Hepatic lymphatic mapping: a pilot study for porta hepatis lymph node identification.

The status of the porta hepatis lymph nodes in patients with hepatic metastases from colorectal cancer affects their prognosis and management. Lymphatic mapping with isosulfan blue dye is well established in breast cancer and melanoma. An animal model consisting of three dogs receiving general anesthesia was utilized. Each dog underwent a laparotomy and increasing doses of isosulfan blue dye were injected into the right medial segment of the liver. Intraoperatively, the presence of blue dye in the porta hepatis region was determined and the lymph node identified. Continuous physiological monitoring was performed. Serum determination of liver function tests, amylase levels, and white blood cell count were performed preoperatively and on postoperative days 1, 2, 4, and 7. The animals were sacrificed on day 7. A portal lymph node was identified in each case and there was no perioperative morbidity or mortality. There were no significant alterations in blood pressure or heart rate in the animals. There was a dose-responsive decrease in the O2 saturation as measured by transcutaneous monitoring, but arterial blood gas analysis showed that pO2 levels remained stable. There were no significant changes in the liver function tests, amylase levels, or white blood cell counts. There was a small increase in alkaline phosphatase, which normalized by postoperative day 7. Hepatic injection of isosulfan blue dye appears to be safe and effective in identifying porta hepatis lymph nodes in the animal model and sets the basis for further study in human subjects.

Preclinical pharmacology, toxicology and efficacy of sphingomyelin/cholesterol liposomal vincristine for therapeutic treatment of cancer.

PURPOSE: To establish the pharmacodynamic relationships between drug biodistribution and drug toxicity/efficacy, a comprehensive preclinical evaluation of sphingomyelin/cholesterol (SM/chol) liposomal vincristine and unencapsulated vincristine in mice was undertaken. METHODS: Pharmaceutically acceptable formulations of unencapsulated vincristine and liposomal vincristine at drug/lipid ratios of 0.05 or 0.10 (wt/wt) were evaluated for toxicity, antitumor activity and pharmacokinetics following intravenous administration. RESULTS: Mice given liposomal vincristine at 2 mg/kg vincristine had concentrations of vincristine in blood and plasma at least two orders of magnitude greater then those achieved after an identical dose of unencapsulated drug. One day after administration of the liposomal vincristine, there were at least tenfold greater drug quantities, relative to unencapsulated vincristine, in the axillary lymph nodes, heart, inguinal lymph nodes, kidney, liver, skin, small intestines and spleen. Increased plasma and tissue exposure to vincristine as a result of encapsulation in SM/chol liposomes was not associated with increased drug toxicities. Treatment of the murine P388 ascitic tumor with a single intravenous dose of unencapsulated drug at 2, 3 and 4 mg/kg, initiated 1 day after tumor cell inoculation, resulted in a 33 to 38% increase in lifespan. In contrast, long-term survival rates of 50% or more were achieved in all groups treated with the SM/chol liposomal vincristine formulations at doses of 2, 3 and 4 mg/kg. At the 4 mg/kg dose, eight of ten and nine of ten animals survived past day 60 when treated with SM/chol liposomal vincristine prepared at the 0.05 and 0.1 drug/lipid ratios, respectively. CONCLUSIONS: Overall, increased and prolonged plasma concentrations of vincristine achieved by liposomal encapsulation were correlated with dramatically increased antitumor activity in comparison with the unencapsulated drug, but no correlations could be established between pharmacokinetic parameters and toxicity.

Characterization of a monoclonal antibody recognizing a 138 kDa glioblastoma-associated antigen.

A monoclonal antibody (A7) was produced which recognizes an oncofetal antigen expressed on glioblastoma multiforme cells, fetal brain, fetal kidney, but not adult tissues. Radioimmunoprecipitates of Zwittergent 3-14 solubilized glioblastoma cells identified a single band at 138 kDa on 8% polyacrylamide sodium dodecylsulfate gels. Distribution studies of A7 in mice demonstrated a tri-phasic serum clearance of t1/2 alpha = 2.1 h, t1/2 beta = 16.7 h and t1/2 gamma = 151.1 h. Tumor localization studies using the U-87 MG xenograft demonstrated the ability of A7 to localize with a tumor:blood ratio of 1.294 +/- 0.094 as compared with 0.293 +/- 0.051 for control antibody AC. A7 does not damage cell membranes and is not internalized when bound to reactive tumor cells.

