Talactoferrin alfa versus placebo in patients with refractory advanced non-small-cell lung cancer (FORTIS-M trial).

BACKGROUND: Talactoferrin alfa is an oral dendritic cell (DC)-mediated immunotherapy (DCMI). We tested whether talactoferrin was superior to placebo in advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: An FORTIS-M trial was an international, multicenter, randomized, double-blind comparison of talactoferrin (1.5 g p.o. BID) versus placebo BID, in patients with stage IIIB/IV NSCLC whose disease had failed two or more prior regimens. Treatment was administered for a maximum of five 14-week cycles. The primary efficacy end point was overall survival (OS); secondary end points included 6- and 12-month survival, progression-free survival (PFS), and disease control rate (DCR). RESULTS: Seven hundred and forty-two patients were randomly assigned (2:1) to talactoferrin (497) or placebo (245). The median OS in the intent-to-treat (ITT) population was 7.66 months in the placebo arm and 7.49 months in the talactoferrin arm [hazard ratio (HR), 1.04; 95% CI, 0.873-1.24; P = 0.6602]. The 6-month survival rates were 59.9% (95% CI, 53.4% to 65.8%) and 55.7% (95% CI, 51.1% to 59.9%), respectively. The 12-month survival rates were 32.2% (95% CI, 26.3% to 38.2%) and 30.9% (95% CI, 26.8% to 35%), respectively. The median PFS rates were 1.64 months and 1.68 months, respectively (HR, 0.99; 95% CI, 0.835-1.16; P = 0.8073). The DCRs were 38.4 and 37.6%, respectively [stratified odds ratio (OR), 0.96; 95% CI, 0.698-1.33; P = 0.8336]. The safety profiles were comparable between arms. CONCLUSIONS: There was no improvement in efficacy with talactoferrin alfa in patients with advanced NSCLC whose disease had failed two or more previous regimens.

Emerging treatment using tubulin inhibitors in advanced non-small cell lung cancer.

INTRODUCTION: Tubulin inhibitors including taxanes and vinca alkaloids are important components of chemotherapy regimens used in advanced non-small cell lung cancer (NSCLC). Despite a treatment paradigm shift due to molecularly-targeted therapies and immunotherapy, a majority of patients will receive chemotherapy during their treatment course. Either used alone or in combination, tubulin inhibitors have demonstrated clinical benefits in different settings of lung cancer management. Areas covered: This review first discusses FDA-approved tubulin inhibitors for NSCLC, such as paclitaxel, docetaxel, vinorelbine, and nab-paclitaxel. The article then provides a summary of novel tubulin inhibitors, including cabazitaxel, eribulin, ixabepilone, patupilone, plinabulin, new colchicine analogues and others. It also discusses new tubulin inhibitor combinations with immunotherapy (PD-1/PD-L1 inhibitors) and molecularly-targeted therapies (e.g. anti-angiogenic agents, mTOR inhibitors, heat shock protein 90 inhibitors, MEK inhibitors, and anti-HER3 agents). Lastly, emerging data on potential resistance mechanisms and predictive biomarkers for tubulin inhibitors are explored. Expert opinion: Tubulin inhibitors will likely continue to play important roles in NSCLC management due to the advent of novel agents and combinations. Through further understanding of tumor biology, investigation of drug resistance, and development of predictive biomarkers, we will be better positioned to incorporate microtubule inhibition into patient specific treatment strategies.

Effects on the monocyte-macrophage system and antitumor activity against L1210 leukemia of cis-bis-cyclopentenecarboxylato-trans-R,R-1,2-diaminocyclohexane -platinum (II) encapsulated in multilamellar vesicles.

Multilamellar vesicles (MLVs) composed of dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol at a molar ratio of 7:3 were used as carriers of cis-bis-cyclopentenecarboxylato-trans-R,R-1,2-diaminocyclohexane-p latinum (II) (CPDP). The encapsulation efficiency of liposomal CPDP (L-CPDP) was 87.6%, and its stability in normal saline at 14 days was 94.4%. The in vitro and in vivo effects on the function of the monocyte-macrophage system and the antitumor activity against L1210 leukemia were investigated in CD-1 and (C57BL/6J X DBA/2J)F1 mice. L-CPDP and cisplatin (CDDP) caused a comparable inhibition of murine-resident peritoneal macrophage (PM) protein and RNA synthesis and superoxide anion release. PM-mediated tumor cell cytotoxicity was completely inhibited at a concentration of 10 micrograms CDDP and L-CPDP/ml but not at concentrations of 1 and 5 micrograms/ml. The differences in plasma clearance of 99mTc-labeled MLV and phagocytic capacity of the liver among animals pretreated with the maximum tolerated doses of L-CPDP (25 mg/kg), empty liposomes, or CDDP (10 mg/kg) were not statistically significant (plasma clearance % of control: 105, 110, and 100, respectively: P greater than .05; liver uptake % of control: 87, 96, and 104, respectively: P greater than .05). At the maximum tolerated doses, the antitumor activity of L-CPDP against L1210 leukemia was similar to that of CDDP when a single dose was administered [median survival of treated mice/median survival of control mice X 100 (%T/C): 181 vs. 175] and slightly higher with the use of a triple-dose schedule (%T/C: 275 vs. 225). L-CPDP is easy to prepare, has a high-encapsulation efficiency and stability, is not more toxic than CDDP to the monocyte-macrophage system, and is at least as effective as CDDP against L1210 leukemia.

