Immunotoxins, hybrid molecules consisting of a monoclonal antibody linked to a polypeptide toxin have shown anti-tumor activity in both animal models and early clinical trials. However, their potential value in the treatment of human cancer may be limited by the development of host antibodies against the conjugate. Such antibodies could potentially alter immunotoxin pharmacokinetics and pharmacodynamics as well as precipitate serum sickness or anaphylaxis. Using a radioimmunoassay we have measured serial anti-ricin A chain (anti-RTA) and anti-murine immunoglobulin (anti-MIG) titers in 22 patients who received the anti-melanoma immunotoxin XomaZymeR-Mel. Significant titers of anti-RTA and/or anti-MIG were detected in 17 of 21 evaluable patients. Of the four patients not developing antibodies, two were likely immunosuppressed secondary to dexamethasone, and CCNU and dexamethasone respectively. Both patients who received immunotoxin at a time when they had detectable anti-immunotoxin antibodies experienced infusion reactions consistent with immune mediated allergic responses. There was a decrease in peak immunotoxin level in the one patient who had serum immunotoxin levels measured at a time when anti-RTA was present. Strategies to suppress the human immune response to immunotoxins are required before repetitive courses of immunotoxin of this design may be administered.
Complexes of albumin with oligosaccharides have been successfully employed in experimental chemotherapy as hepatotropic carriers of antiviral drugs or as vectors of anticancer-agents, but their clinical use is hampered by an immune response they might evoke. In the present experiments we have studied the humoral immunogenicity of conjugates of 9-beta-D-arabinofuranosyl adenine 5'-monophosphate (ara-AMP) with lactosaminated albumin (L-SA), in mice and rats. These complexes were prepared with the aim of increasing the chemotherapeutic index of ara-AMP in the treatment of chronic hepatitis B virus (HBV) infection. L-SA-ara-AMP conjugates prepared with heterologous albumin produced antibodies in mice and rats when repeatedly injected intraperitoneally. The same conjugates prepared with homologous albumin induced only low amounts of antibodies, when given by repeated intraperitoneal injection, whereas they did not evoke antibodies in mice and were tolerogenic in rats when administered repeatedly by the intravenous route. These results suggest that in a clinical use of drug conjugates prepared with oligosaccharide-albumin complexes the risk of an immune response can be reduced by employing human albumin and by injecting the conjugates by the intravenous route.
Methotrexate (MTX), a mainstay in the treatment of acute lymphoblastic leukemia, is associated with both hepatic and neurologic toxicity. Like a folate, MTX is metabolized to polyglutamated derivatives (MTXGlun) with long intracellular half-lives. These metabolites may contribute to MTX toxicity through a direct effect on cellular metabolism or indirectly through a perturbation of folate homeostasis. To better define the effects of chronic MTX treatment, tissue levels of MTX, MTXGlun, and folate were measured in three monkeys treated with weekly intramuscular MTX for 1 year. Greater than 80% of the total tissue MTX found was in the form of polyglutamated derivatives. Most of these derivatives were MTXGlu3-5 but Glu6-7 were easily detectable. Total tissue folates were measured in liver, kidney, brain and testis with MTX treated animals having a 90% loss of total folate in brain tissue. This is of special interest since inborn errors of folate metabolism are often associated with severe neurologic abnormalities.
9-beta-D-Arabinofuranosyladenine 5'-monophosphate (ara-AMP) coupled to lactosaminated human albumin (L-HSA), injected i.v. into rats, selectively enters the liver. The conjugate concentration in parenchymal and sinusoidal hepatic cells, isolated by collagenase perfusion, was found to be practically equal in both cell types. This indicates that the high uptake of L-HSA-ara-AMP complex by the whole liver also corresponds to a high conjugate concentration in hepatocytes where ara-AMP should be targeted in order to increase its chemotherapeutic index in chronic hepatitis B treatment.