Preclinical toxicology study of liposome encapsulated doxorubicin (TLC D-99) given intraperitoneally to dogs.

A preclinical toxicology study of intraperitoneally administered liposome encapsulated doxorubicin and free doxorubicin was carried out in beagle dogs. Dogs received single intraperitoneal infusions of 1.5 mg free or 1.5, 2.25 or 3.37 mg liposomal doxorubicin/kg. One group of four dogs received 1.5 mg liposomal doxorubicin/kg every three weeks for 4 cycles. The dose limiting toxicity of free or liposomal doxorubicin given by the intraperitoneal route was a dose-related chemical peritonitis. This toxicity was more severe in dogs that received by the intraperitoneal route the previously determined maximally tolerated intravenous dose of liposomal doxorubicin (2.25 mg/kg). The abdominal toxicity was characterized by capsular fibrosis and ascites formation. Abdominal toxicity was life threatening after single doses of 3.37 mg liposomal doxorubicin kg, or after multiple (4) doses of 1.5 mg liposomal doxorubicin/kg. Thoracic toxicity (increased fluid, mediastinal edema, thickening of the parietal pleura) was seen after multiple (every 3 weeks for 4 cycles) intraperitoneal doses of 1.5 mg or single doses of 3.37 mg liposomal doxorubicin/kg. Myelosuppression was seen in all groups, but was less severe after intraperitoneal dosage than after intravenous dosage of liposomal doxorubicin.

Liposome encapsulated vincristine: preclinical toxicologic and pharmacologic comparison with free vincristine and empty liposomes in mice, rats and dogs.

A preclinical toxicology study of liposome encapsulated vincristine, free vincristine and empty liposomes was carried out in mice and dogs by single and multiple (daily for 5 days) intravenous injection. Single and multiple dose intravenous injection studies in mice showed the encapsulated form of vincristine to be less toxic than free vincristine. Empty liposomes injected intravenously into dogs were without significant toxicity. In dogs, the toxicities seen with liposomal vincristine were qualitatively similar to those of free vincristine with only minor quantitative differences. The principal toxicities of free and liposomal vincristine in dogs were anorexia, weight loss, pyrexia, myelosuppression and gastrointestinal toxicity. After single high doses of either formulation gastrointestinal toxicity was the dose-limiting toxicity, while either hematologic or gastrointestinal toxicity was dose limiting after multiple dose administration of either drug. Histopathologic lesions of importance were bone marrow atrophy, necrosis and atrophy of the lymphoproliferative tissues, necrosis of gastrointestinal tract mucosa, liver and pancreas, and hemorrhage. Distribution studies in rats showed significantly higher vincristine levels in serum, spleen, liver, trachea, jejunum, cerebrum, lung, ischiatic nerve and heart, and significantly lower levels in colon, stomach, salivary gland, thymus esophagus and pancreas after injection of the liposome-associated agent. No toxicities were seen that should preclude safe clinical trial of liposomal vincristine in man.

Preclinical toxicologic evaluation of DENSPM (N1,N11-diethylnorspermine) in rats and dogs.

A toxicology study of DENSPM was carried out in rats by multiple (once daily for 5 days) intravenous injection. Doses of 12.5, 25 and 50 mg DENSPM/kg were well tolerated. Infusion of 100 mg DENSPM resulted in distressing physical signs, including labored breathing, convulsive movements and acute death. There were no end-organ toxicities induced by this regimen as evaluated by serum chemistry and hematology examinations, and histopathologic exam of all major body organs. Transient hypotension was induced in rats and dogs by rapid infusion of DENSPM; the magnitude of this hypotension was decreased by slow infusion of DENSPM into dogs. Hypotension appears to be the dose-limiting toxicity of this agent when infused rapidly.

