Increased cortisol levels in hair of recent Ecstasy/MDMA users.

Previous research has revealed an acute 8-fold increase in salivary cortisol following self-administrated Ecstasy/MDMA in dance clubbers. It is currently not known to what extent repeated usage impacts upon activity of the hypothalamic-pituitary-adrenal axis over a more prolonged period of time. This study investigated the integrated cortisol levels in 3-month hair samples from recent Ecstasy/MDMA users and non-user controls. One hundred and one unpaid participants (53 males, 48 females; mean age 21.75 years) completed the University of East London recreational drug use questionnaire, modified to cover the past 3-months of usage. They comprised 32 light recent Ecstasy/MDMA users (1-4 times in last 3 months), 23 recent heavy MDMA users (+5 times in last 3 months), and 54 non-user controls. Volunteers provided 3 cm hair samples for cortisol analysis. Hair cortisol levels were observed to be significantly higher in recent heavy MDMA users (mean = 55.0 ± 80.1 pg/mg), compared to recent light MDMA users (19.4 ± 16.0 pg/mg; p=0.015), and to non-users (13.8 ± 6.1 pg/mg; p<0.001). Hence the regular use of Ecstasy/MDMA was associated with almost 4-fold raised hair cortisol levels, in comparison with non-user controls. The present results are consistent with the bio-energetic stress model for Ecstasy/MDMA, which predicts that repeated stimulant drug use may increase cortisol production acutely, and result in greater deposits of the hormone in hair. These data may also help explain the neurocognitive, psychiatric, and other psychobiological problems of some abstinent users. Future study design and directions for research concerning the psychoneuroendocrinological impact of MDMA are also discussed.

Raman spectroscopy and imaging of beta-carotene in live corpus luteum cells.

Raman spectroscopy has been used to identify and locate beta-carotene within individual living luteal cells. The cells were either freshly prepared or cultured; the latter was incubated in the presence or absence of beta-carotene in the form of enriched bovine high-density lipoprotein. Luteal cells were investigated using several Raman spectroscopic and imaging techniques. These techniques did not give accurate concentration levels of beta-carotene within parts of the cell but illustrated the distribution of the molecule. Freshly prepared luteal cells were found to contain an appreciable concentration of beta-carotene. Over a period of several days, the concentration gradually reduced to a nearly undetectable level; similar results were found for cells cultured in the absence of the beta-carotene. For cells cultured in the presence of beta-carotene, the molecular concentration was maintained for as long as 2 weeks. The Raman spectra of fragmented cells showed that the beta-carotene is predominantly localised in the lipid-rich cell components, with the concentration highest in the microsomal fraction. The Raman imaging techniques revealed that beta-carotene was spread over the entire volume of the luteal cells with higher levels occurring at distinct sites, including the surface.

9-Nitrocamptothecin is an effective drug for the treatment of human lung tumors: comparison of in vitro and in vivo studies.

9-Nitrocamptothecin (9NC) results in complete regression of small-cell lung carcinoma (SCLC) and non-SCLC (NSCLC) growing as xenografts in immunodeficient mice. In this study, we have monitored histological changes in the tumors during 9NC-induced regression, and perturbations in the cell cycle of cells derived from these tumors using flow cytometry. In vivo, 9NC treatment induces dramatic changes in the tumor cells, which die by apoptosis and are ultimately eliminated from the normal tissue. In vitro, 9NC treatment resulted in apoptosis and cytostasis of the NSCLC and SCLC cells, respectively. Further, 9NC induced cytostasis in control, normal human lung fibroblasts. Therefore, the studies in vivo have indicated that 9NC acquires a remarkable antitumor activity against both the SCLC and NSCLC types tested, and that results of studies in vitro may not reflect the results observed in vivo.

Preclinical study of the systemic toxicity and pharmacokinetics of 5-iodo-2-deoxypyrimidinone-2'-deoxyribose as a radiosensitizing prodrug in two, non-rodent animal species: implications for phase I study design.