We tested the sensitivity to doxorubicin (DXR) and cisplatin (DDP) of multicellular tumor spheroids (MTS) developed from 2 human lung cancer cell lines; PC-10 squamous cell carcinoma and PC-6 small cell carcinoma. DDP was able to maintain its efficacy in MTS: PC-10 MTS were only 3-fold more resistant to DDP than in monolayer and in PC-6 cells DDP induced cell lethality was essentially unchanged irrespective of cells being in a monolayer or MTS. Atomic absorption spectrometry revealed that DDP uptake was essentially identical, irrespective of cells being in monolayer or MTS. DDP's efficient cell kill effects in MTS seems to be explained by its good penetration into the MTS core. In contrast to DDP, these 2 types of cells responded differently to DXR. Thus, PC-10 MTS became progressively more resistant to DXR when their size increased, whereas the susceptibility of PC-6 MTS tended to increase when the MTS grew larger. Fluorescent microscopic study revealed that prominent DXR fluorescence was observed only at the outer layer of PC-10 MTS. In PC-6 MTS, however, DXR fluorescence was diffusely seen in the entire MTS at low concentrations; nevertheless, owing to PC-6 cells' high sensitivity DXR was able to exert cell lethality. The differences in distribution of DXR fluorescence between PC-10 and PC-6 MTS were corroborated by flow cytometric analysis.
We have previously shown that V79 multicell spheroids are resistant to the anthracenedione mitoxantrone (1). In this paper we demonstrate that this resistance is not solely due to restricted drug penetration into the spheroid, but also to an altered intrinsic resistance of the cells when grown as a three-dimensional structure. We have studied the uptake and toxicity of mitoxantrone in V79-OCF4 monolayers, 100 micron spheroids, 650 micron spheroids, and outer and inner cells of 650 micron spheroids. The LD90 for cells exposed as monolayers to mitoxantrone for two hours was 0.016 microgram/ml, 0.055 microgram/ml for 100 micron spheroids, 1.5 micrograms/ml for outer spheroid cells and 6.2 micrograms/ml for inner spheroid cells. Uptake of [14C]mitoxantrone was linear for all populations with no plateau up to the highest doses used. The uptake of drug required to kill 90% of the cells in a population (UP90) of monolayers was 3.7 ng/10(6) cells, 10.7 ng/10(6) cells for 100 micron spheroids, 169 ng/10(6) cells for outer spheroid cells, and 146 ng/10(6) cells for inner spheroid cells. The relative resistance of spheroids compared to monolayers, based on drug concentration in the medium, was 3.4 for small spheroids, 92 for outer cells, and 390 for inner cells. When cell survival was normalized to drug uptake, the relative resistance of spheroids to monolayers was 2.9 for small spheroids, 46 for outer cells and 40 for inner cells of large spheroids. The data demonstrate that the resistance of multicell spheroids to mitoxantrone is not solely due to restricted drug penetration into the cell aggregate but is also due to a spheroid-induced altered intrinsic resistance of the V79 cells.
Since clinical trials with TNF as a therapeutic agent for cancer are in progress, in this study we have chosen to compare the metabolic characteristics of 125I-labeled and unlabeled RTNF after administration through IM, SC, IP and PO routes. Both RTNF and 125I-RTNF plasma concentration profiles showed an absorption phase and a biexponential decline common to all routes of administration. Moreover the pharmacokinetic analysis indicates that TNF blood levels following SC injection are rather similar to those seen after IM dose. No difference has been found in T max. In contrast, the Kel is apparently increased in the SC route, but the difference is not significant. While a prolonged absorption phase had been obtained after IP injection of RTNF, comparison of t 1/2 beta and Kel between IP and SC or IM route failed to reveal significant differences. Surprisingly some plasma TNF bioactivity has been detected following oral administration.
Attempts were made to reverse the acquired resistance of P388 murine leukemia utilizing non-toxic concentration of nitroxazepine hydrochloride, an antidepressant. The effect of nitroxazepine hydrochloride on the intracellular accumulation of adriamycin and the inhibition of DNA synthesis were studied in these cells. The survival of mice bearing adriamycin-resistant P388 murine leukemia cells treated in vitro with nitroxazepine hydrochloride, adriamycin and a combination of the two drugs was also investigated. The results show that treatment of these cells with nitroxazepine hydrochloride significantly enhanced (1) the intracellular accumulation of adriamycin, (2) inhibition of DNA biosynthesis, and (3) the survival of mice transplanted with adriamycin-resistant P388 murine leukemia cells treated in vitro with a combination of nitroxazepine hydrochloride and adriamycin. The mechanism of restoration of drug sensitivity by nitroxazepine hydrochloride in adriamycin-resistant P388 cells could be due to an enhanced intracellular accumulation of adriamycin. The implications of the present investigations are promising, leaving hope for the utility of nitroxazepine hydrochloride in restoring drug sensitivity in adriamycin-resistant tumors.
