Enhanced therapeutic effects of doxorubicin and paclitaxel in combination with liposome-entrapped ends-modified raf antisense oligonucleotide against human prostate, lung and breast tumor models

Raf-1 protein kinase plays an important role in cell growth, proliferation and cell survival. We have previously described the use of liposome-entrapped antisense raf oligonucleotide (LErafAON) to inhibit Raf-1 expression resulting in tumor growth inhibition and radiosensitization. The present study was undertaken to evaluate the chemosensitization effects of LErafAON in combination with doxorubicin or paclitaxel on a panel of human tumor xenografts. LErafAON (25.0 mg/kg i.v. x 10) displayed significant antitumor activity (P<0.05) when administered as a single agent in prostate (PC-3), lung (A549) and breast (MDA-MB 231) carcinoma models. Doxorubicin (1.0-4.0 mg/kg i.v. per week x 3) and paclitaxel (1.0-4.0 mg/kg i.v. on alternate days x 3) were administered as single agents at non-toxic doses that led to only minimal to moderate antitumor activity. However, a combination of LErafAON with doxorubicin or paclitaxel led to significantly enhanced antitumor activity in all the tumor types tested (PC-3, P<0.03; A549, P<0.035; MDA-MB 231, P<0.045) as compared with LErafAON alone or chemotherapeutic agents alone treated groups. This effect of chemosensitization appeared to be sequence-specific because a mismatch control oligonucleotide continued to show significant tumor growth. Additionally, no inhibition in Raf-1 expression in MDA-MB 231 tumor tissue was observed with mismatch oligonucleotide treatment. On the other hand, LErafAON treatment led to >75% inhibition of Raf-1 expression in tumor tissue. These preclinical observations support the use of LErafAON in combination with chemotherapeutic agents to improve the treatment of human cancers.     

Improved safety, pharmacokinetics and therapeutic efficacy profiles of a novel liposomal formulation of mitoxantrone

We describe here the preclinical studies of a novel formulation of liposome-entrapped mitoxantrone (LEM). The liposome entrapment efficiency of mitoxantrone was 93.4 +/- 2.8%. In vitro cytotoxicity studies in HL60 cells comparing LEM with conventional mitoxantrone (MTO) showed IC50 values of 0.31 +/- 0.05 ng/ml and 0.48 +/- 0.06 ng/ml for LEM and MTO, respectively. In CD2F1 mice, LEM was significantly less toxic as compared with MTO. A single intravenous (i.v.) dose of 15 mg/kg MTO produced 100% mortality in CD2F1 mice by Day 10, whereas a single i.v. dose as high as 35 mg/kg LEM caused no mortality for at least up to Day 60 post-treatment. Multiple doses of MTO (i.v., 5.0 mg/kg, 1x daily, x5) caused 100% mortality by Day 10, whereas a similar dose regimen of LEM caused no mortality in CD2F1 mice. Clinical and histopathology evaluations indicated long-term normal tissue protection in mice treated with relatively high single dose (i.v., 35 mg/kg) or multiple doses of LEM (i.v., 5.0 mg/kg, 1x daily, x5). LEM also demonstrated favourable pharmacokinetic profiles. CD2F1 mice injected with 5 mg/kg i.v. dose of LEM showed plasma levels 51-fold higher than with an equivalent dose of MTO. The area under the plasma concentration-time curve was 200-fold greater with LEM as compared to MTO. The plasma half-lives were 0.96 hours and 0.11 hours for LEM and MTO, respectively. An altered tissue distribution was observed with LEM; cardiac tissue demonstrating at least 2.6-fold lower levels of mitoxantrone with LEM vs. MTO. LEM exhibited significant anti-tumor activity against murine ascitic L1210 leukemia in CD2F1 mice. Treatment with a single dose of 20.0 mg/kg LEM resulted in 100% long-term survivors. LEM 2.5 mg/kg (i.v., x4) had antitumor activity against a human hormone-independent prostate carcinoma (PC-3) grown in athymic mice, while a comparable dose of MTO was too toxic. A significant decrease in toxicity, altered pharmacokinetics, and enhanced efficacy of LEM suggest that LEM may provide a viable alternative to the clinical use of conventional mitoxantrone.     

Delivery of a liposomal c-raf-1 antisense oligonucleotide by weekly bolus dosing in patients with advanced solid tumors: a phase I study.