We have demonstrated previously an improved therapeutic index for oral 5-iodo-2-deoxypyrimidinone-2'-deoxyribose (IPdR) compared with oral and continuous infusion of 5-iodo-2'-deoxyuridine (IUdR) as a radiosensitizing agent using three different human tumor xenografts in athymic mice. IPdR is a prodrug that is efficiently converted to IUdR by a hepatic aldehyde oxidase, resulting in high IPdR and IUdR plasma levels in mice for > or =1 h after p.o. IPdR. Athymic mice tolerated oral IPdR at up to 1500 mg/kg/day given four times per day for 6-14 days without significant systemic toxicities. In anticipation of an investigational new drug application for the first clinical Phase I and pharmacology study of oral IPdR in humans, we studied the drug pharmacokinetics and host toxicities in two non-rodent, animal species. For the IPdR systemic toxicity and toxicology study, twenty-four male or female ferrets were randomly assigned to four IPdR dosage groups receiving 0, 15, 150, and 1500 mg/kg/day by oral gavage x 14 days prior to sacrifice on study day 15. All ferrets survived the 14-day treatment. Ferrets receiving 1500 mg/kg/day showed observable systemic toxicities with diarrhea, emesis, weight loss, and decreased motor activity beginning at days 5-8 of the 14-day schedule. Overall, both male and female ferrets receiving IPdR at 1500 mg/kg/day experienced significant weight loss (9 and 19%, respectively) compared with controls after the 14-day treatment. No weight loss or other systemic toxicities were observed in other IPdR dosage groups. Grossly, no anatomical lesions were noted at complete necropsy, although liver weights were increased in both male and female ferrets in the two higher IPdR dosage groups. Histologically, IPdR-treated animals showed dose-dependent microscopic changes in liver consisting of minimal to moderate cytoplasmic vacuolation of hepatocytes, which either occurred in the periportal area (high dosage group) or diffusely throughout the liver (lower dosage groups). Female ferrets in the highest IPdR dose group also showed decreased kidney and uterus weights at autopsy without any associated histological changes. No histological changes were found in central nervous system tissues. No significant abnormalities in blood cell counts, liver function tests, kidney function tests, or urinalysis were noted. Hepatic aldehyde oxidase activity was decreased to approximately 50 and 30% of control ferrets in the two higher IPdR dosage groups, respectively, after the 14-day treatment period. The % IUdR-DNA incorporation in ferret bone marrow at the completion of IPdR treatment was < or =0.05% in the two lower dosage groups and approximately 2% in the 1500 mg/kg/day dosage group. The % IUdR-DNA in normal liver was < or =0.05% in all IPdR dosage groups. In a pharmacokinetic study in four Rhesus monkeys, we determined the plasma concentrations of IPdR after a single i.v. bolus of 50 mg/kg over 20 min. Using a two-compartment model to fit the plasma pharmacokinetic data, we found that IPdR was cleared in these non-human primates in a biexponential manner with an initial rapid distributive phase (mean T1/2alpha = 6.5 min), followed by an elimination phase with a mean T1/2 of 63 min. The mean maximum plasma concentration of IPdR was 124+/-43 microM with a mean total body clearance of 1.75+/-0.95 l/h/kg. IPdR was below detection (<0.5 microM) in the cerebrospinal fluid. We conclude that there are dose-limiting systemic toxicities to a 14-day schedule of p.o. IPdR at 1500 mg/kg/day in ferrets that were not found previously in athymic mice. However, no significant hematological, biochemical, or histopathological changes were found. Hepatic aldehyde oxidase activity was reduced in a dose-dependent in ferret liver, suggesting partial enzyme saturation by this IPdR schedule. The plasma pharmacokinetic profile in Rhesus monkeys showing biexponential clearance is similar to our published data in athymic mice. These data are being applied

Preclinical toxicity and efficacy study of a 14-day schedule of oral 5-iodo-2-pyrimidinone-2'-deoxyribose as a prodrug for 5-iodo-2'-deoxyuridine radiosensitization in U251 human glioblastoma xenografts.

