Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach.

Tumour-associated macrophages, TAMs, play a pivotal role in tumour growth and metastasis by promoting tumour angiogenesis. Treatment with clodronate encapsulated in liposomes (clodrolip) efficiently depleted these phagocytic cells in the murine F9 teratocarcinoma and human A673 rhabdomyosarcoma mouse tumour models resulting in significant inhibition of tumour growth ranging from 75 to >92%, depending on therapy and schedule. Tumour inhibition was accompanied by a drastic reduction in blood vessel density in the tumour tissue. Vascular endothelial growth factor (VEGF) is one of the major inducers of tumour angiogenesis and is also required for macrophage recruitment. The strongest effects were observed with the combination therapy of clodrolip and a VEGF-neutralising antibody, whereas free clodronate was not significantly active. Immunohistologic evaluation of the tumours showed significant depletion of F4/80+ and MOMA-1+ and a less pronounced depletion of CD11b+ TAMs. Blood vessel staining (CD31) and quantification of the vessels as well as TAMs and tumour-associated dendritic cells (TADCs) in the A673 model showed reduction rates of 85 to >94%, even 9 days after the end of therapy. In addition, CD11c+ TADCs, which have been shown to potentially differentiate into endothelial-like cells upon stimulation by tumour released growth and differentiation factors, were similarly reduced by clodrolip or antibody treatment. These results validate clodrolip therapy in combination with angiogenesis inhibitors as a promising novel strategy for an indirect cancer therapy aimed at the haematopoietic precursor cells that stimulate tumour growth and dissemination and as a tool to study the role of macrophages and dendritic cells in tumorigenesis.

Enhanced heparan sulfate proteoglycan-mediated uptake of cell-penetrating peptide-modified liposomes.

Protein transduction domains (PTDs) are used to enhance cellular uptake of drugs, proteins, polynucleotides or liposomes. In this study, functionalized Antennapedia (Antp, aa 43-58) and HIV Tat (aa 47-57) peptides were coupled to small unilamellar liposomes via thiol-maleimide linkage. Modified liposomes showed higher uptake into a panel of cell lines including tumor and dendritic cells than unmodified control liposomes. Liposome uptake was time and concentration dependent as analyzed by flow cytometry and live-cell microscopy. At least 100 PTD molecules per small unilamellar liposome (100 +/- 30 nm) were necessary for efficient translocation into cells. Cellular uptake of PTD-modified liposomes was 15- to 25-fold increased compared to unmodified liposomes and was inhibited by preincubation of liposomes with heparin. Glycosaminoglycan-deficient CHO cells showed dramatically reduced cell association of PTD-modified liposomes, confirming the important role of heparan sulfate proteoglycans in PTD-mediated uptake. Antp-liposomes used as carriers of the cytotoxic drug N4-octadecyl-1-beta-D-arabinofuranosylcytosine-(5'- 5')-3'-C-ethinylcytidine showed a reduction of the IC50 by 70% on B16F1 melanoma cells compared with unmodified liposomes. PTD-functionalized liposomes, particularly Antp-liposomes, represent an interesting novel carrier system for enhanced cell-specific delivery of a large variety of liposome-entrapped molecules.

Cytotoxic targeting of F9 teratocarcinoma tumours with anti-ED-B fibronectin scFv antibody modified liposomes.

We prepared small unilamellar liposomes derivatised with single chain antibody fragments specific for the ED-B domain of B-fibronectin. This extracellular matrix associated protein is expressed around newly forming blood vessels in the vicinity of many types of tumours. The single chain antibody fragments were functionalised by introduction of C-terminal cysteines and linked to liposomes via maleimide groups located at the terminal ends of poly(ethylene glycol) modified phospholipids. The properties of these anti-ED-B single chain antibody fragments-liposomes were analysed in vitro on ED-B fibronectin expressing Caco-2 cells and in vivo by studying their biodistribution and their therapeutic potential in mice bearing subcutanous F9 teratocarcinoma tumours. Radioactively labelled ((114m)Indium) single chain antibody fragments-liposomes accumulated in the tumours at 2-3-fold higher concentrations during the first 2 h after i.v. injection compared to unmodified liposomes. After 6-24 h both liposome types were found in similar amounts (8-10% injected dose g(-1)) in the tumours. Animals treated i.v. with single chain antibody fragments-liposomes containing the new cytotoxic agent 2'-deoxy-5-fluorouridylyl-N(4)-octadecyl-1-beta-D-arabinofuranosylcytosine (30 mg kg(-1) per dose, five times every 24 h) showed a reduction of tumour growth by 62-90% determined on days 5 and 8, respectively, compared to animals receiving control liposomes. Histological analysis revealed a marked reduction of F9 tumour cells and excessive deposition of fibronectin in the extracellular matrix after treatment with single chain antibody fragments-2-dioxy-5-fluorouridylyl-N(4)-octadecyl-1-beta-D-arabinofuranosylcytosine-liposomes. Single chain antibody fragments-liposomes targeted to ED-B fibronectin positive tumours therefore represent a promising and versatile novel drug delivery system for the treatment of tumours.