PURPOSE: Rapid cleavage in vivo and inefficient cellular uptake limit the clinical utility of antisense oligonucleotides (AON). Liposomal formulation may promote better intratumoral AON delivery and inhibit degradation in vivo. We conducted the first clinical evaluation of this concept using a liposomal AON complementary to the c-raf-1 proto-oncogene (LErafAON). EXPERIMENTAL DESIGN: A dose escalation study was done to determine the maximum tolerated dose and to characterize the toxicities of LErafAON given as weekly intravenous infusion for 8 weeks to adults with advanced solid tumors. Pharmacokinetic analysis and evaluation of c-raf-1 target suppression in peripheral blood mononuclear cells were included. RESULTS: Twenty-two patients received LErafAON (median 7 infusions; range 1-27) at doses of 1, 2, 4, and 6 mg/kg/week. Across all dose cohorts patients experienced infusion-related hypersensitivity reactions including flushing, dyspnea, hypoxia, rigors, back pain, and hypotension. Prolonged infusion duration and pretreatment with acetaminophen, H1- and H2-antagonists, and corticosteroids reduced the frequency and severity of these reactions. Progressive thrombocytopenia was dose-limiting at 6 mg/kg/week. No objective responses were observed. Two patients treated at the maximum tolerated dose of 4 mg/kg/week had evidence of stable disease, with dosing extended beyond 8 weeks. Pharmacokinetic analysis revealed persistence of detectable circulating rafAON at 24 hours in 7 of 10 patients in the highest 2 dose cohorts. Suppression of c-raf-1 mRNA was noted in two of five patients analyzed. CONCLUSIONS: Dose-independent hypersensitivity reactions and dose-dependent thrombocytopenia limited tolerance of LErafAON. Future clinical evaluation of this approach will depend on modification of the liposome composition.     

Phase I study of liposome-encapsulated c-raf antisense oligodeoxyribonucleotide infusion in combination with radiation therapy in patients with advanced malignancies.

PURPOSE: Raf proteins are key elements of growth-related cellular signaling pathways and are a component of cancer cell resistance to radiation therapy. Antisense oligonucleotides to c-raf-1 permit highly selective inhibition of the gene product and offer a strategy for sensitizing cancer cells to radiation therapy. In this dose escalation study, we evaluated the safety of combined liposomal formulation of raf antisense oligonucleotide (LErafAON) and radiation therapy in patients with advanced malignancies. EXPERIMENTAL DESIGN: Patients with advanced solid tumors were treated with LErafAON in a phase I dose escalation study while receiving palliative radiation therapy. Drug-related and radiation-related toxicities were monitored. Pharmacokinetics and expression of c-raf-1 mRNA and Raf-1 protein were determined in peripheral blood mononuclear cells. RESULTS: Seventeen patients with palliative indications for radiation therapy were entered into this study. Thirteen patients received daily infusions of LErafAON and four received twice-weekly infusions. Radiation therapy was delivered in daily 300-cGy fractions over 2 weeks. Patients tolerated radiation, and no unexpected radiation-related side effects were observed. Drug-related reactions (grade > or =2), such as back pain, chills, dyspnea, fatigue, fever, flushing, and hypertension, were observed in most patients and were managed by premedication with corticosteroids and antihistamines. Serious adverse events occurred in five patients, including acute infusion-related symptoms, abnormal liver function tests, hypoxia, dehydration, diarrhea, esophagitis, fever, hypokalemia, pharyngitis, and tachypnea. Twelve of 17 patients were evaluable for tumor response at completion of treatment; four showed partial response, four showed stable disease, and four experienced progressive disease. The intact rafAON was detected in plasma for 30 minutes to several hours. Six patients with partial response or stable disease were evaluable for c-raf-1 mRNA and/or Raf-1 protein expression. Inhibition of c-raf-1 mRNA was observed in three of five patients. Raf-1 protein was inhibited in four of five patients. CONCLUSION: This is the first report of the combined modality treatment using antisense oligonucleotides with radiation therapy in patients with advanced cancer. A dose of 2.0 mg/kg of LErafAON administered twice weekly is tolerated with premedication and does not enhance radiation toxicity in patients. The observation of dose-dependent, infusion-related reactions has led to further modification of the liposomal composition for use in future clinical trials.     