Induction of dog IL-1 by free and liposomal encapsulated doxorubicin.

Liposomal encapsulated doxorubicin, free doxorubicin and a mixture of free doxorubicin plus empty liposomes were observed to induce the production and release of IL-1 from dog peripheral blood lymphocytes (PBLs) in vitro whereas empty liposomes were observed not to induce IL-1 when evaluated with the EL-4/CTLL-1 biological assay. Tumor necrosis factor was not induced as measured by the L-929 biological assay. Maximum IL-1 induction occurred at doxorubicin concentrations of 0.3-0.8 microgram/ml. Dog PBLs can be classified as low, moderate and high responders according to their response to doxorubicin induced IL-1 release. Agarose immobilized doxorubicin does not induce IL-1 indicating that internalization of doxorubicin is required for IL-1 release. Limulus amebocyte lysate testing of doxorubicin indicates that endotoxin contamination is not responsible for this observed release of IL-1.

Comparison of the cardiotoxic effects of liposomal doxorubicin (TLC D-99) versus free doxorubicin in beagle dogs.

The cardiotoxic potential of liposome encapsulated doxorubicin (TLC D-99) prepared by a remote-loading technique was compared with that of free doxorubicin (1.5mg/kg administered every 3 weeks for 8 cycles) in beagle dogs. Both agents were equally myelosuppressive, and all dogs completed both treatments. There were no deaths during the study. Experimental animals were killed between 157 and 164 days after the start of the trial. All of the dogs (n = 6) that received free doxorubicin had either moderate (1 animal) or severe (5 animals) vacuolization of myocardial tissue. None of the dogs treated with liposomal doxorubicin had lesions suggestive of cardiomyopathy. Administration of free doxorubicin was associated with transient anorexia, reduced weight gain, alopecia, and gastrointestinal toxicity. Such adverse reactions were either much less severe or absent in animals that received liposomal doxorubicin. The results of this study demonstrate that TLC D-99 significantly decreases both the myocardial toxicity and other adverse reactions of this potent antineoplastic drug. TLC D-99 is now in Phase II clinical trials.

Preclinical toxicology study of liposome encapsulated doxorubicin (TLC D-99): comparison with doxorubicin and empty liposomes in mice and dogs.

A preclinical toxicology study of intravenously administered liposome encapsulated doxorubicin (TLC D-99), free doxorubicin and empty liposomes was carried out in mice and dogs by single and multiple (daily for 5 days) dose schedules. Single dose intravenous injection studies in mice showed the encapsulated form of doxorubicin to be less toxic (LD50 of 32 mg/kg) than free doxorubicin (LD50 of 17 mg/kg). Toxicity in dogs was evaluated by serial serum chemistry, hematology and EKG analysis, urinalysis, clinical observations, necropsy and histopathologic examination. Empty liposomes injected intravenously into dogs were without significant toxicity. The maximally tolerated dose of free doxorubicin in beagles was 1.5 mg/kg; deaths were seen after a 50% escalation to 2.25 mg/kg. The maximally tolerated dose of liposome encapsulated doxorubicin was higher (2.25 mg/kg); deaths were seen after a 50% escalation to 3.37 mg/kg. A toxicity unique to the encapsulated agent was pyexia (as high as 105.6 degrees F) within twenty four hr of single dosage. This was seen in approximately half of the test animals, was not dose-related, and was not observed in animals that received empty liposomes. The organ specific toxicities seen with TLC D-99 were qualitatively similar to those of free doxorubicin, but less severe.

Prostatic atrophy in dogs after intravenous administration of a ureido-ethylimidazoline derivative (CGP15'720A).