Effective treatment of early endobronchial cancer with regional administration of liposome-p53 complexes.

BACKGROUND: Lung cancer originates in a diffusely damaged bronchial epithelium as a result of sequential and cumulative genetic alterations. We investigated the feasibility of in vivo gene replacement in endobronchial precancerous and cancerous cells by a regionally administered nonviral delivery system. METHODS: After evaluating the in vitro transfection efficiency and cytotoxicity of a variety of cationic liposome-p53 formulations, a specific formulation, DP3-p53, was selected for further in vitro and in vivo evaluation. The ability of DP3-p53 to introduce the p53 gene in the normal bronchial epithelium was studied in transgenic mice that lack the p53 gene. The therapeutic effect of DP3-p53 administered intratracheally was studied in two nude mouse models of endobronchial human lung cancer by use of H358 (p53-null) and H322 (p53-mutant) cells. RESULTS: DP3-p53 was able to effectively introduce and express the p53 gene and induce G1 arrest and apoptosis in H358 cells in vitro and to introduce and transcribe the p53 gene in the bronchial epithelium of transgenic mice that lack the p53 gene in vivo. In therapeutic experiments using groups of four or five mice each, administration of five intratracheal doses of DP3-p53 (2 microg or 8 microg DNA per dose) on days 4, 8, 12, 16, and 20 after intratracheal tumor inoculation significantly inhibited lung tumor formation and prolonged by approximately twofold the survival of mice bearing H358 or H322 endobronchial tumor cells in contrast to the survival among untreated mice and mice treated with the DP3-empty vector (P = .007 [two-sided logrank test] for mice bearing H358 cells and P = .008 [two-sided logrank test] for those bearing H322 cells). CONCLUSIONS/IMPLICATIONS: Liposome-based p53 delivery through the airways is a potentially effective strategy for the treatment of early endobronchial cancer. These results have important implications for the gene therapy and prevention of human lung cancer.

Adenovirus-mediated p53 gene transfer in advanced non-small-cell lung cancer.

BACKGROUND: Preclinical studies in animal models have demonstrated tumor regression following intratumoral administration of an adenovirus vector containing wild-type p53 complementary DNA (Ad-p53). Therefore, in a phase I clinical trial, we administered Ad-p53 to 28 patients with non-small-cell lung cancer (NSCLC) whose cancers had progressed on conventional treatments. METHODS: Patients received up to six, monthly intratumoral injections of Ad-p53 by use of computed tomography-guided percutaneous fine-needle injection (23 patients) or bronchoscopy (five patients). The doses ranged from 10(6) plaque-forming units (PFU) to 10(11) PFU. RESULTS: Polymerase chain reaction (PCR) analysis showed the presence of adenovirus vector DNA in 18 (86%) of 21 patients with evaluable posttreatment biopsy specimens; vector-specific p53 messenger RNA was detected by means of reverse transcription-PCR analysis in 12 (46%) of 26 patients. Apoptosis (programmed cell death) was demonstrated by increased terminal deoxynucleotide transferase-mediated biotin uridine triphosphate nick-end labeling (TUNEL) staining in posttreatment biopsy specimens from 11 patients. Vector-related toxicity was minimal (National Cancer Institute's Common Toxicity Criteria: grade 3 = one patient; grade 4 = no patients) in 84 courses of treatment, despite repeated injections (up to six) in 23 patients. Therapeutic activity in 25 evaluable patients included partial responses in two patients (8%) and disease stabilization (range, 2-14 months) in 16 patients (64%); the remaining seven patients (28%) exhibited disease progression. CONCLUSIONS: Repeated intratumoral injections of Ad-p53 appear to be well tolerated, result in transgene expression of wild-type p53, and seem to mediate antitumor activity in a subset of patients with advanced NSCLC.

Anthracycline antibiotics with high liposome entrapment: structural features and biological activity.