Therapeutic cure against human tumor xenografts in nude mice by a microtubule stabilization agent, fludelone, via parenteral or oral route

Epothilones, 16-membered macrolides isolated from a myxobacterium in soil, exert their antitumor effect, like Taxol, by induction of microtubule polymerization and microtubule stabilization. They are effective against tumor cells that are resistant to Taxol or vinblastine. We recently designed, via molecular editing and total synthesis, a new class of epothilones represented by 26-trifluoro-(E)-9,10-dehydro-12,13-desoxy-epothilone B (Fludelone), which has emerged as a lead candidate for clinical development. Treatment of nude mice bearing MX-1 human mammary carcinoma xenografts (as large as 3.4% body weight) with Fludelone (6-hour i.v. infusion, 25 mg/kg, q3d x 5, q3d x 4) led to complete disappearance and de facto "cure" (i.e., remission without a relapse for over 15% of the average life span of 2 years). The toxicities induced by bolus i.v. injection could be avoided through prolonged i.v. infusion, which allowed for a 10-fold increase in maximal tolerated dose. Complete remission of MX-1 xenografts was achieved with only one third of this maximal tolerated dose. Parallel studies with Taxol and Fludelone [20 mg/kg, 6-hour i.v. infusion (q2d x 4) x3] against HCT-116 human colon carcinoma xenografts revealed that both drugs achieved tumor remission; however, all Taxol-treated mice relapsed in approximately 1.3 months, whereas the Fludelone-treated mice were cured without any relapse for over 7 months. Furthermore, tumor remission was achieved by Fludelone against SK-OV-3 (ovary), PC-3 (prostate), and the Taxol-resistant CCRF-CEM/Taxol (leukemia) xenograft tumors. Most remarkably, p.o. administration of Fludelone (30 mg/kg, q2d x 7, q2d x 9, q2d x 5) against MX-1 xenografts achieved a nonrelapsing cure for as long as 8.4 months. The above results indicate that Fludelone is a highly promising compound for cancer chemotherapeutics.     

Mouse Lewis lung carcinoma and hepatoma ascites treatment by combination of liposome chemotherapy and non-specific immunotherapy

Liposomes have been used as biological carriers of anti-tumor drugs, and their potential use has been tested in mouse Lewis lung carcinoma and hepatoma ascites tumor models. Ara-C3 given by the intraperitoneal i.p. route either at 35 mg/kg in a single dose or at 2.5 mg/kg/dose with 5 doses/day for 3 days had no effect on the average survival time of i.v. implanted LLC. However, the same single dose of Ara-C encapsulated in positively charged MLV significantly improved the average survival time of LLC-bearing mice. MTX was chosen as a test drug for the treatment of hepatoma ascites. Non-encapsulated MTX given at either 3 or 30 mg/kg by the i.p. route had little effect on the average survival of i.p.-implanted hepatoma ascites. However, MTX encapsulated in SUV at a 3 mg/kg dose by the i.p. route significantly improved the average survival time of tumor-bearing mice. A combination of chemotherapy and non-specific immunotherapy has also been tested with these 2 tumor models. Two non-specific microbial immune stimulators, Bacillus Calmette Guérin (BCG) and Corynebacterium parvum (CP) were tested by both the i.v. and i.p. routes. A combination of BCG therapy with non-encapsulated anti-tumor drugs was not effective for either of the tumor models. A combination of BCG therapy with liposome therapy appeared to improve the average survival time of LLC-bearing mice. In particular, BCG treatment by the i.p. route in combination with liposome therapy resulted in a 20% long-term survival rate among treated mice. However, BCG therapy by either route in combination with SUV encapsulated MTX therapy had no effect on the average survival time of hepatoma ascites-bearing mice. Immunostimulation with CP at a given dose appears to be superior to BCG therapy for both tumor models. In the treatment of LLC, injection of CP by either the i.v. or i.p. route appears to be equally effective in combination with liposome therapy. However, for the treatment of hepatoma ascites, CP was only effective by the i.p. route, in combination with liposome therapy.     

Plasma pharmacokinetics, oral bioavailability, and interspecies scaling of the DNA methyltransferase inhibitor, zebularine.

