Monoclonal anitbodies in the lymphatics: toward the diagnosis and therapy of tumor metastases.

Monoclonal antibodies subcutaneously injected into mice track to regional lymph nodes and specifically label target cells there. The lymphatic route of administration can be expected to provide much higher sensitivity, higher target-to-background ratio, faster localization, and lower toxicity than the intravenous route when the aim is to diagnose or treat tumor metastases or lymphoma in the lymph nodes.

Monoclonal antibodies in the lymphatics: selective delivery to lymph node metastases of a solid tumor.

After subcutaneous injection, monoclonal antibodies directed against a tumor can enter local lymphatic vessels, pass to the draining lymph nodes, and bind to metastases there. Lymphatic delivery of antibody to early metastases is more efficient than intravenous administration, and the lymphatic route can be used to image smaller metastatic deposits. Perhaps more important, the lymphatic route minimizes binding of antibodies to circulating tumor antigens and to cross-reactive antigens present on normal tissues. Antibodies inappropriate for intravenous use because of binding to normal tissues may therefore be useful against lymph node metastases when injected subcutaneously or directly into lymphatic vessels.

Carcinogenicity and hepatotoxicity of cycasin and its aglycone methylazoxymethanol acetate in nonhuman primates.

The carcinogenic potential of cycasin and methylazoxymethanol (MAM) acetate was investigated in nonhuman primates. Old-world monkeys (rhesus, cynomolgus, and African green monkeys) received cycasin and/or MAM acetate by oral or ip routes up to 11 years. Eighteen monkeys survived longer than 2 months after initiation of treatment with cycasin (50-75 mg/kg) or MAM acetate (1.5-3.0 mg/kg) given orally 5 days/week; 9 of the animals were necropsied. Histopathologic examination of a liver tumor from 1 of these monkeys revealed well-differentiated hepatocellular carcinoma. A second monkey had multiple tumors, including hepatocellular carcinoma, intrahepatic bile duct adenocarcinoma, renal carcinoma and adenomas, and adenomatous polyps of the colon. Although liver tumors were not observed in the other monkeys, all but 1 monkey had hepatic lesions such as toxic hepatitis and cirrhosis. These monkeys had received cycasin and/or MAM acetate for an average of 57 months (range, 2-133 mo). A group of 10 monkeys received MAM acetate by weekly ip injections (3-10 mg/kg). Six of these animals developed tumors after receiving an average of 6.14 g (range, 3.58-9.66 g) of MAM acetate for an average of 75 months (range, 50-89 mo). Four of the monkeys developed hepatocellular carcinomas, and 2 had multiple primary tumors including hepatocellular carcinomas, renal carcinomas, squamous cell carcinomas of the esophagus, and adenocarcinomas of the small intestine. Our results showed that long-term administration of cycasin and/or MAM acetate by oral and ip routes was hepatotoxic and carcinogenic in old-world monkeys.

Carcinogenicity of aflatoxin B1 in rhesus monkeys: two additional cases of primary liver cancer.

Three of 42 (7%) monkeys given aflatoxin B1 (AFB1) for longer than 2 years have developed primary malignant neoplasms of the liver. Liver biopsies performed at intervals during aflatoxin administration revealed that neoplasia was preceded by pathologic lesions of the liver, including toxic hepatitis, proliferation of pseudotubules, and hyperplastic nodules. Serum alpha-fetoprotein levels, monitored in one of the monkeys by radioimmunoassay, paralleled tumor growth and recurrence of the hepatocellular carcinoma. Normal serum alpha-fetoprotein levels were noted for a monkey with hemangioendothelial sarcoma. Our results implicate AFB1 as a liver carcinogen in monkeys and add additional support to the hypothesis that humans exposed to this substance may be at risk of developing liver cancer.

Long-term feeding of sodium saccharin to nonhuman primates: implications for urinary tract cancer.