We evaluated the entrapment of 21 different water-insoluble aglycones or anthracycline antibiotics in multilamellar liposomes composed of dimyristoyl phosphatidyl choline and dimyristoyl phosphatidyl glycerol at a 7:3 molar ratio. The drug:lipid weight ratio was 1:15 to 1:50. The different analogues tested were modified at position 4 in the aglycone portion (4-demethoxy) and/or positions 2' (halo), 3' (hydroxy, acetoxy), or 4' (epi, acetoxy) in the sugar portion. The entrapment efficiency was assessed by measuring the amount of free drug remaining in the supernatant after centrifugation of the liposomes and by direct examination of the pellets by fluorescent microscopy. Optimal entrapment (greater than 98%) was observed with only four compounds: 4-demethoxyadriamycinone; 2'-iododaunorubicin; 4-demethoxydaunorubicin; and 2'-iodo-3'-hydroxy-4'-epi-4-demethoxydoxorubicin (Compound 22). All other compounds showed significant drug precipitation outside the multilamellar vesicles when observed by fluorescent microscopy. Compound 22, entrapped in liposomes, was evaluated in vivo against i.p. L-1210 leukemia by the i.p. route, and liver metastases of M5076 reticulosarcoma by the i.v. route. In both models, liposome-entrapped Compound 22 was more active than doxorubicin at the optimal dose [median survival (given in percentage) of treated to control animals was for L-1210, greater than 600 versus 212; for M5076, 200 versus 133]. 4-Demethoxy and 2'-iodo are structural modifications that markedly enhance the affinity of anthracycline antibiotics for lipid bilayers without compromising biological activity. These findings will serve as a guideline to obtain liposome-anthracycline preparations, with optimal formulation characteristics, enhanced tumor-targeting properties, and non-cross-resistance with doxorubicin.

Lipophilic cisplatin analogues entrapped in liposomes: role of intraliposomal drug activation in biological activity.

cis-Bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane platinum (II) (NDDP), a lipophilic cisplatin analogue containing two branched leaving groups of 10 carbon atoms, is undergoing clinical evaluation in a liposomal formulation. In previous studies, NDDP entrapped in multilamellar vesicles composed of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) at a 7:3 molar ratio was non-nephrotoxic in humans, not cross-resistant with cisplatin in different in vitro and in vivo systems, and more active than cisplatin against murine models of experimental liver metastases whereas free NDDP was devoid of in vivo antitumor activity at the optimal dose of L-NDDP and barely active at higher doses. To elucidate the mechanisms by which the liposomal carrier enhances the biological properties to this class of antitumor agents, we studied the effect of the liposome composition, size of the branched leaving groups of the platinum compound, and pH and composition of the aqueous phase on the entrapment efficiency, drug leakage, drug stability, and in vivo toxicity and antitumor activity of different liposomal formulations of these agents. In experiments using normal saline as aqueous phase, the presence of DMPG in the lipid bilayer resulted in a decreased stability and an increased biological activity of NDDP, whereas NDDP entrapped in liposomes composed of DMPC alone (not containing DMPG) was stable but devoid of antitumor activity. In studies with structurally related analogues with branched leaving groups of 5, 6, 7, and 9 carbon atoms, similar trends were observed. In addition, the number of carbon atoms in the leaving groups was directly and inversely related to the entrapment efficiency and stability of the analogues, respectively, independently of lipid composition; increasing the size of the branched leaving groups resulted in an increased in situ degradation of the platinum compound and enhanced biological activity and potency. These results suggest that this class of platinum compounds exerts its biological activity through the formation of active intermediates in situ within the lipid bilayers and that the activation reaction is highly dependent on the presence of DMPG and the size of the lipophilic leaving group.

Treatment and prophylaxis of experimental liver metastases of M5076 reticulosarcoma with cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexaneplatinum (II) encapsulated in multilamellar vesicles.

cis-Bis-neodecanoato-trans-R,R-1,2-diaminocyclohexaneplatinum++ +-(II) (NDDP) was encapsulated in multilamellar vesicles composed of dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol at a 7:3 molar ratio. Compared with cisplatin, i.v. administration of an equimolar dose of liposome-encapsulated NDDP (L-NDDP) resulted in 15-fold higher peak platinum levels in the spleen (204.7 versus 13.3 micrograms/g dry tissue), 5-fold higher in the lungs (116.4 versus 21.0 micrograms/g dry tissue), 3-fold higher in the liver (71.6 versus 23.9 micrograms/g dry tissue), and 4-fold higher in the blood (14.8 versus 3.9 micrograms/ml). At the optimal dose and schedule, L-NDDP administered i.p. in mice bearing peritoneal L1210 leukemia resulted in the percentage of median survival time of treated mice divided by median survival time of control mice (%T/C) of 312 versus 225 for cisplatin and free NDDP. When administered i.v., L-NDDP was also more active than cisplatin against L1210 leukemia inoculated i.v. (%T/C 186 versus 142). L-NDDP was markedly active against L1210 leukemia resistant to cisplatin (%T/C, 200 versus 112 for cisplatin). In mice bearing liver metastases of M5076 reticulosarcoma, L-NDDP was significatnly more effective than cisplatin at equimolar doses (mean survival time, 57 +/- 9 (SD) days for L-NDDP versus 42 +/- 3 days for cisplatin, P less than 0.05). L-NDDP was also effective in preventing liver metastases of M5076 when administered up to 24 h prior to tumor inoculation (mean survival, 28 +/- 2 days for L-NDDP versus 22 +/- 2 days for cisplatin, P less than 0.05). L-NDDP is significantly non-cross-resistant with cisplatin and more effective against phagocytic and nonphagocytic murine tumors.