PURPOSE: Zebularine is a DNA methyltransferase inhibitor proposed for clinical evaluation. EXPERIMENTAL DESIGN: We developed a liquid chromatography/mass spectrometry assay and did i.v. and oral studies in mice, rats, and rhesus monkeys. RESULTS: In mice, plasma zebularine concentrations declined with terminal half-lives (t(1/2)) of 40 and 91 minutes after 100 mg/kg i.v. and 1,000 mg/kg given orally, respectively. Zebularine plasma concentration versus time curves (area under the curve) after 100 mg/kg i.v. and 1,000 mg/kg given orally were 7,323 and 4,935 mug/mL min, respectively, corresponding to a total body clearance (CL(tb)) of 13.65 mL/min/kg, apparent total body clearance (CL(app)) of 203 mL/min/kg, and oral bioavailability of 6.7%. In rats, plasma zebularine concentrations declined with t(1/2) of 363, 110, and 126 minutes after 50 mg/kg i.v., 250 mg/kg given orally, and 500 mg/kg given orally, respectively. Zebularine areas under the curve after 50 mg/kg i.v., 250 mg/kg given orally, and 500 mg/kg given orally were 12,526, 1,969, and 7,612 mug/mL min, respectively, corresponding to a CL(tb) of 3.99 mL/min/kg for 50 mg/kg i.v. and CL(app) of 127 and 66 mL/min/kg for 250 and 500 mg/kg given orally, respectively. Bioavailabilities of 3.1% and 6.1% were calculated for the 250 and 500 mg/kg oral doses, respectively. In monkeys, zebularine t(1/2) was 70 and 150 minutes, CL(tb) was 3.55 and 10.85 mL/min/kg after i.v. administration, and CL(app) was 886 and 39,572 mL/min/kg after oral administration of 500 and 1,000 mg/kg, respectively. Zebularine oral bioavailability was <1% in monkeys. Interspecies scaling produced the following relationship: CL(tb) = 6.46(weight(0.9)). CONCLUSIONS: Zebularine has limited oral bioavailability. Interspecies scaling projects a CL(tb) of 296 mL/min in humans.     

GEM 231, a second-generation antisense agent complementary to protein kinase A RIalpha subunit,potentiates antitumor activity of irinotecan in human colon,pancreas, prostate and lung cancer xenografts

GEM 231, a second-generation antisense oligonucleotide targeted against the RIalpha subunit of protein kinase A (PKA) was co-administered with the chemotherapeutic agent irinotecan, a topoisomerase-I inhibitor, to study the antitumor efficacy of the combination in nude mice bearing various human tumor xenografts. The combination treatment of GEM 231 and irinotecan produced enhanced and prolonged tumor-growth inhibition, compared with irinotecan monotherapy, against human colon (HCT-116), pancreas (Panc-1), prostate (PC3) and lung (SKMES) tumors in mice. The extent of tumor-growth inhibition, however, varied among the different tumor models studied. The tumor-growth inhibition depended on the dose of GEM 231 co-administered with irinotecan. The combination of GEM 231 (20 mg/kg, i.p., 5 days on 2 days off x 7) and irinotecan (50 mg/kg, i.v., qwk x 3) produced significantly longer tumor-growth delay than did irinotecan administered alone. Importantly, the co-administration of irinotecan and GEM 231 did not result in higher toxicity compared with monotherapies in the several tumor models tested. These results suggest that the use of irinotecan in combination with GEM 231 may increase the therapeutic index of irinotecan in cancer patients.     

Chemosensitization and radiosensitization of human lung and colon cancers by antimitotic agent, ABT-751, in athymic murine xenograft models of subcutaneous tumor growth

Purpose

ABT-751 is an orally active antimitotic agent that is currently in Phase II clinical trials. This agent binds to the colchicine site on ß-tubulin and inhibits polymerization of microtubules. This disruption of microtubule dynamics leads to a block in the cell cycle at the G₂/M phase, and promotes apoptosis. ABT-751, as a single agent, has antitumor activity against a series of xenograft models including non-small cell lung cancer (NSCLC) and colon cancer. The current studies were conducted to determine whether ABT-751 enhances antitumor activity of standard cytotoxic therapies currently in clinical use.

Methods

Efficacy of ABT-751, in combination with cisplatin, 5-FU, and radiation, was evaluated in the Calu-6 NSCLC, HT-29 colon, and HCT-116 colon carcinoma xenograft models, respectively. Tumor-bearing athymic mice were treated with ABT-751 orally once a day at 75 or 100 mg/kg/day on a 5-days-on, 5-days-off schedule for two cycles.

Results

Efficacy of ABT-751 at 100 mg/kg/day was tested in combination with cisplatin at its maximum tolerable dose (MTD) (10 mg/kg/day, i.p. x1) in Calu-6 tumor-bearing athymic mice. The percent treated/control (%T/C) tumor volume ratios on day 38 were 35, 37, and 6, and the percent tumor growth delay (%TGD) values were 71, 65, and 188 for cisplatin, ABT-751 and the combination groups, respectively. HT-29 colon tumors were used to test ABT-751 in combination with an MTD of 5-FU, 30 mg/kg/day, i.p., q.d. x5. The %T/C ratios on day 38 were 22, 28, and 5 and the %TGD values were 75, 75, and 150 for 5-FU, ABT-751, and the combination groups, respectively. Treatment of HCT-116 colon carcinoma tumors with ABT-751, concurrent with the radiation treatment, was able to both enhance radiation-induced tumor regression, and delay the time to recurrence and progression. Growth curves allowed calculation of enhancement of radiation-induced growth delay (defined as the additional time required for a treated tumor to reach four times its original size) of 2, 9, and 12 days, for ABT-751 alone, radiation alone, and the combination, respectively.