1-(2-[4-Pridyl)-2-imidazoline-1-yl]-ethyl)-3-(4-carboxyphenyl)urea (CGP15'720A) is an experimental antineoplastic agent with marked activity against carcinogen-induced lung tumors in Syrian hamsters and human lung tumor xenografts in nude mice. A preclinical toxicity study of this agent was carried out in mice and dogs which demonstrated the relatively nontoxic nature of the agent. In mice, single intraperitoneal dosage of 12 g/m2 did not produce lethality; however, lethality (30% of treated mice) was seen during treatment with 6 g/m2 daily for 5 days. No hematological, serum-chemistry or histopathological changes were detected in mice after single or five consecutive treatments with 12 g/m2. Dogs were treated with doses ranging from 5 g/m2 to 80 g/m2, with deaths occurring in a non-dose-related fashion after 10, 20, and 40 g/m2. Acute neurological toxicity after infusion was the dose-limiting toxicity in dogs. There were no consistent hematological or serum-chemistry aberrations in the treated dogs. The most consistent histopathological finding was prostatic atrophy, which was detected in 5/12 dogs in this series.

Preclinical toxicity study of streptozocin infused into the internal carotid artery of dogs and baboons.

Mature male and female dogs (10) and male baboons (4) were each given a single dose of streptozocin (STZ) (1000 mg/m2) by infusion directly into the left internal carotid artery over a two-hour time period. Serial hematology and serum chemistry profiles and physical observations were made, the animals necropsied at varying times after dosage, and the major organs examined histologically. In dogs, severe weight loss, neutrophilia, electrolyte disturbances, decreased liver function, diabetes and decreased serum amylase levels were the major toxicities. In baboons, weight loss, hypoglycemic coma, diabetes, decreased liver function and electrolyte disturbances were observed. While systemic toxicities were severe in both animal species, central nervous system toxicity was minimal. Cerebral toxicities consisting of edema in two dogs, and focal necrosis in one dog were observed histopathologically. No cerebral toxicity occurred in baboons. Based upon these findings, a phase I clinical trial of intra-arterial (internal carotid artery) STZ in patients with primary/metastatic brain tumors would appear feasible.

Preclinical toxicity study of mitomycin C infused into the internal carotid artery of beagle dogs.

Young adult male and female beagle dogs were infused with single doses of 0.5 (2 dogs) or 0.25 mg (5 dogs) Mitomycin C (MMC)/kg body weight directly into the internal carotid artery. Serial hematology and serum chemistry profiles, electrocardiograms and physical observations were made, the animals necropsied at varying times after dosage, and the major organs examined histologically. Results indicate that the dose limiting toxicity of this treatment regimen is myelosuppression. No ocular or neurologic toxicity was detected in any test animal. The findings suggest that infusion of MMC can be safely attempted in humans for the treatment of brain tumors that derive their blood supply from the internal carotid artery.

CGP 6809, a sugar-containing nitrosourea derivative: pharmacological and physicochemical properties.

CGP 6809 is a water-soluble nitrosourea derivative with quite distinct chemical and biological properties as compared with the well-known representatives of this class of compounds. It is related to the antibiotic streptozotocin, from which it is distinguished in the structure of the sugar moiety and the position of the methylnitrosourea residue. CGP 6809 possesses practically the same alkylating potential as streptozotocin; however, its carbamoylating activity is comparable with that of CCNU. In contrast to other nitrosourea derivatives, CGP 6809 showed relatively little activity in murine leukemias but was markedly active in solid transplantable melanomas (Harding-Passay, B16), in the 11095 prostate carcinoma, and in a substrain of Yoshida hepatoma (AH 7974) resistant to BCNU and CCNU. In the Ehrlich and Yoshida ascitic tumors complete responses were seen with no toxic death. Dose-dependent activity was found in the human lung carcinoma MBA 9812 and almost complete growth inhibition was achieved in the human melanoma WM 47 by both the oral and parenteral routes of administration. However, mammary tumor lines (Ca 755, 2661/61, R-3230AC), the Guerin-T8 uterus epithelioma, and the Rous sarcoma/S-R proved to be relatively refractory to this drug. This was also the case for the Lewis lung carcinoma implanted i.m. or s.c. However, development of lung metastases was markedly inhibited. Combination therapy using CGP 6809 with cyclophosphamide, 5-fluorouracil, or chlorambucil in the same model led to partial responses of the primary tumor as well as almost total eradication of lung metastases.