Apoptosis induced by anthracycline antibiotics in P388 parent and multidrug-resistant cells.

The effect of the topoisomerase II inhibitor doxorubicin and its non-cross-resistant analogue annamycin on DNA degradation and programmed cell death was examined in murine leukemia P388 cells. P388 parental cells exposed to various concentrations of doxorubicin and annamycin for 24 h displayed dose-dependent DNA cleavage: at 1 microM, both doxorubicin and annamycin were effective in inducing DNA breakdown, but at 10 microM, the effect was markedly decreased or totally absent. In multidrug-resistant P388/Dox cells, doxorubicin did not cause DNA cleavage, while 10 microM annamycin had a significant effect. By agarose gel analysis, drug-induced DNA fragmentation showed the characteristic pattern of internucleosomal ladder. Morphologically, P388 cells treated with 1 microM doxorubicin or annamycin for 24 h showed a reduction in cell volume and condensation of nuclear structures. Similar changes were observed in P388/Dox cells exposed to 10 microM annamycin for 24 h but not in cells exposed to 10 microM doxorubicin. Time course studies demonstrated that DNA fragmentation was detected 12 h after incubation with 1 microM doxorubicin or annamycin, while loss of membrane integrity appeared at 24 h, thus indicating that DNA degradation was a preceding event. DNA fragmentation caused by doxorubicin and annamycin was inhibited by the RNA synthesis inhibitor actinomycin D, the protein synthesis inhibitor cycloheximide, and the endonuclease inhibitor aurintricarboxylic acid. Drug-induced cell death was partially prevented by cycloheximide and aurintricarboxylic acid, thus suggesting that the apoptotic process caused by these drugs requires gene expression, synthesis of new proteins, and activation of endogenous nucleases. In contrast, DNA cleavage was not affected by incubating cells with 1 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, thus indicating that intracellular calcium depletion does not affect anthracycline-induced apoptosis. The results obtained demonstrate that the cell killing effect of anthracyclines is mediated, at least in part, by the induction of apoptosis.

Toxicity and antitumor activity of cis-bis-cyclopentenecarboxylato-1,2-diaminocyclohexane platinum(II) encapsulated in multilamellar vesicles.

The potential of multilamellar vesicles (MLVs) as carriers of cis-bis-cyclopentenecarboxylato-1,2-diaminocyclohexane platinum(II) (CPDP), a lipophilic cisplatin derivative, was assessed. MLVs composed of dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), and cholesterol at different molar ratios were tested. The MLV-CPDP preparation with the highest antitumor activity against L1210 leukemia in vivo was DMPC:DMPG 7:3-CPDP. The encapsulation efficiency of this preparation was 66 +/- 4% (SD); the stability in 0.9% NaCl solution at 4 degrees C was 89% at 14 days and 93% 18 h after incubation in human AB serum at 37 degrees C. The toxicities of DMPC:DMPG 7:3-CPDP and free CPDP (suspended in hydroxypropyl cellulose) administered i.p. were similar (50% lethal dose = 75 versus 91 mg/kg; blood urea nitrogen values 96 h after the administration of the 50% lethal dose = 32.0 versus 34.4 mg/dl). The mean %T/C [(median survival time of treated mice divided by median survival time of control mice) X 100] obtained after a single i.p. injection of the optimal dose of each preparation tested was 215 (range 200 to 232) for DMPC:DMPG 7:3-CPDP, 175 (range 158 to 209) for DMPG-CPDP, 162 (range 136 to 179) for free CPDP, and 178 (range 169 to 189) for cisplatin. Using a multiple i.p. injection schedule (injections on Days 1, 5, and 9), DMPC:DMPG 7:3-CPDP was more active than free CPDP and cisplatin (%T/C: 403, 284, and 253% respectively). DMPC:DMPG 7:3-CPDP is less toxic and more active against L1210 leukemia in vivo than is cisplatin. The encapsulation of CPDP in MLVs composed of DMPC:DMPG 7:3 provides an adequate vehicle for the administration of this lipophilic compound and enhances its antitumor activity against L1210 leukemia.