Conclusion

Collectively, these studies demonstrate that ABT-751 enhanced efficacy of standard cytotoxic therapies in a variety of tumor xenograft models, and that enhancement was at least additive in all systems.     

Therapy of central nervous system leukemia in mice by liposome-entrapped 1-beta-D-arabinofuranosylcytosine

Studies were undertaken to determine the therapeutic effects of liposome-encapsulated 1-beta-D-arabinofuranosylcytosine (lip-ara-C) against intracranial L1210 leukemia. The effects of administration route, drug dosage, liposome type, and tumor load on therapeutic efficacy were also studied. One hundred % mice were cured after a single intracranial 40 mg/kg dose of lip-ara-C, dependent on tumor load. Intracranial lip-ara-C was more effective than i.v. lip-ara-C. A single i.v. dose of lip-ara-C was therapeutically superior to 5-day i.v. infusion of the free drug. Intracranial or i.v. lip-ara-C at therapeutic doses resulted in less systemic toxicity than i.v. infusion of free ara-C, suggesting possible use of lip-ara-C as an adjunct to treatment of central nervous system leukemia.     

Enhancement of the antitumor effect of cyclophosphamide with the hypoxia-selective cytotoxin NLCQ-1 against murine tumors and human xenografts

The antitumor effect of cyclophosphamide (CPM) was investigated against SCCVII murine tumors and PC-3 human xenografts in combination with the hypoxia-selective cytotoxin 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1). The in vivo-in vitro and the tumor regrowth assays were used, respectively, as end points. In certain cases the hypoxia-selective cytotoxin tirapazamine (TPZ) was included for comparison purposes. In the SCCVII/C3H model, bone marrow toxicity studies were performed in parallel by using a modified CFU-GM assay. In the SCCVII/C3H model, when NLCQ-1 (10 mg/kg i.p.) was given 1 h before cyclophosphamide (CPM; 75-200 mg/kg i.p.), dose-modification factors (DMFs) of 1.9 and 1.0 were achieved for the antitumor effect and bone marrow toxicity, respectively. The corresponding DMF values obtained with TPZ (23 mg/kg) given 2.5 h (optimal time) before CPM were 1.3 and 1.0, respectively. Thus, therapeutic indices (T.I.) of 1.9 and 1.3 were achieved with NLCQ-1 and TPZ, respectively. In the PC-3/athymic nude mouse model, NLCQ-1 (10 mg/kg) given 90 min before CPM (36 mg/kg), qd x 4, increased tumor regrowth delay by 8.7 days compared to CPM alone, at 16-fold the original tumor size. The corresponding log cell kill was 0.86 and -0.03 for NLCQ-1 + CPM and CPM alone, respectively. In general, NLCQ-1 in combination with nontoxic but inactive CPM doses (36 or 54 mg/kg, qd x 4) elicited good antitumor activity without subsequent additive systemic toxicity, whereas NLCQ-1 had minimal effect in combination with the active but toxic (> 10% mean net weight loss) CPM dose of 80 mg/kg. These results suggest a potential use of NLCQ-1 in the clinic as an adjuvant to chemotherapy with CPM.     

Therapeutic advantage from combining paclitaxel with the hypoxia-selective cytotoxin NLCQ-1 in murine tumor- or human xenograft-bearing mice