Limited mycobacterial infection of the liver as a consequence of its microanatomical structure causing restriction of mycobacterial growth to professional phagocytes.

Among sites of extrapulmonary growth of Mycobacterium tuberculosis, the liver is the least infected. Our data suggest that this is due to the complete restriction of mycobacterial growth to liver macrophages. Unlike in organs more persistently seeded by M. tuberculosis, in the liver the bacteria do not infect cell types other than professional phagocytes.

In vitro activity of liposomal N4octadecyl-1-beta-D-arabinofuranosylcytosine (NOAC), a new lipophilic derivative of 1-beta-D-arabinofuranocylcytosine on biopsized clonogenic human tumor cells and hematopoietic precursor cells.

N4-octadecyl-1-beta-D-arabinofuranosylcytosine (NOAC) is a new lipophilic derivative of 1-beta-D-arabinofuranosylcytosine (ara-C) with potent antitumor activity against leukemias and solid tumors. In this study the activity of NOAC against freshly explanted clonogenic cells from human tumors was determined and compared with conventional antitumor agents. NOAC was used in two liposomal preparations, a stable lyophilized and a freshly prepared liquid formulation. Both formulations inhibited tumor colony formation equally in a concentration-dependent fashion in both short- (1 h) and long-term (21-28 d) exposure experiments. NOAC (100 microM, long-term exposure) had a significantly better activity compared to the clinically used drugs cisplatin, doxorubicin, 5-fluorouracil, gemcitabine, mitomycin C and etoposide. The comparison of NOAC with ara-C in the long-term exposure experiment showed that ara-C was more effective at 4 and 10 microM, whereas at 1 and 100 microM there was no difference between the two drugs. NOAC was less toxic in a hematopoietic stem cell assay than ara-C and doxorubicin by factors ranging from 2.5 to 200, indicating that this drug is well tolerated at high doses. The antitumor activity of NOAC (NSC 685096) was confirmed by the NCI in vitro drug screening program where the drug was found to be active against several types of human tumors. Further development of NOAC in phase II studies is warranted.

Roles of macrophages in measles virus infection of genetically modified mice.

Knowledge of the mechanisms of virus dissemination in acute measles is cursory, but cells of the monocyte/macrophage (MM) lineage appear to be early targets. We characterized the dissemination of the Edmonston B vaccine strain of measles virus (MV-Ed) in peripheral blood mononuclear cells (PBMC) of two mouse strains expressing the human MV-Ed receptor CD46 with human-like tissue specificity and efficiency. In one strain the alpha/beta interferon receptor is defective, allowing for efficient MV-Ed systemic spread. In both mouse strains the PBMC most efficiently infected were F4/80-positive MMs, regardless of the inoculation route used. Circulating B lymphocytes and CD4-positive T lymphocytes were infected at lower levels, but no infected CD8-positive T lymphocytes were detected. To elucidate the roles of MMs in infection, we depleted these cells by clodronate liposome treatment in vivo. MV-Ed infection of splenic MM-depleted mice caused strong activation and infection of splenic dendritic cells (DC), followed by enhanced virus replication in the spleen. Similarly, depletion of lung macrophages resulted in strong activation and infection of lung DC. Thus, in MV infections of genetically modified mice, blood monocytes and tissue macrophages provide functions beneficial for both the virus and the host: they support virus replication early after infection, but they also contribute to protecting other immune cells from infection. Human MM may have similar roles in acute measles.

Production of functionalized single-chain Fv antibody fragments binding to the ED-B domain of the B-isoform of fibronectin in Pichia pastoris.

