunodeficiency virus (HIV) infection and to establish basic pharmacokinetic data. PATIENTS AND METHODS: In a Phase I dose-escalating trial, seven different single dose applications were studied in 39 patients: 50, 100, 300, 600, 900, 1200 and 1800 mg in capsule and tablet formulations. Inclusion criteria were HIV infection, CD4 count > 100 cells/mm3 and informed consent. Exclusion criteria were active opportunistic manifestations, concomitant zidovudine therapy and neutropenia (< 750 neutrophils/mm3). Safety parameters, 24 h plasma levels and urinary excretion were determined. RESULTS: The tolerance of FZD was excellent up to single doses of 1800 mg. In only one case, a single episode of loose stool was reproducible in a second treatment period and was therefore considered to be a probable drug-related event. In an amendment to the trial, a tablet formulation of FZD did not induce diarrhoea in this patient. FZD was available in measurable concentrations after 2 to 4 h. Maximum concentrations were reached after 4 to 8 h. After normalization for a dose of 100 mg/patient, the mean AUC was 8.6 mg x h/l and the mean Cmax was 1.13 mg/l; t1/2 was 3.78 h. Interestingly, plasma concentrations of zidovudine and zidovudine glucuronide were much lower than with equimolar zidovudine doses. CONCLUSIONS: The zidovudine conjugate FZD is safe and well tolerated at the seven doses tested. Phase II trials are warranted.

Phase I/II trial with fozivudine tidoxil (BM 21.1290): a 7 day randomized, placebo-controlled dose-escalating trial.

BACKGROUND AND OBJECTIVES: A Phase I dose-escalating trial with single doses of fozivudine tidoxil (BM21.1290; FZD), in vitro, and experimental in vivo data indicated that further investigations with this compound were warranted. Advantages of fozivudine tidoxil are the direct delivery of zidovudine monophosphate intracellularly and high concentrations in lymphatic tissues. Our objectives were to evaluate safety, tolerability and efficacy of fozivudine tidoxil in human immunodeficiency virus (HIV)-infected patients. PATIENTS AND METHODS: In a Phase I/II dose-escalating trial, three doses of fozivudine tidoxil (400, 800 or 1200 mg/day) were administered for 1 week. The study was randomized and placebo controlled. Inclusion criteria were HIV infection, CD4 count > 100 cells/mm3 and informed consent. The exclusion criteria were active opportunistic infection and prior antiretroviral treatment. RESULTS: The tolerability of fozivudine tidoxil was excellent in all dose groups. No treatment discontinuations were necessary. Steady-state pharmacokinetics did show slightly higher concentrations as compared with levels after the first dose (20%). Viral load reduction was most pronounced in the 1200 mg/day dose group (-0.64 log). No viral load reduction was seen in the placebo group. CONCLUSIONS: The zidovudine conjugate fozivudine tidoxil is safe and well tolerated at the three doses tested. Based on the differences in molecular weights, the 1200 mg dose is roughly equivalent to 400 mg zidovudine.

In vitro evaluation and characterization of newly designed alkylamidophospholipid analogues as anti-human immunodeficiency virus type 1 agents.

Our laboratories first reported two novel classes of complex synthetic lipids, including alkylamidophosphocholines (PC lipid; CP-51) and alkylamidophosphate ester-linked lipid-AZT conjugates (lipid-AZT conjugates; CP-92), with selective and potent activity against human immunodeficiency virus type 1 (HIV-1). To extend these observations, we synthesized additional PC lipids and lipid-AZT conjugates (INK and INK-AZT conjugate) to evaluate their structure-activity relationships by testing for selectivity against infectious wild-type (wt) and drug-resistant HIV-1 replication, virus fusogenic activity and toxicity for mouse bone marrow cells. PC lipid compounds with medium chain lengths at positions 1 and 2 gave an improved selective index (SI). INK-3, with 12 and 8 carbons and INK-15, with 10 and 12 carbons were among the most selective when evaluated in CEM-SS cells. INK-14, a lipid-AZT conjugate where AZT replaced the choline in PC lipid INK-3, gave the highest SI of > 1250 against both infectious wt HIV-1 replication in CEM-SS cells and a clinical isolate in peripheral blood leukocytes. Notably, the PC lipid compounds INK-3 and INK-15, but not the lipid-AZT conjugate INK-14, were potent inhibitors of matched pairs of AZT-sensitive and AZT-resistant HIV-1 clinical isolates. INK-3 also inhibited replication of HIV-2 and TIBO-resistant HIV-1, and inhibited HIV-1-mediated fusogenic activity by 78, 41 and 9% in a dose-dependent manner. The TC50 for mouse bone marrow cells was > 100 micrograms/ml for INK-3 compared to 9.15-14.17 micrograms/ml for CP-51 and 0.142-0.259 microgram/ml for AZT. These data suggest that optimum PC lipid compounds are significantly less toxic than AZT and have high potential as novel therapeutic agents for AIDS.