Pretreatment effects on the uptake/retention kinetics of L-dopa in Harding-Passey melanoma.

Malignant melanoma cells possess a unique biochemical pathway that converts L-3,4-dihydroxyphenylalanine (L-dopa) to the biopigment melanin. Selective cytotoxic incorporation of exogenous L-dopa into melanoma cells in vivo may provide a means of designing specific chemotherapeutic agents useful in the treatment of this disease. Using the Harding-Passey murine melanotic tumor model, a preferential uptake of [3H]L-dopa by the tumor was characterized. Following pretreatment of the tumor-bearing mice with nonradioactive L-dopa, a significant enhancement (p less than 0.01) of [3H]L-dopa incorporation and retention into melanoma for a period of 24 h was observed, when compared with the concomitant tissue distribution and clearance of radioactivity in the control animals. This finding suggests that by initial pretreatment of melanoma with nonradioactive L-dopa, the subsequent selective accumulation of [3H]L-dopa in tumor may provide a useful tool in testing new modalities of therapy in malignant melanoma.

Therapeutic and diabetogenic potential of two newly synthesized nitrosoureido sugars.

Two newly synthesized nitrosoureido sugars have been evaluated for their antitumor activity and diabetogenic potential in a number of in vitro and in vivo preclinical tumor model systems. 2-Amino-2-deoxy-N'-methyl-N'-nitrosoureido-1,3,4,6-tetra-O-acetyl-alpha- D- mannopyranose (MAZ), a lipophilic mannosamine derivative, and ethyl-6-deoxy-3,5-di-O-methyl-6-(3-methyl-3-nitrosoureido)-alph a- D-glucofuranoside (EDOMEN or CGP 6'809), were both found to inhibit L1210 leukemia cell growth in vitro by 50% at approximately 5.0 X 10(-5) M. At these concentrations, little effect was noted immediately on L1210 cell radiolabeled precursor incorporation; however, at higher concentrations, EDOMEN inhibited [3H]leucine and [3H]mannose incorporation, while MAZ specifically decreased L1210 cell [3H]thymidine and [3H]leucine incorporation. Inhibition of Lewis lung carcinoma and B16 melanoma cell growth by 50% in vitro was achieved at higher concentrations of these agents (10(-4) to 10(-3) M). Since the currently available nitrosoureido sugars, streptozotocin and chlorozotocin, have been observed by us to be diabetogenic, EDOMEN and MAZ were evaluated for their specific toxicity to rat pancreatic beta-cells in vitro. Cytotoxicity in beta-cell cultures was monitored both by phase-contrast microscopy and the release of insulin into the culture medium. beta-Cells were found to be 10-fold more sensitive to the toxic effects of MAZ than were pancreatic fibroblasts. EDOMEN, on the other hand, did not damage beta-cells preferentially and therefore was not considered diabetogenic. Both MAZ and EDOMEN had moderate activity as antileukemic agents in mice. At 50 mg/kg/day i.p. for 5 days, MAZ increased the life span of female DBA/2J mice with L1210 leukemia by over 50%. Similarly, doses of EDOMEN at 125 to 250 mg/kg/day i.p. for 5 days increased L1210 leukemic life span by nearly 60%. At these doses, no effect of MAZ was observed on primary Lewis lung carcinoma growth or life span of tumor-bearing C57BL/6 mice. EDOMEN, however, increased life span in Lewis lung carcinoma mice by up to 33% and caused an apparent antimetastatic effect. These studies indicate that EDOMEN may have enhanced value as a cancer chemotherapeutic agent due to its therapeutic effectiveness, lack of diabetogenic potential, and other favorable formulation properties (water solubility) as compared with other clinically available nitrosoureas.

Functional and chemical markers of PCNU activity.