Adult mongrel dogs were used to compare hepatic arterial infusion and arterial occlusion-infusion with regard to local and systemic Adriamycin levels. The drug was infused into the right proper hepatic artery of each dog after occlusion of the gastroduodenal artery. Blood and tissue samples were collected at regular intervals for Adriamycin determination. Four weeks later, each dog again received the same agent in the right proper hepatic artery, but this time arterial flow was blocked with an inflated balloon during drug infusion (arterial occlusion-infusion). Blood and tissue drug levels were determined and compared with those obtained using infusion without occlusion. Results indicate that when Adriamycin is administered into the hepatic artery of dogs, occlusion-infusion produces significantly greater hepatic venous drug levels, drug uptake, and drug metabolism than does infusion alone.
Although cisplatin may have intrinsic activity against astrocytoma, limited penetration into the central nervous system may severely curtail delivery of adequate amounts of drug to the tumor. In an attempt to increase the dose of delivered drug without markedly increasing toxicity, cisplatin was administered by 5-day continuous intravenous infusion at a dose of 28 mg/m2/day (total dose 140 mg/m2) to 15 evaluable patients (5 with astrocytoma Grade III and 10 with astrocytoma Grade IV). Median age was 39 years, median Karnofsky Performance Status was 80%, and all patients had been previously treated with other modalities. One patient (7%) achieved Partial Response as demonstrated by increased strength of paretic extremities, increased Karnofsky Performance Status, and decrease of enhancing tumor mass on CT scan. Although nephrotoxicity was minimal, nausea and vomiting (usually mild) was seen in 14 patients. Myelosuppression was also common (anemia in 7 patients, leukopenia in 4 patients and thrombocytopenia in 3 patients). Ototoxicity was seen in 5 patients and may represent a combined modality toxicity with prior cranial radiotherapy. Routes and schedules which allow a higher peak serum concentration of cisplatin (such as subselective intracerebral artery infusion) may be necessary to achieve greater central nervous system drug penetration and to maximally exploit cisplatin in the treatment of astrocytoma.
Methotrexate was administered by IV infusion, 2g (1.19 +/- 0.05 g/m2) over 24 hours, to a homogeneous group of patients undergoing treatment for breast cancer. Three courses were given at three week intervals. Methotrexate and 7-hydroxy-methotrexate plasma and urine pharmacokinetics were investigated. The average terminal half-lives of methotrexate and 7-hydroxy-methotrexate in plasma were 15.02 and 15.19 hours respectively. The area under concentration-time curve was 723.8 +/- 196.4 microM x h for methotrexate and 598.1 +/- 212.5 microM x h for 7-hydroxy-methotrexate. The total average urinary excretions of methotrexate and 7-hydroxy-methotrexate over a 96 hour period were 52% and 5.4% respectively. Urinary clearance of methotrexate was 3.46 +/- 1.4 1/h. In contrast, urinary excretion of 7-hydroxy-methotrexate was not linear. These results confirm the protein binding of metabolite to serum albumin and may suggest that distribution of 7-hydroxy-methotrexate is different from unchanged drug or that the metabolite can be eliminated by another route, such as bile.
This study was performed to determine the effect of treatment with cyclophosphamide (CY) on the formation of antimurine and anti-ricin A chain antibodies in rats treated with a murine monoclonal antimelanoma antibody-ricin A chain immunotoxin (IT). Animals received treatment with either IT alone or IT plus CY. IT treatment consisted of daily IV injections at a dose of 1 mg/kg/day on days 0, 1, 2, 3, 4. CY treatment consisted of a 25 mg/kg IP dose on day -1 followed by daily IP doses of 5 mg/kg/day on days 0, 1, 2, 3, 4. Antibody binding activities in treated animals were measured by enzyme-linked immunosorbent assay and reported as optical density values. Rats treated with IT plus CY had lower binding activity on day 7 (0.09 vs 0.6; p = .02), day 14 (0.42 vs 1.22; p = .001), and day 21 (0.11 vs 1.3; p = .001) compared to rats treated with IT alone. Lower levels of anti-ricin A chain binding activity were observed in CY treated rats on day 14 (0.35 vs 1.25; p = .001), but not on day 7 or day 21. These results indicate that treatment with CY can abrogate the immune response to murine antibody and partially abrogate the immune response to ricin A chain.