In anticipation of an initial clinical Phase I trial in patients with high-grade gliomas of p.o. administered 5-iodo2-pyrimidinone-2'-deoxyribose (IPdR) given daily for 14 days as a prodrug for 5-iodo-2'-deoxyuridine (IUdR)-mediated tumor radiosensitization, we determined the systemic toxicities and the percentage IUdR-DNA incorporation in normal athymic mouse tissues and a human glioblastoma xenograft (U251) after this dosing schedule of IPdR. Using a tumor regrowth assay of s.c. U251 xenografts, we also compared radiosensitization with this IPdR-dosing schedule to radiation therapy (XRT) alone (2 Gy/day for 4 days) or to XRT after continuous infusion IUdR for 14 days at the maximum tolerated dose in mice (100 mg/kg/day). Athymic mice with and without U251 s.c. xenografts tolerated 750 or 1500 mg/kg/day of p.o. IPdR (using gastric lavage) for 14 days without weight loss or activity level changes during treatment and for a 28-day posttreatment observation period. The percentage IUdR-DNA incorporation in U251 tumor cells was significantly higher after p.o. IPdR (750 and 1500 mg/kg/day) for 14 days (3.1 +/- 0.2% and 3.7 +/- 0.3%, respectively) than continuous infusion IUdR for 14 days (1.4 +/- 0.1%). Compared to XRT alone, a significant sensitizer enhancement ratio (SER) was found with the combination of p.o. IPdR (1500 mg/kg/d) + XRT (SER = 1.31; P = 0.05) but not for the combination of continuous infusion IUdR + XRT (SER = 1.07; P = 0.57) in the U251 xenografts. The percentage IUdR-DNA incorporation after IPdR at 1500 mg/kg/day for 14 days in normal bone marrow, normal small intestine, and normal liver were 1.2 +/-0.2%, 3.3 +/- 0.3%, and 0.2 +/- 0.1%, respectively. We conclude that a 14-day p.o. schedule of IPdR at up to 1500 mg/kg/day results in no significant systemic toxicity in athymic mice and is associated with significant radiosensitization using this human glioblastoma multiforme xenograft model. Based on these data and our previously published data using shorter IPdR dosing schedules, which also demonstrate an improved therapeutic index for IPdR compared to IUdR, an initial clinical Phase I and pharmacokinetic study of p.o. IPdR daily for 14 days is being designed.

LEX 032: a novel recombinant human protein for the treatment of ischaemic reperfusion injury.

Reperfusion injury is defined as the enhancement of the damage that occurs in ischaemic cells during the reperfusion period. Cellular damage to the brain occurs not only during the ischaemic period, but also during the reperfusion period. Such injury occurs when blood flow is restored to heart, brain or other tissue after flow has been blocked. Several mechanisms appear to play a role in the generation of reperfusion injury. To a greater or lesser extent, most involve neutrophils. The infiltration of neutrophils into the previously ischaemic area has been implicated as playing major role following reperfusion. Microscopic examination of tissue has shown a direct correlation between the duration of oxygen deprivation with the amount of damage, and the extent of activated neutrophil recruitment. Activated neutrophils are responsible for the release of serine proteases, which directly lead to tissue damage. Activated neutrophils also contain a newly assembled enzyme that produces tissue damaging free radicals. However, a preliminary and necessary step is to attach the activated neutrophil on to the lining of the blood vessels, a process requiring proteolytic activity. Administration of a drug that prevents neutrophil transmigration would reduce reperfusion injury. SuperGen is developing a drug, LEX 032, with a unique spectrum of activities, including the ability to inhibit binding of neutrophils to the vascular surface by blocking this proteolytic activity. In addition, this drug inhibits free radical production by neutrophils, and inhibits the activity of released serine proteases. Therefore, LEX 032 is expected to prevent or minimise neutrophil mediated reperfusion injury. Blockade of all three destructive inflammatory responses should limit the amount of damaged tissue and save viable tissue. A drug with these capabilities might find use in the treatment of myocardial infarction, shock-resuscitation, replantation surgery, frostbite, burns and organ transplantation. Since LEX 032 has no inhibitory activity against thrombin and plasmin, it represents an ideal drug for use in the treatment of ischaemic stroke. Recently, data have been published demonstrating that ischaemic stroke patients given the thrombolytic drug tPA were at least 30% more likely to have minimal or no disability at three months, as measured by outcome scales, when compared to placebo-treated patients. Presumably, this action was because of the hastening of brain reperfusion, and may have been limited due to reperfusion injury. The FDA approved the use of tPA for the limited treatment of acute ischaemic stroke. Since LEX 032 has been shown to limit neutrophil mediated reperfusion damage, it may find use either alone, to ameliorate damage occurring spontaneously during ischaemic stroke, or in combination therapy with tPA to reduce reperfusion injury secondary to thrombolytic therapy. This unique approach may have broad therapeutic potential in the treatment of neutrophil mediated diseases since, unlike a monoclonal antibody for example, it is independent of the specific adhesion molecule(s). These diseases include inflammatory diseases which are, at least in part, caused or exacerbated by excessive neutrophil proteases, such as acute pancreatitis, arthritis, allograft rejection, sepsis, meningitis, acute pulmonary inflammation, psoriasis and damage caused by burns. This is in addition to reperfusion-related diseases such as myocardial infarction, stroke, shock-resuscitation, replantation surgery, frostbite, burns and organ transplantation.