The Pichia pastoris expression system was used to produce functionalized single-chain antibody fragments (scFv) directed against the ED-B domain of the B-fibronectin (B-Fn) isoform which was found to be present only in newly formed blood vessels during tumor angiogenesis. Therefore, scFv antibody fragments recognizing the ED-B domain are potential markers for angiogenesis. We constructed four functionalized scFv antibody fragments for direct labeling with radioactive molecules or toxins or for attachment to liposomes serving as carriers for cytotoxic or antiangiogenic compounds. The C-termini of the scFv antibody fragments contain 1-3 cysteine residues that are separated by a hydrophilic linker (GGSSGGSSGS) from the binding domain and are accessible for site-specific functionalization with thiol-reactive reagents. Plasmid expression, culture conditions, and purification were optimized in 1-L cultures. The scFv antibody fragments were purified by anion exchange chromatography. The yields were 5-20 mg/L culture medium. The large-scale production of one scFv antibody fragment in a 3.7-L fermenter gave a yield of 60 mg. The reactivity of the cyteines was demonstrated by labeling with the thiol-reactive fluorescent dye ABD-F. The four scFv antibody fragments bound specifically to ED-B-modified Sepharose and binding was further confirmed by immunofluorescence on cell cultures using ED-B-positive human Caco-2 tumor cells. Furthermore, we could demonstrate specific binding of scFv-modified liposomes to ED-B-positive tumor cells. Our results indicate that the P. pastoris expression system is useful for the large-scale production of cysteine-functionalized alpha-ED-B scFv antibody fragments.

Induction of cell cycle-dependent cytotoxicity and apoptosis by new heterodinucleoside phosphate dimers of 5-fluorodeoxyuridine in PC-3 human prostate cancer cells.

Fluorodeoxyuridine (5-FdUrd) is an antineoplastic agent with clinical activity against different types of solid tumours. To enhance the effectiveness of this drug, we have synthesised new heterodinucleoside phosphate dimers of 5-FdUrd. These dimers were compared to 5-FdUrd for their cytotoxic effect and the cell cycle dependence of cytotoxicity, as well as for their capacity to induce apoptosis and inhibit thymidylate synthetase (TS) in androgen-independent human PC-3 prostate tumour cells. Incubation of the cells with the dimers N(4)-palmitoyl-2'-deoxycytidylyl-(3'-->5')-5-fluoro-2'-deoxyuri din e (dCpam-5-FdUrd) and 2'-deoxy-5-flourouridylyl-(3'-->5')-2'-deoxy-5-fluoro-N(4)-octa decylc ytidine (5-FdUrd-5-FdC18) resulted in a marked cytotoxicity with IC(50) values of 4 microM, similar to 5-FdUrd. In contrast to 5-FdUrd, 100% toxicity was achieved with concentrations of 100-200 microM 5-FdUrd-5-FdC18. Flow cytometric analysis revealed an increase in the cell population in S-phase after treatment with 5-FdUrd, 5-FdUrd-5-FdC18, and dCpam-5-FdUrd from 36 to 63%, 50%, and 77%, respectively. dCpam-5-FdUrd was more potent than 5-FdUrd in arresting the cell cycle. Significant S-phase arrest was indicated by a decreased proportion of cells in G1- and G2/M-phases. Cell cycle arrest and inhibition of cell proliferation were followed by apoptosis, as shown by a 6- to 8-fold increased binding of Apo2.7 antibody, a 9- to 11-fold increase in caspase-3 activity, DNA fragmentation, and by cell morphology showing the appearance of apoptotic bodies. Importantly, 5-FdUrd-5-FdC18 increased the number of apoptotic cells to 160% compared to 5-FdUrd under the same conditions. As with 5-FdUrd, the two dimers also inhibited TS in a time- and concentration-dependent manner, although requiring 100-fold higher concentrations. In conclusion, dCpam-5-FdUrd and 5-FdUrd-5-FdC18 exert stronger cytotoxicity and induce more S-phase arrest and apoptosis than does 5-FdUrd in PC-3 cells, suggesting their potential role in the treatment of human prostate cancer.

The novel heterodinucleoside dimer 5-FdU-NOAC is a potent cytotoxic drug and a p53-independent inducer of apoptosis in the androgen-independent human prostate cancer cell lines PC-3 and DU-145.