At the time of analysis, the first 30 patients with Ommaya reservoirs (OR) at the Children's Orthopedic Hospital and Medical Center in Seattle, Washington had had 32 reservoirs for a mean duration of 28 months. In all, the reservoir chambers had been punctured for either diagnostic or therapeutic purposes a total of 1,287 times with a mean of 40 injections per reservoir and 1.4 injections per month. Six reservoir infections were diagnosed in five patients - a rate of one infection for every 153 reservoir-months. Four infections were attributed to reservoir use - a rate of one infection for every 322 reservoir entries, and less than one infection in 900 entries when a standard aseptic protocol for sampling and injection was applied. There was no correlation between infectious complications and the frequency with which reservoirs were injected, but there was evidence that some of the infections resulted from incomplete compliance with recommended technique for skin preparation and reservoir entry. Four infections were treated successfully with intravenous and intra-reservoir antibiotics without reservoir removal. Only one reservoir had to be removed because of persistent infection. Two other reservoirs were removed because of trauma and malfunction. Twenty-seven patients (90%) retained their original reservoir, up to 9.5 years after implantation. Intraventricular chemotherapy via an indwelling subcutaneous reservoir is a preferred method for delivery of intrathecal chemotherapy, with an acceptable infection risk relative to the benefits of patient comfort and therapeutic efficacy.
The pharmacology of hepatic regional administration of CisplatinR (DDP) was examined in a rabbit model. Routes and modes of administration were: IV, hepatic arterial infusion (HAI), HAI with stopflow, HAI with microembolic material [collagen for embolization (CFE)], and portal vein (PV). DDP was rapidly administered, blood samples were drawn over 45 minutes, and hepatic tissue was obtained. Filterable plasma DDP levels were measured by HPLC. Hepatic DDP levels were determined by atomic absorption spectroscopy. All modes of regional administration yielded significantly higher hepatic DDP levels when compared to tissue levels following IV administration. Only the PV and HACFE routes resulted in significantly less systemic drug exposure (AUC) when compared to IV administration. These data indicate a relative pharmacologic advantage of 1.8 for HAI, 3.4 for PV, 1.8 for HAI stopflow, and 4.3 for HACFE compared to IV DDP administration. This pre-clinical study demonstrates substantial pharmacologic advantage for PV and HACFE routes of DDP administration and suggests that clinical trials based on this information be considere d.
The purpose of these studies was to characterize liposomes containing the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) and to examine their interaction with mouse peritoneal macrophages. Because of its hydrophobic properties, FMLP can be readily incorporated into both phosphatidylcholine (PC) and PC/phosphatidylserine (PS) (7:3, mol ratio) multilamellar vesicles (MLV). The association of FMLP with the MLVs is stable in medium with or without serum. The size distribution and aqueous trap volume of MLV consisting of PC or PC/PS that contain FMLP at a 10:1 molar ratio do not differ from that of MLV without FMLP. MLV containing equimolar phospholipid-to-FMLP ratios are smaller but have larger aqueous trap volumes. MLV consisting of PC and FMLP (10:1 molar ratio) are phagocytosed more efficiently than PC MLV, and their phagocytosis is mediated via the FMLP receptors on macrophages. In contrast, MLV consisting of PC/PS or PC/PS with FMLP (7:3:1 molar ratios) are phagocytosed to the same extent, and so the FMLP receptors on macrophage surface do not influence this process. The delivery of FMLP in liposomes to macrophages is very efficient: up to 25-fold more FMLP delivered by MLV is internalized into macrophages than when it is in free form. Once associated with the macrophages in either free form or via liposomes, some FMLP is degraded and released into the medium, but the majority of FMLP molecules remain with the macrophages to be degraded at a steady rate. The present findings may explain how liposome-FMLP produces tumoricidal properties in macrophages.
Levels of dihydrofolate reductase (DHFR) were measured in livers from man and a variety of animals and birds to explore possible causes for the remarkably low levels of DHFR previously reported in fresh human tissues, including liver (Cancer Drug Delivery 2, 133 (1985)). While levels were lower in wild than domesticated animals and birds, suggesting a possible role for folic acid supplements fed to the latter, levels in both were far higher than in humans. Hepatic DHFR levels were similar in adult and young animals, birds and humans. In primates, monkey livers had DHFR levels similar to those of other animals, while levels in great apes were much lower and closer to levels in man. These findings suggest that low levels of DHFR are a feature peculiar to man, which is shared with the primates closest to man, but which distinguishes him from most animals and birds.
The effect of recombinant human tumor necrosis factor-alpha (rhTNF-alpha) on rodent hematopoietic precursors and hepatocytes was evaluated. rhTNF-alpha appears to have minimal toxic effects on isolated hepatocytes and on granulocyte-macrophage colony-forming units (CFU-GM) in vitro. It does, however, show a dose- and schedule-dependent inhibition of pluripotent bone marrow stem cells in mice. This was most evident with prolonged continuous infusion schedules. In contrast, single daily injections of rhTNF-alpha did not depress bone marrow or peripheral blood counts, although slight elevations in hepatic enzyme levels were apparent. These studies suggest that continuous infusions of rhTNF-alpha in man may produce enhanced bone marrow suppression over equivalent bolus doses.