PURPOSE: The antitumor effect of paclitaxel was investigated against murine tumors and human xenografts in combination with the hypoxia-selective cytotoxin NLCQ-1. METHODS: The tumor regrowth assay was used as the endpoint and an optimal administration schedule was followed, based on previous studies. In certain cases the hypoxia-selective cytotoxin tirapazamine (TPZ) was included for comparison. NLCQ-1 was given i.p. in saline, whereas paclitaxel was given i.p. (C3H) or i.v. (athymic mice) in an appropriately formulated vehicle. RESULTS: In the SCCVII/C3H model, when NLCQ-1 (10 mg/kg) was given 90 min after paclitaxel (8 mg/kg) twice a day 4 h apart on days 0 and 9, tumor regrowth delay was increased by 10.3 days compared to paclitaxel alone, at fivefold the original tumor size. This corresponds to 1.51 log cell kill. In the same study, TPZ resulted in 4.6 days of extra delay compared to paclitaxel alone, which corresponds to 0.91 log cell kill. Paclitaxel alone resulted in 3.9 days of tumor growth delay compared to control, or 0.42 log cell kill, but this delay was not statistically significant ( P<0.2). In the FSaIIC/C3H model, when NLCQ-1 (10 mg/kg) was given 90 min after paclitaxel (12 mg/kg) on day 0, tumor regrowth delay was increased by 5.8 days compared to paclitaxel alone, at 20-fold the original tumor size. In athymic nude mice bearing PC-3 prostate xenografts, NLCQ-1 (10 mg/kg) given 90 min before paclitaxel (8 mg/kg) for five consecutive days, increased tumor regrowth delay by 5.6 days compared to paclitaxel alone, at threefold the original tumor size. This corresponds to 0.95 log cell kill whereas the log cell kill for paclitaxel alone was 0.52. No improvement was observed in the tumor regrowth delay at any lower paclitaxel doses given in combination with NLCQ-1. No concurrent enhancement in paclitaxel-induced toxicity was observed in any of the combination treatments or in any of the models tested. NLCQ-1 alone was ineffective at the doses given. CONCLUSIONS: These results suggest that an enhancement in tumor growth delay can be achieved both in murine tumors and in human xenografts due to a synergistic interaction between NLCQ-1 and paclitaxel.     

Preventive effect of matrix metalloproteinase inhibitor, R-94138, in combination with mitomycin C or cisplatin on peritoneal dissemination of human gastric cancer cell line TMK-1 in nude mice.

R-94138, a matrix metalloproteinase inhibitor, was examined for the ability to prevent peritoneal dissemination of a human gastric cancer xenograft, TMK-1. When the supernatant of a co-culture of TMK-1 cells and human normal fibroblast cells was subjected to gelatin zymography, it was clear that the protein expression of MMP-2 had been inhibited by R-94138. When TMK-1 was injected intraperitoneally (i.p.) into nude mice at 5 x 10(5) cells/body, the resulting peritoneal dissemination mimicked clinical carcinomatous peritonitis. When the maximum tolerated dose of mitomycin C (MMC) or cisplatin (DDP) was given 12 h after the tumor inoculation, peritoneal dissemination was completely inhibited, while the effect of R-94138 was limited when it was given i.p. at a dose of 20 mg/kg in a schedule of q.d. x 5 starting 12 h after tumor injection. MMC and DDP also suppressed peritoneal dissemination when they were administered 1 week after the tumor inoculation at a single dose of 2 and 3 mg/kg i.p., respectively. R-94138 inhibited peritoneal dissemination when it was administered i.p. at a dose of 30 mg/kg in a schedule of q.d. x 5 starting from 1 week after tumor injection. The combination of MMC and R-94138 increased the preventive effect on peritoneal dissemination. R-94138 seems to be a promising candidate to prevent peritoneal dissemination of gastric cancer.     

Treatment of intravenously implanted Lewis lung carcinoma with liposome-encapsulated cytosine arabinoside and non-specific immunotherapy

Liposomes have been used as biological carriers of antitumor drugs, and their potential use has been tested using various mouse tumors. In this study, we describe a potential role of liposome-encapsulated 1-beta-D-arabinofuranosylcytosine (Ara-C) with a mouse solid lung tumor model. Non-encapsulated Ara-C at 25 mg/kg dose by the intraperitoneal (i.p.) route on days 1, 4 and 7 had no improving effect on the average survival time of tumor-bearing mice compared to untreated control mice. However, the same dose of Ara-C encapsulated in multilamellar liposomes (MLV) improved the average survival of tumor-bearing mice by 60 to 80%. Ara-C was encapsulated more efficiently when DSPC or DPPC MLV were prepared at temperatures below their respective transition temperatures. DSPC and DPPC MLV prepared at 25 degrees C and DPPC MLV prepared at 50 degrees C were equally effective for in vivo therapy, while DSPC MLV prepared at 60 degrees C were not as effective. Non-specific immunotherapy using BCG (Bacillus Calmette-Guérin, Mycobacterium tuberculosis) and CP Corynebacterium parvum) was effective, particularly when injected by the intravenous (i.v.) route, in prolonging the average survival of tumor-bearing mice. A combination of either i.v. BCG or i.p. CP with liposome therapy gave no further improvement in the average survival of tumor-bearing mice. However, a combination of either i.p. BCG or i.v. CP with liposome therapy was somewhat more effective than either liposome therapy or immunotherapy alone.     