Paclitaxel-induced apoptosis is associated with expression and activation of c-Mos gene product in human ovarian carcinoma SKOV3 cells.

The c-Mos gene product is a component of the cytostatic factor and, as such, stabilizes the maturation-promoting factor causing cell-cycle blockade at metaphase II in unfertilized eggs. The potential role of c-Mos in regulating cell-cycle progression and cell death in somatic cells remains unknown. We studied whether paclitaxel-induced M-phase arrest and apoptosis are associated with c-Mos gene expression and activation in SKOV3 ovarian carcinoma cells. The first cellular effect observed with continuous exposure to 50 ng/ml paclitaxel (ID50) was mitotic arrest with an increase in the accumulation of cyclin B1 and stimulation of cdc2/cyclin B1 kinase in a time-dependent manner during a 36-h incubation. DNA fragmentation determined by agarose gel electrophoresis and quantitation of [3H]thymidine-prelabeled genomic DNA was a later event, first detected at 24 h and peaking at 48 h (later time points were not studied). Induction of the c-Mos gene expression and activation were determined by Western blot analysis, immunoprecipitation using a polyclonal anti-mos antibody, reverse transcription-PCR assay, and 32P-ATP incorporation into c-Mos protein or the substrate of glutathione S-transferase mitogen-activated protein kinase kinase, respectively. Both induction and activation were clearly detected after 24 h of exposure to paclitaxel concentrations of >50 ng/ml, coinciding with drug-induced apoptosis. Mitogen-activated protein kinase activation preceded c-Mos gene induction. Paclitaxel-induced c-Mos gene expression was completely abrogated by cycloheximide and actinomycin D. Mos gene expression was also induced in SKOV3 cells that were treated with vinblastine but not in those that were treated with camptothecin, etoposide, or cisplatin. We concluded that tubulin-disturbing agents induce c-Mos gene expression and activation in SKOV3 ovarian carcinoma cells and that such an effect occurs after mitotic blockade and coincides with drug-induced apoptosis.

Increased cytotoxicity and reversal of resistance to cis-diamminedichloro-platinum(II) with entrapment of cis-Bis-neodecanoato-trans-R,R-1,2-diaminocyclohexaneplatinum (II) in multilamellar lipid vesicles.

The role of liposome entrapment in modulating the cytotoxicity of a lipophilic cisplatin derivative was assessed. cis-Bis-neodecanoato-trans-R,R-1,2-diaminocyclohexaneplatinum++ +(II) (NDDP) was tested in suspension (free NDDP) or entrapped in multilamellar vesicles composed of dimyristoylphosphatidyl choline and dimyristoylphosphatidyl glycerol (L-NDDP). Against LoVo colon carcinoma cells sensitive to cisplatin, L-NDDP was two times more cytotoxic in vitro than free NDDP and cisplatin (Do 7 microM for L-NDDP, 15 microM for free NDDP, and 16 microM for cisplatin). Against LoVo cells resistant to a concentration of 3 micrograms/ml of cisplatin, L-NDDP was three times more cytotoxic than free NDDP and cisplatin (Do 14 microM for L-NDDP, 45 microM for free NDDP, and 48 microM for cisplatin). In in vivo studies, free NDDP was less potent and less active than L-NDDP against i.p. L-1210 leukemia (free NDDP, optimum %T/C 148 at a dose of 75 mg/kg; L-NDDP, optimum %T/C 185 at a dose of 25 mg/kg) and i.p. L1210/PDD leukemia (free NDDP, optimum %T/C 128 at a dose of 50 mg/kg on Days 1, 5, and 9; L-NDDP, optimum %T/C 200 at a dose of 12.5 mg/kg on Days 1, 5, and 9). Free NDDP administered i.v. was inactive against liver metastases of M5076 reticulosarcoma (%T/C 102) while L-NDDP showed significant activity (%T/C 140). The single dose i.v. LD50 in mice of free NDDP and L-NDDP were similar (79.4 mg/kg for free NDDP and 64.5 mg/kg for L-NDDP). These studies show that NDDP is a liposome-dependent drug since it can only be satisfactorily formulated in the liposomal form and since the liposomal carrier plays a crucial role in determining its antitumor activity.

Antitumor activity of free and liposome-entrapped annamycin, a lipophilic anthracycline antibiotic with non-cross-resistance properties.