Intrathecal busulfan treatment of human neoplastic meningitis in athymic nude rats.

The current study was designed to evaluate the toxicity and activity of Spartaject Busulfan, a microcrystalline preparation of busulfan, following its intrathecal administration into a nude rat model of human neoplastic meningitis. Animals were treated through permanent indwelling subarachnoid catheters. Human glioma D-456 MG growing in the subarachnoid space was treated with 8.1 micromol of intrathecal Spartaject Busulfan. Single-dose therapy was also subsequently compared with 4 doses of 8.1 and 2.0 micromol busulfan, respectively, against D-456 MG neoplastic meningitis. Additional experiments evaluated a saline control versus 8.1 micromol x 1, 6.2 micromol x 4 and 4.1 micromol x 4, respectively, against D-456 MG. A single dose of 8.1 micromol of intrathecal Spartaject Busulfan resulted in an increase in median survival of 61.7% compared with the saline control. In experiment 2, all busulfan treatments showed increases in median survival of 142.8% (8.1 micromol x 1), 52.3% (2.0 micromol x 4), and 23% (8.1 micromol x 4) (p < 0.001 for all groups) compared with the saline control. These results suggest that a narrow therapeutic dose range for both toxicity and activity has been defined for intrathecal busulfan in the treatment of human neoplastic meningitis in athymic nude rats. Although busulfan has only limited activity against solid tumors, the high doses achievable in the CSF following intrathecal administration coupled with the steep dose-response relationships of alkylating agents, provide rationale for further evaluation of this agent.

Effect of serine proteinase inhibitors on neutrophil function: alpha-1-proteinase inhibitor, antichymotrypsin, and a recombinant hybrid mutant of antichymotrypsin (LEX032) modulate neutrophil adhesion interactions.

Circulating serine proteinase inhibitors (serpins) regulate a number of proteinases that participate in the inflammatory process. In this study, we investigated possible modulatory effects of serpins on neutrophil adhesion. Antichymotrypsin (ACT), alpha1-protease inhibitor (alpha1-PI), and LEX032, a recombinant hybrid of ACT and alpha1-PI were shown to inhibit neutrophil adhesion to fibronectin (FN)-coated surfaces and, to a lesser extent, adhesion to other extracellular matrix proteins. The inhibitory effect of serpins on neutrophil adhesion to FN was found to be related to inhibition of FN proteolysis based on the following observations: (1) elastase treatment of FN-coated plates, but not of neutrophils, resulted in enhanced neutrophil adhesion; and (2) serpins inhibited FN proteolysis by neutrophil proteases. Serpins also inhibited neutrophil spreading, as well as shedding of neutrophil CD43, but not L-selectin, CD18, or CD29. We conclude that serpins modulate neutrophil adhesion both by inhibiting proteolytic processing of extracellular matrix proteins and proteolytic shedding of CD43.

Biodistribution and pharmacokinetics of 131I-labeled LEX 032, a recombinant hybrid of antichymotrypsin.