Antineoplastic activity of two taxol derivatives on an ovarian tumor xenografted into nude mice

In order to compare the antineoplastic activities of taxol A, taxol B, a mixture of the two (taxol A 72%) and vinblastine, a human ovarian tumor serially transplanted into 104 female athymic mice was used. In the first experiment (11th passage), the antineoplastic activities of taxol A, taxol B and the mixture taxol AB were tested. The same dose was used in each case (12.5 mg/kg i.e. 1/20 of the evaluated LD50 value). It was administered subcutaneously for 5 consecutive days. Three courses of treatment were performed, with 2 rest periods of 1 week in between. All the taxol derivatives produced a statistically significant delay in the tumor growth. However, taxol B had the lowest chemotherapeutic response. In the second experiment (18th passage), different dose levels were administered (mixture 12.5 mg/kg/day x 4 - taxol A 8.8. mg/kg/day x 4 - taxol B 3.5 mg/kg/day x 4 - vinblastine 0.5 mg/kg/day x 2). For all the taxol derivatives 4 treatment courses with 3 rest periods of 4 days were used, and for vinblastine 4 treatment courses with 3 rest periods of 1 week. At the end of the second experiment, vinblastine, taxol A and a mixture of the two showed similar significant activity, whereas no objective antitumor response was observed following the taxol B treatment at the dose level chosen. The experimental results obtained clearly demonstrate that, in the taxane system, the greatest degree of antineoplastic activity can be attributed to taxol A.     

Verapamil enhancement of antitumor effect of cis-diamminedichloroplatinum(II) in nude mouse-grown human neuroblastoma

The antitumor effect of combined use of cis-diamminedichloroplatinum(II) (CDDP) and verapamil, a calcium influx blocker, was examined in neuroblastoma transplanted to BALB/c athymic mice. The response of the tumor to CDDP was related to the dose administered. Regression of the tumor was observed when CDDP was administered at 4.2 mg/kg/injection. The retardation of tumor growth was observed in the group to which CDDP was administered at 2.1 mg/kg/injection. When verapamil was administered with CDDP, regression of the tumor was observed in the group treated with CDDP at 2.1 mg/kg/injection, and the retardation of tumor growth was observed in the group treated with CDDP at 1.4 mg/kg/injection. These results indicate that verapamil enhances the antitumor effect of CDDP against transplanted neuroblastoma in BALB/c athymic mice.     

Toxicity and antitumoral activity of two new complex compounds of rhenium

Toxicity and antitumor action of two new complex compounds of rhenium (K2 [ReCl6] and [ReCl(4).2C6H4N2]) were investigated. LD50 of K2 [ReCl6], i.v., was 136 +/- 37 mg/kg; i.p.,--272 +/- 74 mg/kg. LD50 of [ReCl(4).2C6H4N2], i.v., was 543 +/- 148 mg/kg; i.p., 600 mg/kg was tolerable. Single maximum tolerable dose of 200 mg/kg K2 [ReCl6], i.p., did not influence the growth of ascitic tumor of Ehrlich, nor did daily intravenous dose of 50 mg/kg inhibit solid tumor of Ehrlich. No inhibition by [ReCl(4).2C6H4N2] was registered when 25 or 50 mg/kg were administered.     

Enhancement of antitumor activity of polyethylene glycol-coated liposomal doxorubicin with soluble and liposomal interleukin 2.

Polyethylene glycol-coated liposomal doxorubicin (Doxil) has a sustained release profile and a mild myelosuppressive effect that may enable a beneficial interaction with lymphocyte-activating cytokines, such as interleukin 2 (IL-2). Previous studies have shown that liposome entrapment of IL-2 potentiates its immunomodulatory effects and reduces the need for frequent dosing. We assessed the therapeutic effect of Doxil (8 mg/kg) followed by free or liposomal IL-2 (50,000 Cetus Units x 3) in mice bearing M109 lung adenocarcinoma transplanted i.v. or i.p. Doxil was always administered i.v., whereas IL-2 was given i.v. in the i.v. M109 model and i.p. in the i.p. M109 model. The optimal combined treatment was significantly more effective than liposomal chemotherapy alone, producing tumor-free, long-term survivors in 100% (i.v. M109) and 94% (i.p. M109) of the mice, compared with 50% and 56%, respectively, for Doxil alone. The efficacy boost of IL-2 appeared to be formulation dependent, with free IL-2 and IL-2 in small unilamellar vesicles most active in the i.v. tumor model, and IL-2 in multilamellar vesicles most active in the i.p. tumor model. The combination of Doxil with free or liposomal IL-2 was devoid of any conspicuous toxicity. Cytokine treatment without chemotherapy was completely ineffective. Liposome-based chemoimmunotherapy is a synergistic and highly efficacious approach to eradicate metastatic and regionally spread tumors.     