Using growth delay and clonogenic cell survival as end points, we have shown that the 3-dimensional structure of human lung tumour spheroids confers a degree of resistance to the anthracyclines doxorubicin and 4'-deoxydoxorubicin, relative to cells grown as monolayer. 4'-deoxydoxorubicin induces a longer growth delay and greater clonogenic cell kill than doxorubicin in spheroids, although it is no more cytotoxic in monolayer. Using fluorescent microscopy we have demonstrated, qualitatively, that lipophilic anthracycline analogues partition into the spheroid more rapidly and to a greater degree than doxorubicin. It is apparent that penetration is an important aspect of anthracycline drug resistance in spheroids, and the spheroid model may represent a better in vitro system for testing lipophilic analogues of cytotoxic drugs.
Adriamycin (ADM) is an effective antineoplastic drug. However, the amount of ADM that can be administered must be limited because of the risk of developing a severe dose-dependent myocardiopathy. Prenylamine (PNL), a calcium antagonistic drug, provided partial protection against ADM-induced cardiotoxicity in mice and in the rabbit. Thus, it was considered important to evaluate the cardioprotective potential of PNL in patients given ordinary doses of ADM. Twenty-six patients were selected and randomized in two groups, and a double-blind trial was begun. Group A (n = 13): patients received ADM, i.v. at standard oncological doses up to 550 mg/m2, plus placebo, orally. Group B (n = 13): ADM was administered as in Group A, but PNL 200 mg/day was given instead of placebo. Standard ECG and chest radiographs were performed at the beginning of treatment and every two months. Mode-M echocardiograms and 24-hour ambulatory ECGs were obtained previously to the beginning of the ADM treatment and two months after the administration of the last dose of the drug. In Group A, three patients died from oncological causes, total ADM dose was 359 +/- 100 mg/m2, and the mean age was 59.7 years. One patient in this group developed a congestive myocardiopathy while another patient developed a severe supraventricular arrhythmia. In Group B, four patients died from oncological causes, total ADM dose was 367 +/- 132 mg/m2, and the mean age was 63.8 years. No myocardiopathy was found in this group. These findings suggest that simultaneous administration of PNL may mitigate ADM cardiotoxicity, but larger trials are needed to draw definite conclusions.
The present paper is concerned with the question whether cells of the mononuclear phagocyte system (MPS) function as a transport system for doxorubicin (DXR)-containing liposomes into solid tumors. The investigations were performed in solid IgM immunocytoma bearing Lou/M Wsl rats. In this tumor system macrophage accumulation was observed during DXR and cisplatin induced tumor regression. Tumor-bearing rats were treated i.v. with 1 mg/kg DXR (either free or entrapped in liposomes) for 4 consecutive days. Changes in the cellular composition of the tumor were studied by histology and cytology comparing free and liposomal DXR treatment. Therapy with DXR-liposomes (lip DXR) induced macrophage accumulation similar to free DXR. No differences in cellular changes in the tumor between both types of treatment were found. The DXR-content of tumor-associated cells was measured at various times during treatment with free or lip DXR by means of flow cytometry. Most DXR-fluorescence was associated with dead cells and cellular debris. No clear-cut differences in DXR-content were found between macrophages isolated from tumors of free DXR treated animals and those isolated from lip DXR treated animals. In order to investigate whether DXR-liposomes are taken up by tumor tissue (either via macrophages or directly by the tissue itself), [3H]inulin-labeled DXR-liposomes were administered to tumor-bearing rats. The distribution of radiolabeled lip DXR in non-treated rats was compared with that in rats which were pretreated with non-radiolabeled lip DXR to induce macrophage accumulation. In both untreated and pretreated animals most of the administered 3H-dose was recovered in liver and spleen. Only a minor fraction was recovered from the tumor and other tissues. This fraction was not higher than that obtained when a comparable amount of free [3H]inulin was injected. In conclusion, this study suggests that DXR-liposomes do not enter the solid IgM immunocytoma, even when during therapy macrophages pass the endothelial barrier. Therefore, it is unlikely that macrophage-mediated transportation of DXR-liposomes into the tumor is involved in the mechanism of tumor regression induced by liposome-entrapped DXR.