The lipophilic anthracycline antibiotic annamycin (Ann) was entrapped in liposomes of different size [median diameter: 1.64 microns, multilamellar liposomal Ann (L-Ann); 0.030 micron, small unilamellar Ann (S-Ann)] with > 90% entrapment efficiency and tested in vitro against four pairs of sensitive and multidrug-resistant (MDR) tumor cell lines and in vivo by the i.v. route in five tumor models: advanced s.c. B16 melanoma; s.c. M5076 reticulosarcoma; lung metastases of Lewis lung carcinoma; and s.c. KB and KB-V1 xenografts in nude mice. Predetermined optimal doses of the different formulations were used and the results were compared with doxorubicin (Dox). In vitro, Ann, either in suspension in 10% dimethyl sulfoxide (F-Ann) (1 mg/ml) or entrapped in liposomes, was able to partially overcome resistance in all four pairs of sensitive and MDR KB, 8226, P388, and CEM cell lines (resistance indexes 63, 269, 333, and 356 for Dox versus 4, 5, 19, and 8.7 for L-Ann, respectively). In vivo, both F-Ann and liposome-entrapped Ann were slightly more effective than Dox in inhibiting the growth of advanced s.c. B16 melanoma tumors. L-Ann was markedly more effective than Dox and moderately more effective than F-Ann in prolonging the life span of animals bearing s.c. M5076 and lung metastases of Lewis lung carcinoma tumors. All drugs were equally effective at optimal doses in delaying the growth of s.c. KB xenografts, whereas all Ann formulations were markedly more effective than Dox in delaying the growth of s.c. KB-V1 (MDR) xenografts. In all in vivo experiments, S-Ann was consistently more effective than L-Ann and L-Ann was more effective than F-Ann. These results indicate that (a) Ann is more effective than Dox by the i.v. route against several tumor models and that MDR tumors are partially not cross-resistant to Ann both in vitro and in vivo, (b) liposomes enhance the in vivo antitumor properties of Ann, and (c) small liposomes are more effective than large liposomes in enhancing Ann antitumor activity.

Cellular pharmacology of liposomal cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexaneplatinum(II) in A2780/S and A2780/PDD cells.

We studied the cytotoxicity, cellular accumulation, and DNA interactions induced by liposome-entrapped NDDP (L-NDDP) and cisplatin in A2780 human ovarian carcinoma cells sensitive (A2780/S) and resistant (A2780/PDD) to cisplatin. L-NDDP was 2-fold more cytotoxic than cisplatin against A2780/S cells with 5-h or 24-h drug exposure. Both cell lines were equally sensitive to L-NDDP, while A2780/PDD cells were 4-fold resistant to cisplatin (resistance indexes, 1.30-1.85 and 4.02-4.50, respectively). Using a drug exposure time of 24 h, L-NDDP cytotoxicity was independent of the liposome composition used, whereas with shorter drug exposure (5 h), the cytotoxicity of L-NDDP was directly related to the relative content of DMPG in the liposome carrier. However, changes in liposome composition or drug exposure time did not alter the resistance index of L-NDDP in this cell system. The cellular accumulation of L-NDDP was similar in both cell lines and 2- to 5-fold higher than that of cisplatin in A2780/S cells, whereas the cellular accumulation of cisplatin was reduced by 2- to 3-fold in A2780/PDD cells. The presence of DMPG in the lipid bilayer enhanced by 2-fold the cellular accumulation of L-NDDP, in good correlation with the direct relation between cytotoxic potency of L-NDDP and the presence of DMPG in the liposome carrier. Pt/DNA levels were determined at different time points after drug exposure for 1 h. Peak Pt/DNA levels were observed at 6 h for cisplatin and at 9 h for L-NDDP. Peak Pt/DNA levels and Pt/DNA over time of L-NDDP were about 1.5- and 3-fold higher than those of cisplatin in A2780/S and A2780/PDD cells, respectively, when equimolar concentrations of both drugs were used. Cisplatin induced significant DNA interstrand and DNA-protein cross-links in A2780/S cells, and a good correlation was observed between cytotoxicity against both cell lines and both types of lesions. In contrast, equimolar concentrations of L-NDDP induced only minimal DNA interstrand cross-links in either cell line.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro cytotoxicity, cellular pharmacology, and DNA lesions induced by annamycin, an anthracycline derivative with high affinity for lipid membranes.

Annamycin (AN) is an anthracycline antibiotic with high affinity for lipid membranes which is being developed for clinical studies formulated in liposomes. We studied the in vitro cytotoxicity, cellular pharmacology, and DNA damage induced by AN in P388 cells sensitive and resistant to doxorubicin (DOX). AN was as cytotoxic as DOX against P388-sensitive cells and about 50 times more cytotoxic than DOX against P388-resistant cells (resistance index 5 for AN versus 250 for DOX). Cellular uptake of AN by sensitive cells was 2-3-fold higher than that of DOX. In resistant cells, cellular uptake of AN and DOX was approximately 65% and 30%, respectively, of the cellular uptake in sensitive cells. As a result, cellular uptake of AN by resistant cells was higher than uptake of DOX by sensitive cells. DOX was fully retained in sensitive cells while it was effluxed rapidly from resistant cells. In contrast, efflux of AN was similar in sensitive and resistant cells, thus suggesting that it is not mediated by P-glycoprotein. AN was more effective than DOX in inducing single DNA breaks, double DNA breaks, and DNA-protein cross-links, both in sensitive and resistant cells, although DNA damage was lower in resistant cells than in sensitive cells. DNA lesions induced by AN in resistant cells were similar to or greater than those induced by DOX in sensitive cells. These studies indicate that the lack of cross-resistance between DOX and AN appears to be related, at least in part, to the relatively higher cellular uptake of AN compared with DOX and is associated with the ability of AN to induce significant DNA damage in resistant cells.