BACKGROUND: We analyzed the cytotoxic properties of the new heterodinucleoside phosphate dimer 5-FdU-NOAC, which is composed of the cytotoxic drugs 5-FdU and N(4)-octadecyl-1-beta-D-arabinofuranosylcytosine (NOAC) against human prostate tumor cells. METHODS: 5-FdU-NOAC effects on cell proliferation, cell cycle distribution, thymidylate synthase activity, and apoptosis were investigated in vitro in the two human prostate carcinoma cell lines DU-145 and PC-3 and compared to cells treated with the corresponding single drugs 5-FdU and NOAC. RESULTS: Treatment of the cells with 5-FdU-NOAC resulted in IC(50) values of 3.9-5 microM and in a complete inhibition of cell proliferation at 200 microM after 96 hr compared to 5-FdU, where 10% of the cells remained resistant. Flow cytometric analysis revealed cell cycle perturbations in S-phase only in the DU-145 cells. 5-FdU-NOAC caused 50% inhibition of thymidylate synthase after 90 min at 0.6 microM in both cell lines. Apoptotic cell fractions in DU-145 (66%) and in PC-3 (34%) cells were found after treatment with 5-FdU-NOAC for 96 hr. DNA fragmentation further confirmed the induction of apoptosis. CONCLUSIONS: 5-FdU-NOAC inhibits thymidylate synthase and cell cycle progression causing proliferation arrest and apoptosis in DU-145 and PC-3 cells, suggesting a potential role of 5-FdU-NOAC for the treatment of prostate cancer.

In vivo antigen loading and activation of dendritic cells via a liposomal peptide vaccine mediates protective antiviral and anti-tumour immunity.

Initiation of antiviral and anti-tumour T cell responses is probably achieved mainly by dendritic cells (DC) transporting antigen from the periphery into organised lymphoid tissues. To develop T cell vaccines it is, therefore, important to understand the accessibility of the antigen to DC in vivo and whether DC are activated by vaccination. Here we have evaluated the immunogenicity of a liposomal vaccine formulation with antigenic peptides derived from the glycoprotein of the lymphocytic choriomeningitis virus. Liposome-encapsulated peptides were highly immunogenic when administered intradermally and elicited protective antiviral immunity. After intradermal injection, liposomes formed antigen depots which facilitated long-lasting in vivo antigen loading of dendritic cells almost exclusively in the local draining lymph nodes. The immunogenicity of the liposomal peptide vaccine was further enhanced by incorporation of immunostimulatory oligonucleotides leading to activation of DC. This optimised liposomal peptide vaccine elicited also anti-tumour immunity and induced CTL responses comparable to adoptively transferred, peptide-presenting DC. Thus, our data show that liposomal formulations of peptide vaccines are highly effective at direct in vivo antigen loading and activation of DC leading to protective antiviral and anti-tumour immune responses.

Cytotoxicity, cell-cycle perturbations and apoptosis in human tumor cells by lipophilic N4-alkyl-1-beta-D-arabinofuranosylcytosine derivatives and the new heteronucleoside phosphate dimer arabinocytidylyl-(5'-->5')-N4-octadecyl-1-beta-D-arabinofuranosylcytosi ne.

The arabinofuranosylcytosine (AraC) derivative N4-octadecyl-1-beta-D-arabinofuranosylcytosine (NOAC) and its (5'-->5')-heterodinucleoside phosphate analog NOAC-AraC were compared with AraC for cytotoxicity, cell-cycle dependence, phosphorylation by deoxycytidine (dC) kinase and apoptosis induction in native, AraC- or NOAC-resistant HL-60 cells. NOAC was cytotoxic in all cells with three to seven-fold lower IC50 concentrations than those of NOAC-AraC or AraC. In contrast to NOAC-AraC, the lipophilic monomer NOAC overcame AraC resistance, inducing apoptosis in more than 80% of native and AraC-resistant HL-60 cells. This suggests that NOAC-AraC may be cleaved intracellularly only at very slow rates to AraC and NOAC or to the 5'-monophosphates, whereas NOAC exerts different mechanisms of action from AraC. In vitro the dimer was cleaved by phosphodiesterase or human serum to NOAC, AraC and AraC monophosphate. In contrast to AraC, N4-alkylated AraC derivatives with alkyl chains ranging from 6-18 C atoms were not substrates for dC kinase. Furthermore, treatment of the multidrug-resistant cell lines KB-ChR-8-5 and KB-V1 with the N4-hexadecyl-AraC derivative NHAC did not induce P-170 glycoprotein expression, suggesting that the N4-alkyl-AraC derivatives are able to circumvent MDR1 multidrug resistance. The in vivo activity of liposomal NOAC in a human acute lymphatic leukemia xenograft model confirmed the antitumor activity of this representative of the N4-alkyl-arabinofuranosylcytosines.