BACKGROUND: It was observed in the early 1970s that saccharin produced bladder cancer in rats. However, it has been unclear whether sodium saccharin when consumed by humans poses a substantial carcinogenic hazard. Numerous epidemiologic studies have not shown any evidence of increased urothelial proliferation associated with ingestion of sodium saccharin. PURPOSE: Our purpose was to determine the effects of long-term feeding of sodium saccharin to three species of nonhuman primates. METHODS: Twenty monkeys of three species (six African green, seven rhesus, six cynomolgus, and one hybrid [of rhesus male and cynomolgus female parentage]) were treated with sodium saccharin (25 mg in the diet/kg body weight daily for 5 days a week) beginning within 24 hours after birth and continuing for up to 24 years. Sixteen monkeys (seven rhesus and nine cynomolgus) served as controls. During their last 2 years of life, urine was collected from selected treated and control animals and evaluated for various urinary chemistries and for the presence of calculi, microcrystalluria, and precipitate. Urinary bladders were examined by light microscopy and by scanning electron microscopy. RESULTS: Sodium saccharin treatment had no effect on the urine or urothelium in any of these monkeys. There was no evidence of increased urothelial cell proliferation, and there was no evidence of formation of solid material in the urine. CONCLUSION: Although the dose of sodium saccharin administered to these monkeys was only five to 10 times the allowable daily intake for humans, the results provide additional evidence that sodium saccharin is without a carcinogenic effect on the primate urinary tract.

Lymphatic absorption and tissue disposition of liposome-entrapped [14C]adriamycin following intraperitoneal administration to rats.

The lymphatic absorption and tissue distribution of free [14C]Adriamycin, "empty" [3H]liposomes, free [14C]Adriamycin plus empty [3H]liposomes, and [14C]Adriamycin entrapped into [3H]liposomes have been examined at intervals after i.p. injection into rats. Following treatment with empty [3H]liposomes, almost 30% of the liposomal lipid marker was recovered in 24-hr thoracic duct lymph, but when [14C]Adriamycin was added to or encapsulated in liposomes, this value was reduced to 10%. Conversely, only 1% of free [14C]Adriamycin was recovered in 24-hr lymph, but liposomal encapsulation produced a six-fold increase in this value. Studies on the tissue distribution of the liposomal lipid marker after dosing with empty liposomes revealed uptake by diaphragm, liver and spleen, but the highest tissue concentrations were noted in lymph nodes. Liposomal encapsulation of Adriamycin altered its tissue disposition, chiefly by increasing the concentration of drug equivalents in diaphragm, liver and spleen. Although free Adriamycin was accumulated by lymph nodes to some extent, this lymph node accumulation was markedly enhanced by liposomal encapsulation and was present only in those nodes through which lymph draining the peritoneal cavity passes. This finding, together with the observation that diaphragm and thoracic duct lymph contain relatively high levels of liposomal lipid and Adriamycin equivalents, indicates that liposomes are selectively absorbed from the peritoneal cavity by lymphatics and are retained by certain lymph nodes. The results of this study suggest that i.p. administration of liposome-encapsulated drugs may provide a means of selectively concentrating anti-tumor agents in lymphatic channels and lymph nodes.

Carcinogenic and other adverse effects of procarbazine in nonhuman primates.

The carcinogenic potential of procarbazine is under investigation in three species (Macaca mulatta, Macaca fascicularis, and Cercopithecus aethiops) of nonhuman primates. A total of 55 monkeys have received procarbazine s.c., p.o., or i.p. for periods of up to 12 years. Eleven of the 42 monkeys (26%) necropsied thus far have had malignant neoplasms, 6 of which were acute leukemia. Two monkeys developed osteogenic sarcomas, two monkeys developed hemangiosarcomas, and a single case of lymphocytic lymphoma was found. The average total dose of procarbazine received by the monkeys developing cancers was 36.0 g; the average duration of procarbazine treatment was 75 months. A number of the toxic effects of procarbazine seen in humans were also noted in this series of monkeys, including vomiting and myelosuppression. Another striking toxic effect was on the reproductive system of the males. The majority of the adult males necropsied to date have had testicular atrophy with complete aplasia of the germinal epithelium.

Induction of osteogenic sarcomas and tumors of the hepatobiliary system in nonhuman primates with aflatoxin B1.