Pharmacokinetic and biodistribution studies were conducted in rats on a novel serine protease inhibitor, LEX 032, that was radiolabeled with 131I by the Bolton-Hunter reagent. LEX 032, a genetically engineered recombinant human nonglycosylated serpin, has been shown to have antiinflammatory properties in a number of animal models of inflammation and reperfusion injury. When 131I-LEX 032 was injected intravenously, a rapid whole body clearance of radioactivity was seen. Blood clearance followed a similar pattern. Forty-eight hours postinjection, 2.00 +/- 0.65 of the administered dose remained in the body. Greater than 59% of the radio-activity was excreted in the urine within the first 24 hr. Little radioactivity was found in the feces. With the exception of the thyroid, no significant organ-related uptake was noted. Radioactivity in the liver peaked at 20 min postinjection, with 1.00 +/- 0.13% administered dose/g and approximately 10% administered dose in the whole liver. At 1 hr, uptake in the kidney (9.30 +/- 1.52% administered dose/g) was the highest among all tissues, except for the thyroid. Gamma camera images were consistent with the biodistribution pattern. Pharmacokinetics and biodistribution were not affected by the dose of LEX 032 and were quite different from those of glycosylated wild type antichymotrypsin. These data indicate that LEX 032 exhibits the pharmacokinetics expected of a nonglycosylated 45 kDa protein.

Inhibition of human leukocyte proteinase 3 by a novel recombinant serine proteinase inhibitor (LEX032).

The interaction of a bioengineered serpin (LEX032) with human leukocyte proteinase 3 (PR 3) has been investigated. LEX032 was found to be a time-dependent inhibitor of PR 3, forming a highly-stable enzyme-inhibitor complex (Ki 12 nM).

Biodistribution and metabolism of internally 3H-labeled oligonucleotides. II. 3',5'-blocked oligonucleotides.

The pharmacokinetics and metabolism of four radiolabeled phosphodiester oligonucleotides with 3'- and 5'-blocked ends were studied in mice and compared with previously studied, unblocked, all-phosphodiester and all-phosphorothioate oligonucleotides. The radiolabel was a tritiated methyl group enzymatically attached at an internal cytidine. The ends of the blocked phosphodiester oligonucleotides were protected by cyclization or by incorporation of either phosphorothioate or methylphosphonate linkages. Although these modifications protected the blocked oligonucleotides from degradation by exonucleases present in mouse serum, degradation initiated by endonucleases was 50% complete in 0.5-5 hr. After intravenous injection, the blocked oligonucleotides were much less stable than the all-phosphorothioate oligonucleotide and only marginally more stable than the previously studied, unblocked phosphodiester oligonucleotide. Even a "chimeric" blocked oligonucleotide with 16 phosphorothioate linkages and eight contiguous phosphodiester linkages was rapidly degraded. Despite the favorable serum binding, tissue accumulation, and stability observed with phosphorothioate oligonucleotides, these properties did not provide the chimeric oligonucleotide access to a compartment where its phosphodiester linkages were stable. In other respects, the blocked and chimeric phosphodiester oligonucleotides also resembled the unblocked phosphodiester oligonucleotide; radiolabel was cleared rapidly from the blood, there was little evidence of tissue accumulation, high performance liquid chromatographic analysis of tissue extracts showed extremely rapid degradation to mononucleotides, and only mononucleotide metabolites were present in urine. In summary, blocked phosphodiester oligonucleotides are rapidly attacked by endonucleases present in mice. Unless this problem is less serious in primates, such blocked oligonucleotides will be relatively unattractive candidates for drug development.

Biodistribution and metabolism of internally 3H-labeled oligonucleotides. I. Comparison of a phosphodiester and a phosphorothioate.

Biodistribution and metabolism of oligonucleotides were determined using a 3H-labeled 20-nucleotide phosphodiester and its phosphorothioate analog. The oligonucleotides were radiolabeled by 3H-methylation of an internal deoxyctidine with HhaI methylase and S- [3H]adenosylmethionine. Biodistribution studies were conducted after intravenous injection of 6 mg/kg (5 muCi) oligonucleotide. Metabolism of the oligonucleotides was determined by paired-ion high performance liquid chromatography. After phosphodiester injections, radiolabel rapidly cleared the blood. Relative initial concentrations were as follows: kidney > blood > heart > liver > lung > spleen. Radiolabel in spleen peaked at 1 hr and remained elevated for 24 hr. At 2 hr the concentration in all organs, except spleen, was equal to that in blood. High performance liquid chromatographic analysis of the kidney, liver, and spleen extracts and urine indicated extremely rapid metabolism to monomer. Results of studies after the injection of phosphorothioate oligonucleotide differed from those using the phosphodiester. Despite its rapid clearance from blood, phosphorothioate accumulated rapidly in all tissues, especially the kidney. Kidney uptake increased over time, remaining very high for 24 hr. Ratios of organ to blood concentrations at 2 hr for all organs were 5:1 or greater. Kidney and liver ratios were 84:1 and 20:1, respectively. Analysis of the kidney and liver extracts and urine indicated that slow metabolism occurred. These data suggest that phosphodiester oligonucleotides would have limited therapeutic utility. The stability and organ distribution of the phosphorothioate oligonucleotide imply that such oligonucleotides may have therapeutic potential.