Antileishmanial activity of the ether phospholipid ilmofosine.

The ether phospholipid ilmofosine (BM 41.440) was active in vitro against amastigotes of Leishmania donovani and an antimony-resistant line of L. infantum in mouse peritoneal macrophages with ED50 values of 3.7 microM and 3.5 microM respectively. Ilmofosine was also active against L. donovani in BALB/c mice following oral and subcutaneous dosing, with an ED50 value of 10.5 mg/kg x 5 by the oral route.

Cytotoxic activity of the thioether phospholipid analogue BM 41.440 in primary human tumor cultures.

The inhibitory effect of the new thioether alkyl lysophospholipid analogue 1-hexadecylmercapto-2-methoxy-methyl-rac-glycero-3-phosphocholine (BM 41.440, Ilmofosine) on colony formation of different spontaneous human tumors was studied in vitro using a methyl cellulose monolayer assay. The most sensitive tumors were lung (small cell, squamous cell and adenocarcinomas), gastrointestinal and ovarian cancers and hyernephromas. On the basis of the current definition of sensitivity by the National Cancer Institute, Bethesda, MD, i.e. more than 70% inhibition of colony formation at an arbitrary concentration of 10 micrograms/ml, 34 out of 64 malignancies tested were susceptible to BM 41.440.

Synthesis of thioether phosphocholine analogues.

The synthesis of thioether phospholipids, which represent a new class of antitumor agents, is reported here. In particular, the route of synthesis of 3-hexadecylmercapto-2-methoxymethylpropyl-2'-trimethylammoni o-ethyl phosphate (BM 41.440, Ilmofosine), one of the most potent cytostatic/cytotoxic derivatives, is described in detail. Starting with diethyl bis-hydroxymethylmalonate, ethyl 2-phenyl-1,3-dioxane-5-carboxylate is formed via diethyl 2-phenyl-1,3-dioxane-5,5-dicarboxylate and 5-ethoxycar-bonyl-2-phenyl-1,3-dioxane-5-carboxylic acid. Reduction of ethyl 2-phenyl-1,3-dioxane-5-carboxylate with LiAlH4 affords 5-hydroxymethyl-2-phenyl-1,3-dioxane. Alkylation with dimethyl sulfate gives 5-methoxymethyl-2-phenyl-1,3-dioxane. The ring structure then is opened by N-bromosuccinimide, resulting in the formation of 3-bromo-2-methoxymethylproply benzoate. Reaction of 3-bromo-2-methoxymethylpropyl benzoate with the sodium salt of hexadecanethiol leads to 3-hexadecylmercapto-2-methoxy-methylpropanol, which is reacted with a cyclic chlorophosphate to give the corresponding phosphorylated 3-hexadecylmercapto-2-methoxymethylpropanol. Treatment with trimethylamine yields BM 41.440. This compound already has been tested in clinical phase I/II trials in West Germany.

Pharmacokinetics of the thioether phospholipid analogue BM 41.440 in rats.