Enhanced therapeutic effect against liver W256 carcinosarcoma with temperature-sensitive liposomal adriamycin administered into the hepatic artery.

The antitumor activity of Adriamycin encapsulated in temperature-sensitive liposomes combined with local hyperthermia (HT) was tested in rats bearing well-developed liver W256 carcinosarcoma tumors. Two h after rats received Adriamycin encapsulated in temperature-sensitive liposomes via either the hepatic artery (i.a.) or the femoral vein (i.v.) or free Adriamycin i.a., liver HT was applied at 42 degrees C for 6 min. In animals treated with liposomal Adriamycin i.a., HT resulted in a 38% reduction in the tumor volume ratio and a 2.2-fold increase in the life span of the animals. In animals treated with liposomal Adriamycin i.v. or free Adriamycin i.a., HT did not alter the tumor volume ratio or life span of the animals. Administration i.a. of liposomal Adriamycin markedly increased the tumor drug levels (4-14-fold), reduced the systemic distribution of the drug, and slowed the drug decrease from both the tumor and liver compared with animals treated i.v.. Liver HT in animals treated with liposomal Adriamycin i.a. further increased tumor drug levels by 1.5-2.6-fold, further slowed the drug decrease from the tumor, and resulted in a dissociation of the parallel decrease of drug and lipid from the tumor. This latter effect was not observed in the other groups. These pharmacological findings combined with the lack of beneficial effect from HT in animals treated with free Adriamycin i.a. or liposomal Adriamycin i.v. suggest that i.a. administration of Adriamycin encapsulated in temperature-sensitive liposomes results in a significant retention of intact liposomes in the tumor vasculature that are able to release the encapsulated drug into the tumor cell compartment upon raising the temperature to the phase transition level.

Cellular pharmacology of liposomal cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexaneplatinum (II) in mouse resident peritoneal macrophages, Kupffer cells, and hepatocytes.

The in vitro and in vivo interaction of liposomal cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexaneplatinum++ + (II) (L-NDDP) with mouse resident peritoneal macrophages (RPM), Kupffer cells (KC), and hepatocytes was studied. The peak in vitro uptake of L-NDDP by RPM was 12.5 ng elemental platinum/100 micrograms cell protein and constituted 0.2% of the platinum available for phagocytosis. The subsequent release of platinum by RPM was rapid initially, with a 20-fold increase over the first 4 h, followed by a plateau; ultrafilterable (free) platinum constituted 50% of the total platinum released at 24 h. The retained intracellular platinum in RPM at 24 h was close to 50% of that initially present. The peak in vitro uptake of L-NDDP by KC was 11.3 ng platinum/100 micrograms cell protein and amounted to 0.2% of the platinum available for phagocytosis. The release of platinum by KC was detectable only after 4 h of incubation and increased 3-fold over the next 14 h. The ultrafilterable platinum released by KC at 18 h was 40% of the total platinum released. The retained intracellular platinum in KC at 18 h was 33% of that initially present. The peak in vitro uptake of L-NDDP by hepatocytes was almost 50 ng platinum/100 micrograms cell protein and constituted 0.8% of the platinum available for intake. Following the i.v. injection of L-NDDP, hepatocytes contained up to 6-fold higher platinum concentrations than KC. This observation was supported by transmission electron microscopy showing a higher concentration of multilamellar vesicles within hepatocytes than in KC, 5 min after i.v. injection of L-NDDP. These findings suggest that L-NDDP becomes available to the liver following i.v. injection, that both macrophages and hepatocytes play a role in the metabolism of L-NDDP, and that Kupffer cells could mediate a sustained release of platinum in the liver following the interaction with L-NDDP, indicating the potential of L-NDDP for the treatment of tumors in the liver.

3-(Iodoacetamido)-benzoylurea: a novel cancericidal tubulin ligand that inhibits microtubule polymerization, phosphorylates bcl-2, and induces apoptosis in tumor cells.