The carcinogenicity of aflatoxin B1 (AFB1) has been under evaluation in nonhuman primates for the past 13 years. A total of 47 Old World monkeys, chiefly rhesus and cynomolgus, have received AFB1 i.p. (0.125 to 0.25 mg/kg) and/or p.o. (0.1 to 0.8 mg/kg) for 2 months or longer, and 12 are currently alive and without evidence of tumor. Thirteen of the 35 monkeys necropsied to date (37%) developed one or more malignant neoplasms, yielding an overall tumor incidence of 28%. Five of the neoplasms were primary liver tumors (2 hepatocellular carcinomas and 3 hemangioendothelial sarcomas), and 2 cases of osteogenic sarcoma were found. Other tumors diagnosed were 6 carcinomas of the gall bladder or bile duct, 3 tumors of the pancreas or its ducts, and one papillary Grade I carcinoma of the urinary bladder. The tumors developed in animals receiving an average total AFB1 dose of 709 mg (range, 99 to 1354 mg) for an average of 114 months (range, 47 to 147 months). Fifteen of the 22 necropsied monkeys (68%) without tumor showed histological evidence of liver damage, including toxic hepatitis, cirrhosis, and hyperplastic liver nodules. These animals had received an average total AFB1 dose of 363 mg (range, 0.35 to 1368 mg) for an average of 55 months (range, 2 to 141 months). Our results indicate that AFB1 is a potent hepatotoxin and carcinogen in nonhuman primates and further support the hypothesis that humans exposed to this substance may be at risk of developing cancer.

Targeting of murine radiolabeled monoclonal antibodies in the lymphatics.

The tissue localization of a radiolabeled monoclonal antibody directed against a mouse Class I major histocompatibility antigen has been determined in mice following i.v. and s.c. administration. When labeled antibody was given s.c., radioactivity rapidly accumulated in regional lymph nodes draining the injection site, allowing visualization of the nodes by gamma camera imaging within minutes of injection. At 2 h after s.c. injection, radioactivity in regional nodes was present largely as intact antibody, but considerable degradation of antibody present in nodes was noted by 12 h after injection. Since little of the radioactivity reached the blood stream, visualization of regional nodes was possible for long periods after dosing. In contrast, antibody given i.v. showed no significant accumulation in lymph nodes at any time after dosing.

Optimization of monoclonal antibody delivery via the lymphatics: the dose dependence.

After interstitial injection in mice, antibody molecules enter local lymphatic vessels, flow with the lymph to regional lymph nodes, and bind to target antigens there. Compared with i.v. administration, delivery via the lymphatics provides a more efficient means for localizing antibody in lymph nodes. An IgG2a (36-7-5) directed against the murine class I major histocompatibility antigen H-2Kk has proved useful for studying the pharmacology of lymphatic delivery. The antibody specifically binds to most cells in Kk-positive strains of mice and to none in Kk-negative mice. At very low doses, most of the antibody remains at the injection site in Kk-positive animals. As the dose is progressively increased, most effective labeling occurs first in nodes proximal to the injection site and then in the next group of nodes along the lymphatic chain. At higher doses, antibody overflows the lymphatic system and enters the blood-stream via the thoracic duct and other lymphatic-venous connections. Once in the blood, antibody is rapidly cleared, apparently by binding to Kk-bearing cells. These findings indicate that the single-pass distribution of monoclonal antibodies in the lymphatics can be strongly dose dependent, a principle which may be of clinical significance in the improvement of immunolymphoscintigraphic imaging, especially with antibodies directed against normal and malignant lymphoid cells. Monoclonal antibodies directed against normal cell types in the lymph node may be useful for assessing the integrity of lymphatic chains by immunolymphoscintigraphy or, more speculatively, for altering the status of regional immune function. The results presented here indicate that a low or intermediate antibody dose may optimize the signal:noise ratio for imaging. In Kk-negative animals, the percentage of dose taken up in the major organs was essentially independent of the dose administered; there was no evidence for saturable sites of nonspecific binding. These findings provide background for attempts to use antitumor antibodies via the lymphatic route. Specific binding to target cells (and any cross-reaction with normal tissues) would presumably be superimposed on the nonspecific pharmacology of the antibody in vivo.

Effects of long-term oral administration of DDT on nonhuman primates.

Because of reports on tumorigenic activity in different animal species exposed to DDT a decision was made in 1969 to evaluate the long-term effects of DDT on 24 cynomolgus and rhesus monkeys. DDT (20 mg/kg) was given in the diet for 130 months, followed by an observation period that ended in 1994. The two cases of malignant tumor detected in the DDT group included a metastatic hepatocellular carcinoma in a 233-month-old male and a well-differentiated adenocarcinoma of the prostate in a 212-month-old monkey. Benign tumors detected in the DDT group included three cases of leiomyoma, two of which were uterine and one, esophageal. No tumor was detected in the control group of 17 monkeys. Fatty changes in the liver were observed in 52.9% of the DDT group and 29.4% of the control group. More specific signs of hepatotoxicity were documented microscopically in seven DDT monkeys. Severe tremors and histological evidence of CNS and spinal cord abnormalities were observed in six DDT monkeys. The present findings show clear evidence of hepatic and CNS toxicity following long-term DDT administration to cynomolgus and rhesus monkeys. However, the two cases involving malignant tumors of different types are inconclusive with respect to a carcinogenic effect of DDT in nonhuman primates.