Cell-cycle arrest and p53-independent induction of apoptosis in vitro by the new anticancer drugs 5-FdUrd-P-FdCydOct and dCydPam-P-FdUrd in DU-145 human prostate cancer cells.

PURPOSE: Current therapies have limited impact on the progression of metastatic hormone-refractory prostate cancer. Therefore, we investigated the utility of new heterodinucleoside phosphate dimers of 5-fluorodeoxyuridine (5-FdUrd) in p53-mutated and androgen-independent DU-145 human prostate tumour cells. METHODS: The effects of the dimers were assessed in vitro by a cell proliferation assay for cytotoxicity, flow cytometry for cell cycle distribution, confocal laser scanning microscopy for the detection of apoptotic bodies, poly(ADP-ribose) polymerase cleavage for caspase 3 activity and by a thymidylate synthetase assay. RESULTS: The new dimers N4-palmitoyl-2'-deoxycytidylyl-(3'-->5')-5-fluoro-2'-deoxyuridine (dCydPam-P-FdUrd) and 2'-deoxy-5-fluorouridylyl-(3'-->5')-2'-deoxy-5-fluoro-N4-octade cylcytidine (5-FdUrd-P-FdCydOct) caused marked cytotoxicity with IC50 values of 3-4 microM. 5-FdUrd-P-FdCydOct at 200 microM was capable of eradicating 100% of tumour cells whereas 10% of the cells were resistant to 5-FdUrd. Cytotoxicity was caused by a dramatic S-phase arrest, resulting in an increase of this cell population from 34% to 85% with 5-FdUrd-P-FdCydOct and to 81% with dCydPam-P-FdUrd. S-phase arrest was followed by apoptosis, as shown by 85% of the cells staining positive for Apo 2.7 antibody, a six- to eight-fold increased caspase 3 activity and DNA fragmentation. Thymidylate synthase activity was inhibited by 50% at 0.6-0.7 microM dimer concentration. The dimers were hydrolysed in vitro by phosphodiesterase I and human serum to the corresponding nucleosides and nucleoside monophosphates. CONCLUSIONS: The new dimers dCydPam-P-FdUrd and 5-FdUrd-P-FdCydOct are effective prodrugs of 5-FdUrd and have potential value for the treatment of p53-mutated and hormone-independent human prostate carcinomas.

Protective antiviral cytotoxic T cell memory is most efficiently maintained by restimulation via dendritic cells.

Dendritic cells (DC) play a key role in the initiation of T cell-mediated immune responses and may therefore be successfully used in antiviral and antitumor vaccination strategies. Because both strength and duration of an immune response determines the outcome of a vaccination protocol, we evaluated the life span of DC-induced antiviral CTL memory against systemic and peripheral challenge infections with lymphocytic choriomeningitis virus (LCMV). We found that expansion and activation of CTL by DC was transient. Protection against systemic LCMV infection after DC immunization was relatively long-lived (>60 days), whereas complete protection against peripheral infection via intracerebral infection or infection into the footpad with LCMV, where rapid recruitment of effector T cells to the site of infection and elimination of viral pathogen plays a major role, was short-lived (<30 days). Protective immunity was most efficiently restored by administration of antigenic peptides via DC, rather than in combination with IFA or in liposomes. These results suggest that Ag presentation by DC may be crucial for both initiation and maintenance of protective CTL-mediated immunity against viruses infecting solid organs or against peripheral mesenchymal or epithelial tumors.

Synthesis, in vitro anti-HIV and anti-hepatitis B activities and pharmacokinetic properties of amphiphilic heterodinucleoside phosphates containing ddC and AZT.

Amphiphilic heterodinucleoside phosphates containing AZT and ddC as antiviral monomer were synthesized according to the hydrogenphosphonate method and evaluated in vitro against HIV. dT-N4-pamddC was the most active (IC50 = 40 microM, EC50 = 80 nM) and least toxic (TI = 524) dimer and it exhibited also strong antiviral effects against eight AZT-resistant HIV strains. The ddC-containing heterodimers additionally inhibited HBV replication by 50-80% at 50 microM in Hep G2 2.2.15 cells.