BM 41.440 (1-hexadecylmercapto-2-methoxymethyl-rac-glycero-3-phosphocholine) is a cytotoxic thioether phospholipid analogue that recently has entered phase I trials in cancer patients. The objective of this study was to evaluate the pharmacokinetics of this compound in female rats after administration of a single oral dose (15 mg/kg body weight [bw] ). Furthermore, BM 41.440 serum concentrations were determined under a daily oral treatment of up to 13 weeks. Blood samples were obtained via permanent catheters from the femoral arteries before and after drug administration for a total of 120 hr. Urine was collected in 24 hr-intervals for 120 hr; the volume was measured, and aliquots were stored at -20 C until analytical determination of the thioether derivative. BM 41.440 was assayed in serum and urine by means of a specific, newly developed reverse-phase high pressure liquid chromatography technique. Mean maximum serum concentrations (1.7 micrograms/ml, n = 4 animals) were attained after seven hr. A terminal half-life of ca. 27 hr was calculated from the rate constant for the terminal elimination phase (lambda z approximately 0.026/hr). The mean serum BM 41.440 concentration-time-area-under-the-curve was 52.9 mg X hr/l. The ratio of total body clearance to absorption fraction was 4.7 ml/min X kg bw. Only a small amount of the drug was found in the urine. The quantity excreted in the urine during a 24 hr-interval never exceeded 1.5% of the administered dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Antitumor activity of Ilmofosine (BM 41.440) in the 3Lewis-lung carcinoma model.

Ilmofosine (1-hexadecylthio-2-methoxymethyl-1,3-propanediol-phosphocholine, BM 41.440) is a thioether phospholipid with cytostatic/cytotoxic properties. The antineoplastic activity of this compound was investigated in vivo in the 3Lewis-lung carcinoma system. 3Lewis lung tumor-bearing C57Bl/6 mice were treated with 0.625 to 40 mg Ilmofosine/kg per day p.o. either from days 1 to 9 or from days 11 to 28 after intrafoot-pad tumor cell inoculation. Ilmofosine caused a significant dose-related response on tumor growth and metastases, expressed in terms of tumor diameter, tumor weight, survival time and number of metastases-free animals as compared to sham-treated and positive (cyclophosphamide) controls. The results suggest that direct cytostatic/cytotoxic effects, rather than immune-modulatory mechanisms, preferentially contribute to the antitumor activity of Ilmofosine in vivo.

Synergistic enhancement of the antiproliferative activity of cis-diamminedichloroplatinum(II) by the ether lipid analogue BM41440, an inhibitor of protein kinase C.

The new phospholipid analogue 3-hexadecylmercapto-2-methoxy-methyl-propyl-1-phosphocholine inhibits the phospholipid-calcium-dependent protein kinase, partially purified from Walker carcinoma cells with a Ki value of 0.56 microM. The compound inhibits the phorbol ester stimulated phosphorylation of the ribosomal protein S6 indicating that the depression of Ca2+-phospholipid-dependent protein kinase by the alkyl phospholipid also occurs in intact cells. The dose effect curve for the inhibition of cell proliferation by 3-hexadecylmercapto-2-methoxy-methyl-propyl-1-phosphocholine in Walker cells exhibits a close correlation to the dose effect curve for the depression of Ca2+-phospholipid-dependent protein kinase activity. Although alternative mechanisms cannot be excluded, the data suggest that the growth inhibitory activity of 3-hexadecylmercapto-2-methoxy-methyl-propyl-1-phosphocholine correlates with the inhibition of Ca2+-phospholipid-dependent protein kinase. The antiproliferative activity of 3-hexadecylmercapto-2-methoxy-methyl-propyl-1-phosphocholine is synergistically enhanced by cis-diamminedichloroplatinum(II).

Phase I trial of the thioether phospholipid analogue BM 41.440 in cancer patients.

BM 41.440 (1-hexadecylmercapto-2-methoxymethyl-rac-glycero-3-phosphocholine) is a new thioether phospholipid, which has been shown to possess antineoplastic, antimetastatic, anti-invasive and immunomodulating properties in several tumor models. The mechanism whereby this compound exerts its direct antineoplastic effect is thought to be related to specific interference with the normal phospholipid metabolism, preferentially of neoplastic cells. BM 41.440 was evaluated in a multicenter phase I study in patients (pts) with refractory cancers. In phase I A, 34 pts were orally treated with doses ranging from 0.5 to 7.0 mg/kg body weight (bw). Three different formulations were tested. The maximum-tolerated dose (MTD) was ca. 5 mg/kg bw. The limiting side effects were nausea and vomiting. There was no evidence for systemic toxicities like myelosuppression, nephro-, neuro-, hepatotoxicity or hematological side effects. The current phase I B is designed to determine the MTD of BM 41.440 administered orally on a daily schedule for at least eight weeks. So far, 19 pts have entered this trial at dose levels ranging from 1.0 to 5.0 mg/kg bw/day. Some pts receiving 1.0 and 2.5 mg/kg bw/day, respectively, have been treated, up to now, for more than nine months. Clinical progress was followed with at-least-weekly blood counts, chemistry profiles, urine analysis, liver function tests and recordings of side effects. Tumor parameters were evaluated at eight-week intervals. In parallel, pharmacokinetic investigations were performed in some pts in phase I A and IB. First results on tolerability and therapeutic efficacy of the long-term BM 41.440 treatment are reported in this intermediate evaluation.