3-(Iodoacetamido)-benzoylurea (3-IAABU) is a newly synthesized antitubulin compound with a molecular weight of 347. 3-IAABU exhibited anticancer activity in a variety of tumor cell lines with ID90 in the range of 0.015-0.29 microM for leukemic cells and 0.06-0.92 microM for solid tumors. Higher selectivity against malignant cells was observed with 3-IAABU than that with vinblastine and paclitaxel. It inhibits microtubule assembly in tubulin systems either with or without microtubule-associated proteins (ID50 was 0.1 microM and 1.2 microM, respectively) and microtubule depolymerization was not affected, indicating an inhibition of polymerization by binding of 3-IAABU to the heterodimeric subunit of tubulin. 3-IAABU was shown to inhibit the binding of colchicine, a subunit binding compound, but did not inhibit binding of vinblastine and guanosine 5'-triphosphate/guanosine 5'-diphosphate, indicating that colchicine site corresponds to the site that 3-IAABU locates. Tumor cells treated with 3-IAABU showed scattered chromosomes in metaphase. Normal microtubule architecture or spindle apparatus was absent in these cells; instead, punctuated aggregates of tubulin were found by an immunofluorescent staining. Cell cycle analyses showed an accumulation of tumor cells at M phase after a 4-h treatment with 3-IAABU. The phosphorylated bcl-2 representative of an inactivated form of the oncoprotein was found in the cells 12 h after treatment with 3-IAABU. These cells progressed to apoptosis within 16 h. As a new tubulin ligand, 3-IAABU could be a promising agent in cancer chemotherapy.

Phase I clinical and pharmacological study of liposome-entrapped cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane platinum(II).

cis-Bis-neodecanoato-trans-R,R-1,2-diaminocyclohexaneplatinum++ +(II) (NDDP) is a liposome dependent cisplatin analogue since the liposome carrier is required for its i.v. administration and for its biological activity. A Phase I study of liposome entrapped NDDP (L-NDDP) was performed using a single i.v. injection every 4 weeks. L-NDDP was prepared and characterized at M. D. Anderson Cancer Center. The maximum tolerated dose of L-NDDP was 312.5 mg/m2. The dose-limiting toxicity was myelosuppression, affecting all three blood cell lineages. The granulocyte nadir occurred on days 14-18, and the platelet nadir consistently earlier (days 11-12). The median day of recovery of blood cell counts was day 21 (range, 18-32). Other toxicities included grade 2 nausea and vomiting, fever consisting of a single temperature spike in most patients, grade 1 diarrhea after 60% of courses, and grade 1-2 malaise lasting for 5-10 days after the infusion in 73% of courses. Transient alanine aminotransferase elevations without clinical relevance were common. No signs of renal dysfunction or ototoxicity were observed. One patient with a preexisting peripheral neuropathy showed some progression of the neuropathy after a cumulative dose of 1605 mg/m2. Except for fever and transient liver dysfunction, no liposome related side effects were observed in spite of the high doses of lipid administered. The blood clearance of L-NDDP fits a two-compartment model at lower doses and a single-compartment model at the maximum tolerated dose, suggesting that saturation of the reticuloendothelial organs occurs at the maximum tolerated dose. Two minimal responses were observed. L-NDDP has a toxicity profile similar to that of carboplatin. Phase II studies to address the issue of how the therapeutic index of platinum compounds is affected by liposome entrapment are being planned.

Superoxide anion (O-2) production by peripheral blood monocytes in Hodgkin's disease and malignant lymphoma.

Superoxide anion (O-2) is the first metabolite of the monocyte oxygen burst pathway, which plays an important role in the monocyte microbicidal function. The capacity of peripheral blood monocytes to produce O-2 was studied in 63 patients with Hodgkin's disease (31 with active disease and 32 in complete remission), 15 patients with active malignant lymphoma, and 57 normal control subjects. O-2 release was quantified by evaluating superoxide dismutase-inhibitable reduction of cytochrome c after stimulation of monocytes with phorbol myristate acetate. Results were expressed in nanomols O-2 per mg protein per hour. O-2 production was lower than normal in patients with active Hodgkin's disease (163.3 v 214.5, P less than .05). It was normal in patients with Hodgkin's disease in complete remission (216.2 v 214.5, P greater than .05) and high in patients with malignant lymphomas (317.9 v 214.5, P less than .01). Within the group with active Hodgkin's disease, patients in relapse after therapy had a lower O-2 production than those previously untreated (99.8 v 181.8, P less than .01). Stage of disease was unrelated to the defect. The presence of B symptoms and a decreased delayed type hypersensitivity to recall skin test antigens were associated with normal O-2 production. The results obtained suggest that monocyte dysfunction is part of the immune dysregulation associated with active Hodgkin's disease. The O-2 determination is a relatively easy test to perform and may be useful in identifying the patients with Hodgkin's disease who have an increased risk of opportunistic infections.