Low density lipoprotein and liposome mediated uptake and cytotoxic effect of N4-octadecyl-1-beta-D-arabinofuranosylcytosine in Daudi lymphoma cells.

Low density lipoprotein (LDL) receptor-mediated uptake and cytotoxic effects of N4-octadecyl-1-beta-D-arabinofuranosylcytosine (NOAC) were studied in Daudi lymphoma cells. NOAC was either incorporated into LDL or liposomes to compare specific and unspecific uptake mechanisms. Binding of LDL to Daudi cells was not altered after NOAC incorporation (K(D) 60 nM). Binding of liposomal NOAC was not saturable with increasing concentrations. Specific binding of NOAC-LDL to Daudi cells was five times higher than to human lymphocytes. LDL receptor binding could be blocked and up- or down-regulated. Co-incubation with colchicine reduced NOAC-LDL uptake by 36%. These results suggested that NOAC-LDL is taken up via the LDL receptor pathway. In an in vitro cytotoxicity test, the IC50 of NOAC-LDL was about 160 microM, whereas with liposomal NOAC the IC50 was 40 microM. Blocking the LDL receptors with empty LDL protected 50% of the cells from NOAC cytotoxicity. The cellular distribution of NOAC-LDL or NOAC-liposomes differed only in the membrane and nuclei fraction with 13% and 6% respectively. Although it is more convenient to prepare NOAC-liposomes as compared to the loading of LDL particles with the drug, the receptor-mediated uptake of NOAC-LDL provides an interesting rationale for the specific delivery of the drug to tumours that express elevated numbers of LDL receptors.

Metabolism of the new liposomal anticancer drug N4-octadecyl-1-beta-D-arabinofuranosylcytosine in mice.

Metabolism and excretion of the new antitumor drug N4-octadecyl-1-beta-D-arabinofuranosylcytosine (NOAC) was investigated in mice. Mice were injected i.v. with tritium-labeled liposomal NOAC (4 micromol/mouse). Analysis of HPLC-purified extracts of liver homogenates by liquid chromatography coupled with mass spectrometry revealed only the presence of unmetabolized drug. To study the excretion of the administered drug, mice were injected with tritium-labeled liposomal NOAC or as comparison with 1-beta-D-arabinofuranosylcytosine (ara-C; 4 micromol/mouse) and housed up to 48 h in metabolic cages. Urine and feces were collected at different time points and the kinetics of excreted radioactivity were determined. After 48 h, 39% of the injected [5-3H]NOAC radioactivity was excreted in urine and 16% in feces, whereas ara-C radioactivity was only found in urine with 48% of the injected dose. Feces extracts and urine were purified by HPLC and radioactive fractions were further analyzed by liquid chromatography coupled with mass spectrometry. The radioactivity of feces extracts of NOAC-treated mice was composed of unmetabolized NOAC, hydroxylated NOAC (NOAC + OH), its sulfated derivative (NOAC + OSO3H), and unidentified metabolites, whereas in urine, the hydrophilic molecules ara-C and ara-U were found. During the period of 48 h only 2% of the injected NOAC was eliminated in its unmetabolized form, whereas 25% was identified as main metabolite ara-C. Urine collected during 48 h in ara-C-treated mice contained 33% of the injected dose as unmetabolized drug and 13% as the main metabolite ara-U. Thus, NOAC is metabolized by two major pathways, one leading to the hydrophilic metabolites ara-C and ara-U and the other to hydroxylated and sulfated NOAC.

Toxicity of liposomal 3'-5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine in mice.

Toxicities of 5-fluoro-2'-deoxyuridine (FUdR) and its liposome incorporated dipalmitoyl derivative (FUdR-dipalmitate) to mouse bone marrow, spleen, liver and ileum were compared after treatment for 6 consecutive days. The applied doses of the two formulations, which were shown earlier to have equal antitumor activity in mouse tumor models, were 600 and 2 mumol/kg respectively. When applied in these doses, toxicity to the hemopoietic system, measured as a decreases in progenitor and precursor cells of the erythroid and granuloid/macrophage lineage in bone marrow and spleen, was more severe for FUdR than for liposomal FUdR-dipalmitate. In the liver, mitotic figures, as indicators of cell division, were absent for both drugs while in control livers the number of cells in mitosis was approximately 2%. Toxicity to the ileum was more severe for liposomal FUdR-dipalmitate than for FUdR and was manifested by granulocyte infiltration, the presence of cell debris, loss of columnar epithelial cells and enlarged nuclei with prominent nucleoli in these cells. Thus, by prolonging the retention time of FUdR in vivo, using liposomes as a vehicle and FUdR-dipalmitate as a lipophilic prodrug, the dose-limiting toxicity appears to shift from bone marrow to the gastrointestinal tract.