A study was conducted to elucidate functional or chemical parameters that may be useful for in vivo toxicologic and pharmacokinetic analysis of PCNU. The growth-inhibitory and DNA-related parameters of this agent and its serum breakdown products in murine leukemia L1210 cells were carried out in direct comparison with BCNU. In human and dog sera, the T1/2 of intact PCNU (5-12 min) was similar to BCNU (5-16 min). However, much larger T1/2 values for PCNU (140 min) and BCNU (110 min) were determined for drug incubated in dog brain homogenates. Uptake of 14C-labeled PCNU in L1210 cells was rapid, and tenfold higher than in CCRF-CEM, a human leukemia cell line naturally resistant to BCNU and PCNU. BCNU was shown to induce DNA strand damage in L1210 and to inhibit radioactive thymidine incorporation into DNA and cross-link proteins with DNA in L1210. PCNU, by comparison, only weakly inhibited thymidine incorporation and did not induce DNA strand breakage or produce DNA-protein cross-links in L1210 at reasonable concentrations. The compounds are further differentiated in fetal calf serum by the breakdown products derived from initial concentrations of intact drug that are equipotent; those of BCNU inhibit L1210 growth whereas those of PCNU do not. PCNU, which is rapidly broken down into inactive metabolites, may have a selective therapeutic advantage if infused directly to the target site.

A fluorescence enhancement assay for cellular DNA damage.

A fluorescence procedure is described for quantitative measurement of DNA damage in mammalian cells. The technique is based upon the time-dependent partial alkaline unwinding of cellular DNA followed by determination of duplex:total DNA ratios with bisbenzamide, which has a differential molar fluorescence with single-stranded and duplex DNA. The method is rapid, does not require radioactive labeling of DNA, and is sufficiently sensitive to detect damage induced with 100 rads of X-irradiation. This method is standardized with respect to the alkaline unwinding unit, Mn0, and the unwinding constant, beta. Results obtained with this new technique and with hydroxylapatite chromatography for physical separation of single- and double-stranded DNA were confirmatory. The utility of the technique was demonstrated by detection of dose-related damage with X-irradiation and a variety of antineoplastic agents in unlabeled murine leukemia cells.

Tourniquet infusion versus hyperthermic perfusion.

The tourniquet infusion method was compared with hyperthermic perfusion in canine limbs by using Adriamycin, actinomycin-D, and melphalan. Tourniquet infusion provided comparable tissue levels with Adriamycin and significantly higher levels with actinomycin-D and melphalan in the treated extremity than hyperthermic perfusion with the same drugs and dosages. Higher systemic leak was observed, more so with melphalan, with the tourniquet infusion method. Tourniquet infusion has caused complete regression of four malignant tumors involving extremities (one malignant melanoma, two Kaposi's sarcomas, one squamous cell carcinoma) and partial greater than 50% regression of nine tumors (three malignant melanomas, three squamous cell carcinomas, one malignant schwannoma, one malignant fibrohistiocytoma, one liposarcoma) followed by excision of residual tumor. Five patients with extremity sarcomas precluding adequate surgical margins were treated preoperatively with the this method. Longer follow-up is needed, as is a larger number of patients for a valid comparison of tourniquet infusion with hyperthermic perfusion.

DNA damage by anthracycline drugs in human leukemia cells.

Leukemia cells from 4 acute myelocytic leukemia (AML) and 1 acute lymphocytic leukemia (ALL) patients were incubated with a set of 6 anthracycline agents: Adriamycin (Am), 4'-epi-Adriamycin (4'-epi-Am), daunorubicin (Dm), 4-demethoxy-daunorubicin (4-dDm), carminomycin (Cm) and N-trifluoroacetyl-Am-14-valerate (AD32). Cells were assayed for drug uptake after incubation for 2 h, and for DNA damage and drug retention 4 h later. Uptake and retention patterns were characteristic for each agent and fairly uniform for the different cell populations. In contrast, profiles of the amount of DNA damage produced reflected striking differences in each population of cells. These individual responses raise the possibility that leukemic cells resistant to one anthracycline may yet be sensitive to another.