Tissue localization of methotrexate-monoclonal-IgM immunoconjugates: anti-SSEA-1 and MOPC 104E in mouse teratocarcinomas and normal tissues.

Methotrexate (MTX) was coupled to the tumor-targeting monoclonal IgM, anti-SSEA-1 and the non-targeting myeloma IgM, MOPC 104E. At 24-h intervals following injection, drug deposition in MH-15 teratocarcinomas and in several normal tissues was followed by immunoperoxidase microscopy using the M16 monoclonal antibody to MTX. MTX-anti-SSEA-1 was deposited on the surface and in the interior of living tumor cells 24 h after injection; at 48 h and after, only low-level binding to necrotic tissue was found. There was no significant gradation in staining from the outside to the interior of the tumors. In tumors, the control MOPC 104E immunoconjugate was detectable only in necrotic tissue. Binding to SSEA-1-expressing normal tissues was undetectable, except for pericryptal fibroblasts in the small intestine. No significant pathology was found in normal tissues that are SSEA-1 positive. High levels of the immunoconjugate were detected in the liver, where MTX was found predominantly in Kupffer cells and possibly in hepatocytes; again, no significant morphological changes were associated with this retention. Thus tumor-associated antigens can be suitable targets for antibody-drug conjugates even when present in normal tissues and in large quantities, provided that the antigens in normal tissues are inaccessible. Moreover, deposition in viable tumor tissue can be assessed using monoclonal antibodies to methotrexate.

Pharmacokinetics of 99mTc-metallothionein-B72.3 and its F(ab')2 fragment.

The radionuclide of choice for use in diagnosis in most nuclear medicine diagnostic procedures is 99mTc. It is important, therefore, to establish whether there is potential clinical efficacy of an antitumor antigen directed monoclonal antibody labeled with 99mTc. We have investigated the potential use of a 99mTc labeled antibody complex which uses conjugation of the metal binding protein, metallothionein (MT), to bind the radiolabel. The stability and pharmacokinetics of the conjugates in normal and tumor bearing mice were compared to radioiodinated controls. Measurements done in CD-1 mice comparing either 99mTc-MT-B72.3 with 125I-B72.3 or 99mTc-MT-F(ab')2 with 125I-F(ab')2 indicated that the 99mTc-MT-B72.3 cleared at a rate which was faster than 125I-B72.3 while the two radiolabeled F(ab')2 fragments cleared at similar rates. The 99mTc from both labeled IgG and F(ab')2 was found in the kidneys and urine. While all the 99mTc in the urine was in the form of low molecular weight compounds, the serum contained radioactivity comigrating on size exclusion chromatography with the injected monoclonal antibody. With the exception of kidneys, organ values for 99mTc-MT-B72.3 were consistently lower than the 125I values, while the 99mTc-MT-F(ab')2 cleared other organs at similar rates to those for the iodinated monoclonal antibody. Both 99mTc-MT-B72.3 and F(ab')2 showed higher 24-h tumor:blood ratios than the iodinated proteins. Due to the pharmacokinetic properties of 99mTc-MT-F(ab')2 and the half-life of 99mTc, 99mTc-MT-F(ab')2 is the agent best suited for imaging.

Uptake and metabolism of 111In-labeled monoclonal antibody B6.2 by the rat liver.