Pharmacokinetic studies of mitoxantrone and one of its metabolites in serum and urine in patients with advanced breast cancer.

OBJECTIVE: Mitoxantrone (MTO) was administered to patients with advanced breast cancer either as free MTO (f-MTO) or liposomal MTO (1-MTO). The intra- and interindividual variations in serum pharmacokinetics of MTO were analysed. In addition, the excretion of MTO and its metabolite mitoxantrone dicarboxylic acid (MTOD) in urine was determined. METHODS: The concentration of MTO was measured by high-performance liquid chromatography in serum over a period of 24 h and the amount of MTO and the metabolite MTOD excreted in urine over 18 h was determined. Pharmacokinetic parameters of f-MTO and 1-MTO were calculated. RESULTS: 1-MTO had a significantly longer half-life of distribution in the deep (third) compartment and thus a larger area under the curve (AUC) than f-MTO. No difference was found with respect to distribution in the peripheral (second) compartment. The kinetics of MTO in serum did not significantly differ between patients. In four patients repeated pharmacokinetic analyses gave superimposable results. Thus, there was no enzyme induction during therapy. By contrast, two patients with oedema had a much longer mean residence time (MRT) and AUC for MTO in serum. Despite the altered pharmacokinetics of f-MTD and 1-MTO, no toxic adverse effects occurred in these two patients. CONCLUSIONS: f-MTO and 1-MTO exhibited different distribution patterns in the deep compartment with a significantly increased half-life for 1-MTO. There is no need to monitor MTO for treatment of breast cancer patients with f-MTO. In patients with oedema, the MRT of MTO is prolonged. The clinical relevance of this observation is as yet unclear.

In vitro anti-human immunodeficiency virus and anti-hepatitis B virus activities and pharmacokinetic properties of heterodinucleoside phosphates containing AZT or ddC.

In vitro activities, against human immunodeficiency virus (HIV)- and hepatitis B virus (HBV)-infected cells, of four amphiphilic heterodinucleoside phosphates containing 3'-azido-2',3'-dideoxythymidine (AZT) or 2',3'-dideoxycytidine (ddC) as antiviral monomers were evaluated. The four compounds were N4-hexadecyl-2'-deoxyribocytidylyl-(3'-->5')-3'-azido-2',3'-deoxyt hymidine (N4-hxddC-AZT), N4-palmitoyl-2'-deoxyribocytidylyl-(3'-->5')-3'-azido-2',3'-deoxyt hymidine (N4-pamdC-AZT), N4-hexadecyl-2'-deoxycytidylyl-(3'-->5')-2',3'-dideoxycytidine (N4-hxddC-ddC) and 2'-deoxythymidylyl-(3'-->5')-N4-palmitoyl-2',3'-dideoxycytidine (dT-N4-pamddC). All four dimers were active against HIV, dT-N4-pamddC being the most active and least toxic. dT-N4-pamddC also exhibited strong antiviral effects against a panel of eight AZT-resistant HIV strains. The ddC-containing heterodimers incorporated in liposomes additionally inhibited HBV replication by 50-80% in HepG2 2.2.15 cells. AZT and the AZT-containing dimers were ineffective. Differences in pharmacokinetic properties between the antiviral monomers and the heterodimers were evaluated using liposomal formulations of 3H-labelled AZT heterodimers as model compounds. The cellular distribution of AZT in H9 cells was predominantly cytoplasmic, whereas the amphiphilic dimers were distributed more evenly throughout the cytoplasm, nuclear membranes and microsomes. Blood levels of the heterodimers decreased at a rate two- to threefold slower than AZT and the areas-under-the-curves were five- to sevenfold higher for N4-pamdC-AZT and N4-hxddC-AZT, respectively. Compared to AZT, the peak levels of the dimers were three to four times higher in blood and five to six times higher in the liver. Analysis of blood samples showed that 34% of N4-pamdC-AZT was metabolized to AZT, whereas only 9% of N4-hxddC-AZT released AZT. Considering the antiviral potency and the favourable pharmacokinetic properties of the heterodimers, these compounds merit further exploration as antiviral drugs.