Long-term toxicity and carcinogenicity study of cyclamate in nonhuman primates.

Twenty-one monkeys (cynomolgus, rhesus, African green) were fed cyclamate (100 mg/kg and 500 mg/kg) in the diet five times per week from a few days after birth and continuing for up to 24 years. Malignant tumors were diagnosed in three 24-year-old cyclamate monkeys; these were metastatic colon carcinoma (rhesus; 500 mg/kg), metastatic hepatocellular carcinoma (cynomolgus; 500 mg/kg), and a small, well differentiated adenocarcinoma of the prostate (cynomolgus; 100 mg/kg). Benign tumors were found at necropsy in three females; these were adenoma of the thyroid gland (rhesus; 100 mg/kg) and two cases of leiomyoma of the uterus (rhesus; 100 mg/kg and 500 mg/kg). No tumors were detected in an age-matched control group of 16 monkeys. Examination of the testes revealed complete testicular atrophy in one of the old cyclamate monkeys, and focal germ cell aplasia (Sertoli-only tubules) in two other cyclamate monkeys. Focal spermatogenic interruption (maturation arrest) at various germ cell levels mixed with normal spermatogenesis was observed in both the cyclamate-treated and the control monkeys, all of which were over 20 years old. Measurements of terminal cyclohexylamine concentrations showed that three of the males dosed with cyclamate at 500 mg/kg were high converters, with plasma concentrations comparable to the levels that produce testicular atrophy in rats. However, only one of the three high converters showed histologic evidence of irregular spermatogenesis. The overall conclusion is that the testicular abnormalities and the sporadic cases of different malignancies found after more than 20 years of dosing do not provide clear evidence of a toxic or carcinogenic effect of sodium cyclamate in monkeys.

Accumulation of nicotine in the uterine fluid of the six-day pregnant rabbit.

In 6-day pregnant New Zealand White rabbits dosed intravenously with 3H-nicotine, the 3H-activity in the uterine fluid was approximately 5 to 11 times greater than that in the plasma at the corresponding times; 3H-nicotine itself accounted for most of this radioactivity. Dichlorodiphenyltrichloroethane (DDT) also accumulated in the uterine luminal fluid of 6-day pregnant rabbits, but to a lesser extent. However, nicotine or DDT accumulation did not occur in similarly treated, nonpregnant rabbits. The radioactivity in the uterine fluid of rabbits treated with 14C-isoniazid, salicylic acid, barbital, antipyrine, and caffeine was not different from that in the plasma (uterine fluid to plasma radioactivity ratios ranged between 0.67 and 1.85) in both 6-day pregnant and nonpregnant rabbits. No differences in regard to nicotine metabolism, volume of distribution, plasma disappearance, plasma protein binding, or urinary excretion were found between 6-day pregnant and nonpregnant rabbits. Accumulation of nicotine took place in the uterine luminal fluid of nonpregnant does pretreated with either progesterone or human chorionic gonadotropin, but did not occur in does pretreated with estrogen. It is possible that the accumulation of nicotine in uterine fluid of pregnant does and in human chorionic gonadotropin- or progesterone-pretreated nonpregnant does is due to the binding of nicotine to specific uterine fluid proteins.

The lymphocytic absorption of p,p'-DDT and some structurally-related compounds in the rat.

The lymphatic absorption of a series of 14C-labelled compounds structurally related to p,p'-DDT has been examined in thoracic duct-cannulated rats. The compounds (p,p'-DDT, p,p-DDT, P,P'-DDD, p,p-DDD, DDE, DDA and 2,4-D) varied markedly in lipid solubility, as well as in extent of lymphatic absorption, urinary and biliary excretion, and localization in body fat. More than 12% of the dose was recovered in 24-hour thoracic duct lymph of rats treated with the p,p'- and o,p-isomers of DDT and DDD and with DDE, and this percentage was increased by concomitant fat absorption. A strict parallel between lipid solubility and lymphatic absorption was not observed in this series of compounds, possibly because of differences in their rates and routes of excretion.