When 111In-labeled murine monoclonal antibodies are used in radio-scintigraphic diagnostic procedures, a large fraction of the injected radionuclide is sequestered by the liver. Neither the cells responsible for the uptake nor the mechanism of uptake are known. Little is known about either the site within the liver of antibody metabolism or the form of the products of metabolism. In these studies, the uptake and metabolism of a monoclonal antibody, B6.2 radiolabeled with 111In or 125I [either intact B6.2 or F(ab')2] were determined in rats. One h after injection of either 125I- or 111In-diethylenetriaminepentaacetic acid (111In-DTPA)-labeled B6.2, the predominant liver cell in which the radionuclide was found was the parenchymal cell. At this time, the absolute uptake of 125I in the liver was 0.23 +/- 0.06% (SD) of the injected dose compared to 0.61 +/- 0.06% when the radionuclide was 111In. Removal of the Fc portion of the antibody reduced the absolute liver uptake of 125I to 0.10 +/- 0.01 and the absolute uptake of 111In to 0.16 +/- 0.06. Both radionuclides were still associated predominantly with the parenchymal cell. Using size exclusion high performance liquid chromatography analysis of liver supernatants the metabolism of radiolabeled B6.2 was followed for 24 h. Of the radioactivity recovered, 47.9% of the 125I was precipitable by centrifugation (and presumed bound to cell membranes) while 15.4% was attached to B6.2 found in the cytosol. In contrast, when 111In-DTPA-B6.2 was administered, 16.0% of 111In recovered from the liver was precipitable by centrifugation, and 6.5% was attached to B6.2 found in the cytosol. Sixty % of the 111In was recovered as a low molecular weight (less than 1000) component in the cytosol. This metabolite was not immunoreactive, nor did it comigrate with ferritin, and was resolved into four components by ion exchange high performance liquid chromatography. Of these, only a minor component cochromatographed with an 111In-DTPA standard. These data suggest that the large accretion of radionuclide by the liver is due to uptake of monoclonal antibodies by an Fc receptor-mediated mechanism and the subsequent accumulation of low molecular weight metabolites, presumably 111In-DTPA, attached to one or more amino acids. The reasons for the entrapment of metabolites in the liver are under investigation.

Intraperitoneal immunoconjugates.

Intracavitary instillation of radioantibodies has been proposed as therapy for anatomically confined malignant disease. To evaluate this therapeutic strategy, a monoclonal antibody reactive with human transferrin receptor (7D3) was evaluated for localization in a human malignant mesothelioma transplanted i.p. in athymic nude mice. This antibody was purified and labeled with 131I, 125I, or 111In. Radiolabeled antibody was administered i.p. or i.v. to tumor-bearing mice. Three h after injection, the percentage of injected dose/g (ID/g) of tumor was higher in free-floating ascites tumor cells (31.0%/g tumor cell pellet) after i.p. injection than after i.v. injection (12.0%). However, localization of radiolabel in i.p. solid tumors was similar (5.37% ID/g i.p. versus 4.73% of ID/g i.v.), and by 24 h both routes of administration produced similar localization of radiolabel in both free-floating ascites cells and solid tumors. In contrast, uptake of radiolabel into liver, kidney, and to a lesser extent bone and bone marrow, was less with i.p. than with i.v. administration. In clinical studies with 111In and 90Y antibodies administered i.p. to patients with ovarian cancer, confined biodistribution of the radioantibody was again seen, although interpatient variability of rate of egress of the radiolabel was documented. Therefore, both preclinical and clinical data indicate that i.p. therapy with immunoconjugates may be advantageous for cancer confined to the peritoneal cavity. This advantage stems primarily from reduced localization of isotope in organs of catabolism or toxicity (liver, kidney, bone, and bone marrow), rather than greatly increased levels of isotope in tumor. Unresolved problems include degree of antibody penetration into solid tumors, microdosimetry, and radioantibody effectiveness for tumor killing.

Experimental studies of radioimmunodetection of cancer: an overview.

During the past 8 years numerous patients have been treated by injection of radiolabeled monoclonal antibodies for both diagnosis and treatment of cancer. It has become common to refer to this approach by such terms as "antibody guided delivery" or "antibody guided targeting." In general the results, while somewhat promising, have failed to fulfill our initial expectations. It is now clear that there are many physiological barriers that antibodies face in their travel toward their tumor associated antigen. The papers in the "Experimental Studies of Radioimmunodetection" section of the symposium describe in detail these barriers. We must remember that antibody conjugates are no more "guided" than classical drugs and hormones and are subject to the same physiological principals.