Drugs in autoimmune diseases.

Autoimmune diseases arise when autoimmunity or the loss of self tolerance results in tissue damages. Many mechanisms have been proposed for the origin of autoimmunity, including immunologic, viral, hormonal and genetic factors. All known parts of the immunological network are involved in causing immunopathologic symptoms. Therefore, more or less specific immunosuppressants are widely used in the treatment of autoimmune disorders which range from organ-specific, i.e. Hashimoto's thyroiditis, to non-organ-specific or systemic diseases, i.e. systemic lupus erythematosus. Unspecifically acting cytostatics do not only suppress autoimmune reactions but also create severe side-effects due to the impairment of immune responses against foreign antigens, leading, for example, to an increased risk of infections. Moreover, the genotoxic activity of cytostatics might induce malignancies. Corticosteroids are clinically well known and very active agents for the management of acute symptoms but different side-effects limit their use in the treatment of chronic diseases. Cyclosporin A has been an important step forward to a more specific prevention of organ transplant rejections and to the therapy of some autoimmune disorders. Modern approaches to immunosuppression include monoclonal antibodies directed against a variety of different determinants on immunocompetent cells. Ciamexone and Leflunomide which are in early clinical and preclinical development, respectively, might be interesting new drugs. Future immunopharmacologic drug research and development should lead to more specific, low molecular weight, orally active and chemically defined immunosuppressive compounds with good tolerability under long-term treatment of autoimmune diseases.

Cytotoxic ether phospholipids. Different affinities to lysophosphocholine acyltransferases in sensitive and resistant cells.

Alkyllysophospholipids (ALP) which are 1-O-alkyl analogs of the cell membrane component 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC) represent a family of new antitumor drugs. Susceptibility of cells to ALP is correlated to a selective inhibition of fatty acid incorporation into 1,2-diacyl-sn-glycero-3-phosphocholine in intact cells. This report examines oleoyl-CoA-1-acyl-GPC acyl-transferase activities in cell-free systems of ALP-sensitive methylcholanthrene-induced fibrosarcoma cells (MethA cells) and ALP-resistant bone marrow-derived murine macrophages (BMM phi). The specific activities for the oleoyl-CoA-1-acyl-GPC acyltransferases were 1.05 +/- 0.06 nmol X mg-1 X min-1 and 2.98 +/- 0.27 nmol X mg-1 X min-1, respectively. The kinetic parameters for 1-palmitoyl-GPC were Km = 16.6 microM, Vmax = 4.3 nmol X mg-1 X min-1 (BMM phi) and Km = 7.6 microM, Vmax = 2.0 nmol X mg-1 X min-1 (MethA cells). In the presence of 1-O-octadecyl-2-O-methyl racemic glycero-3-phosphocholine (ET-18-OCH3), one of the most potent cytotoxic ALP, the acyltransferase was dose dependently inhibited in MethA cells with a 50% inhibition concentration at 40 micrograms/ml. The BMM phi-acyltransferase was not affected up to 80 micrograms of ET-18-OCH3/ml. The kinetic parameters (Km' = 15.4 microM, Vmax' = 2.2 nmol X mg-1 X min-1) suggest that ET-18-OCH3 is a competitive inhibitor in MethA cells. Inhibitor constants for ET-18-OCH3, calculated from Dixon plots, were found to be 423 microM (BMM phi) and 13 microM (MethA cells) indicating a 33-fold larger affinity of ET-18-OCH3 to the MethA cells than to the BMM phi acyltransferase. From these data we assume that the inhibition of oleic acid incorporation into cellular phosphocholine during the antineoplastic action of ALP may be due to different affinities of the inhibitor to the 1-acyl-GPC acyltransferases in different cell types.

BM 41.440: a new antineoplastic, antimetastatic, and immune-stimulating drug.