Antiangiogenic and antitumor activity of a selective PDGFR tyrosine kinase inhibitor, CP-673,451

CP-673,451 is a potent inhibitor of platelet-derived growth factor beta-receptor (PDGFR-beta) kinase- and PDGF-BB-stimulated autophosphorylation of PDGFR-beta in cells (IC(50) = 1 nmol/L) being more than 450-fold selective for PDGFR-beta versus other angiogenic receptors (e.g., vascular endothelial growth factor receptor 2, TIE-2, and fibroblast growth factor receptor 2). Multiple models have been used to evaluate in vivo activity of CP-673,451 and to understand the pharmacology of PDGFR-beta inhibition and the effect on tumor growth. These models include an ex vivo measure of PDGFR-beta phosphorylation in glioblastoma tumors, a sponge model to measure inhibition of angiogenesis, and multiple models of tumor growth inhibition. Inhibition of PDGFR-beta phosphorylation in tumors correlates with plasma and tumor levels of CP-673,451. A dose of 33 mg/kg was adequate to provide >50% inhibition of receptor for 4 hours corresponding to an EC(50) of 120 ng/mL in plasma at C(max). In a sponge angiogenesis model, CP-673,451 inhibited 70% of PDGF-BB-stimulated angiogenesis at a dose of 3 mg/kg (q.d. x 5, p.o., corresponding to 5.5 ng/mL at C(max)). The compound did not inhibit vascular endothelial growth factor- or basic fibroblast growth factor-induced angiogenesis at concentrations which inhibited tumor growth. The antitumor efficacy of CP-673,451 was evaluated in a number of human tumor xenografts grown s.c. in athymic mice, including H460 human lung carcinoma, Colo205 and LS174T human colon carcinomas, and U87MG human glioblastoma multiforme. Once-daily p.o. x 10 days dosing routinely inhibited tumor growth (ED(50) < or = 33 mg/kg). These data show that CP-673,451 is a pharmacologically selective PDGFR inhibitor, inhibits tumor PDGFR-beta phosphorylation, selectively inhibits PDGF-BB-stimulated angiogenesis in vivo, and causes significant tumor growth inhibition in multiple human xenograft models.     

Development, characterization and therapy of a disseminated model of childhood neuroblastoma in SCID mice

PURPOSE: To develop a highly reproducible model of disseminated childhood neuroblastoma in mice to allow secondary evaluation of therapeutics against microscopic disseminated disease. METHODS: CB17/Icr SCID were injected i.v. with 10(3) to 5 x 10(6) human NB-1691 neuroblastoma cells. NB-1691 cells were detected by PCR for synaptophysin and tyrosine hydroxylase in peripheral blood, and bone marrow. Therapeutic studies evaluated topotecan and vincristine as single agents or in combination. Topotecan was administered i.v. daily for 5 days on two consecutive weeks. Courses were repeated every 21 days for three cycles. Vincristine (1 mg/kg) was administered i.v. every 7 days for nine consecutive weeks. Treatment started 11-21 days after tumor cell inoculation. RESULTS: Following injection of > or = 1 x 10(5) cells 100% of mice developed disease. Mice inoculated with 10(7) cells survived a median of 42 days. Survival time was a linear function of the cell inoculum. At autopsy, gross tumor was routinely detected in many organs in particular liver, ovaries, kidneys and adrenals. NB-1691 cells were detected by PCR in peripheral blood, and bone marrow. Immunohistochemical staining showed that lesions were strongly positive for synaptophysin, chromogranin A and negative for leukocyte common antigen. Topotecan (0.6 mg/kg) alone extended median survival from 44 days (controls) to 95 days. When treatment was started 21 days after inoculation of NB-1691 cells, topotecan extended median survival from 39 days (controls) to 91 and 99 days at dose levels of 0.3 and 0.6 mg/kg, respectively. Vincristine (1 mg/kg) extended survival by a median of 9.5 days. In combination with vincristine (1 mg/kg), median survival was increased to 141 days (topotecan 0.6 mg/kg) and 159 days (topotecan 1.0 mg/kg). CONCLUSION: This model of disseminated neuroblastoma is highly reproducible. As this model may more closely simulate childhood disease it may be a valuable adjunct in developing new approaches to advanced stage, poor prognosis neuroblastoma.