Alkyllysophospholipids are analogs of the cell membrane component lysophosphocholine. The thioether lysophospholipid BM 41.440 (1-hexadecylmercapto-2-methoxymethyl-rac-glycero-3-phosphocholine) is already in use in phase I and II trials in human cancer therapy. A direct antitumor effect of this new compound has been shown in vitro using 35 different cell types of murine and human origin. All normal cells investigated were not affected in the concentration range (1-10 micrograms/ml) that was cytotoxic for most tumor cells studied. In vivo, antimalignant and antimetastatic actions have been documented in the Meth A sarcoma, L1210 leukemia, B 16 melanoma and the 3Lewis-lung carcinoma tumor models, respectively. Murine, bone marrow-derived macrophages (M phi), preincubated with BM 41.440, showed an increased cytotoxicity in vitro. Addition of syngeneic spleen cells and low doses of BM 41.440 to this system enhanced tumor cell destruction 20- to 100-fold compared to controls dependent on the target cells used (YAC, ABLS-8.1, L1210, and P815). In vivo, Meth A sarcoma growth was dose and time dependently reduced in CB6F1 mice under therapeutic IV application of BM 41.440-activated M phi. The mean survival time of DBA mice, treated once IP with BM 41.440 4 days before L1210 challenge, increased from 24 to 38 days.

Role of gastrointestinal tract and liver in acetate metabolism in rat and man.

Net acetate uptake/release by various tissues was studied in vivo in fed, starved and Paromomycin-treated rats and in patients with cirrhosis of the liver. In humans the portal vein, hepatic vein and hepatic arterial blood flow rates were determined simultaneously. In rats acetate is only intestinally produced and released into the portal vein. Intestinal production is decreased by 33% in starved and Paromomycin-treated rats compared to fed animals. Portal vein hepatic vein acetate differences are linearly related to the portal vein acetate concentration (r = 0.92). Acetate uptake from the portal vein by the liver was found when the portal venous concentration exceeded 180 mumol l-1. In humans the hepatic net acetate uptake from the portal vein/net acetate release into the hepatic vein, measured as mmol min-1, is linearly related to the portal vein acetate concentration (r = 0.96). The data indicate that the liver may homeostatically regulate the systemic acetate concentration in rat and man.

Treatment of human clonogenic tumor cells and bone marrow progenitor cells with bleomycin and peplomycin under 40.5 degrees C hyperthermia in vitro.

Tumor cells derived from 13 different individual human tumors were plated in a colony forming monolayer assay. The effect of bleomycin and peplomycin on colony formation was assessed in normothermic conditions and after a hyperthermic treatment at 40.5 degrees C for 2 h at the beginning of the culture. In three out of the 13 tumor samples (two colon carcinomas, one malignant melanoma), hyperthermic incubation resulted in a thermal enhancement of the effects of bleomycin and peplomycin. In addition, human bone marrow progenitor cells (CFU-C) were subjected to the same procedure. Peplomycin proved to be less toxic to CFU-C than bleomycin. In samples from eight different donors, homogeneous dose-response curves were observed. There was no difference between normo- and hyperthermic incubation.

Preparative continuous separation of biological particles by means of free-flow magnetophoresis in a free-flow electrophoresis chamber.

For sorting, cells or cellular components can specifically be labeled by antibody-coated magnetic beads. We have developed a device for continuous magnetic sorting based on the flow-chamber of a free-flow electrophoresis system. Magnetically labeled particles are injected into a given continuously flowing chamber buffer and pass an inhomogeneous magnetic field, configurated perpendicular to the flow direction. According to its magnetic moment, the magnetic material is deviated into the direction of the magnetic forces, while nonmagnetic material passes the field without interaction. The magnetic forces can be changed with the electrical current of the solenoids producing the magnetic field. As in the free-flow electrophoresis system, the particle fractions are collected in different vials. On-line control of the experiments can be performed by an optical scanning system. Experiments with model particles achieved a sorting purity of more than 99% at a rate of up to 5 X 10(8) particles per hour. In experiments with blood cells, a high enrichment of either B-or-T-lymphocytes was obtained. In contrast to free-flow electrophoresis, there is no limitation, in principle, regarding the type of chamber buffer to be used. This allows an optimal adaptation of the buffer conditions to the requirements of vital sorting. The preliminary results so far confirm this conclusion.