Evaluation of 11C-colchicine for PET imaging of multiple drug resistance.

UNLABELLED: Overexpression of P-glycoprotein (P-gp) can confer multiple drug resistance (MDR) phenotype on cancer cells and tumors by reducing intracellular accumulation of various cytotoxic agents. Early diagnosis of MDR in the clinic will serve to improve the efficacy of chemotherapeutic intervention and the quality of life of patients. In this article we describe use of a positron-emitting MDR tracer, 11C-colchicine (CHC), to evaluate MDR by PET imaging. Unlike existing MDR tracers such as 99mTc-sestamibi, this compound is electroneutral, with biodistribution not affected by perturbations of membrane potential. METHODS: In vitro studies showed that resistance to CHC is correlated to resistance to Taxol (paclitaxel). The results of biodistribution experiments were found to be consistent with previously reported experiments with CHC labeled with other isotopes. On the basis of in vitro experiments with a series of drug-resistant variants of the human neuroblastoma BE (2)-C cell line, a mathematic model of 11C-CHC distribution in tumors was formulated. Dynamic PET 11C-CHC imaging experiments were performed with nude rats xenografted with the BE (2)-C-sensitive and -resistant strains. Each scan was accompanied by a transmissions scan and a static FDG scan. These scans allowed improved image localization. RESULTS: We observed an approximately 2-fold difference between 11C-CHC accumulation in sensitive and resistant tumors. Imaging data were analyzed using the mathematic model, and various parameters characterizing resistance could be identified and estimated. In particular, the parameter r, proportional to the level of resistance of the tumors, was obtained. We showed that the ratio of these r parameters determined from the sensitive and resistant tumors was identical to the ratio of CHC accumulation in the corresponding sensitive and resistant cell lines used for xenografting. CONCLUSION: These in vivo experiments provided additional evidence for the indirect effect of P-gp action on CHC-to-tubulin binding, which in turn determines CHC uptake in tumors. The significance of these findings and future plans is discussed.

Pharmacokinetics and dosimetry of an alpha-particle emitter labeled antibody: 213Bi-HuM195 (anti-CD33) in patients with leukemia.

UNLABELLED: Data from nine patients with leukemia participating in a phase I activity-escalation study of HuM195, labeled with the alpha-particle emitter 213Bi (half-life = 45.6 min), were used to estimate pharmacokinetics and dosimetry. This is the first trial using an alpha-particle emitter in humans. The linear energy transfer of alpha particles is several hundredfold greater than that of beta emissions. The range in tissue is approximately 60-90 microm. METHODS: The activity administered to patients ranged from 0.6 to 1.6 GBq. Patient imaging was initiated at the start of each injection. Thirty 1-min images followed by ten 3-min images were collected in dynamic mode; a 20% photopeak window centered at 440 keV was used. Blood samples were collected until 3 h postinjection and counted in a gamma counter. Contours around the liver and spleen were drawn on the anterior and posterior views and around a portion of the spine on the posterior views. No other organs were visualized. RESULTS: The percentage injected dose in the liver and spleen volumes increased rapidly over the first 10-15 min to a constant value for the remaining hour of imaging, yielding a very rapid uptake followed by a plateau in the antibody uptake curves. The kinetic curves were integrated to yield cumulated activity. The mean energy emitted per nuclear transition for 213Bi and its daughters, adjusted by a relative biologic effectiveness of 5 for alpha emissions, was multiplied by the cumulated activity to yield the absorbed dose equivalent. Photon dose to the total body was determined by calculating a photon-absorbed fraction. The absorbed dose equivalent to liver and spleen volumes ranged from 2.4 to 11.2 and 2.9 to 21.9 Sv, respectively. Marrow (or leukemia) mean dose ranged from 6.6 to 12.2 Sv. The total-body dose (photons only) ranged from 2.2 x 10(-4) to 5.8 x 10(-4) Gy. CONCLUSION: This study shows that patient imaging of 213Bi, an alpha-particle emitter, labeled to HuM195 is possible and may be used to derive pharmacokinetics and dosimetry. The absorbed dose ratio between marrow, liver and spleen volumes and the whole body for 213Bi-HuM195 is 1000-fold greater than that commonly observed with beta-emitting radionuclides used for radioimmunotherapy.

MR of CNS sarcoidosis: correlation of imaging features to clinical symptoms and response to treatment.

BACKGROUND AND PURPOSE: Sarcoidosis is an idiopathic systemic granulomatous disease, recognized in a patient when clinical and radiologic findings are confirmed by histopathologic analysis. The objective was to identify a relationship between MR imaging and clinical findings in CNS sarcoidosis. METHODS: The clinical charts of 461 patients with biopsy-proved sarcoidosis were reviewed retrospectively. Criteria for including patients in the study included those with symptoms referable to the CNS, excluding those with another explanation for their symptoms, those with headaches or other subjective complaints without accompanying objective findings, and those with peripheral neuropathy other than cranial nerve involvement or myopathy without CNS manifestations. Thirty-four of 38 patients whose conditions met the criteria for CNS sarcoidosis underwent a total of 82 MR examinations. The positive imaging findings were divided into categories as follows: pachymeningeal, leptomeningeal, nonenhancing brain parenchymal, enhancing brain parenchymal, cranial nerve, and spinal cord and nerve root involvement. Treatment response, clinical symptomatology, and any available histopathologic studies were analyzed with respect to imaging manifestations in each of the categories. RESULTS: Eighty-two percent of the patients with sarcoidosis with neurologic symptoms referable to the CNS had findings revealed by MR imaging. However, eight (40%) of 20 cranial nerve deficits seen at clinical examination of 13 patients were not seen at contrast-enhanced MR imaging, and 50% of the patients with symptoms referable to the pituitary axis had no abnormal findings on routine contrast-enhanced MR images. In contradistinction, 44% of 18 cranial nerves in nine patients with MR evidence of involvement had no symptoms referable to the involved cranial nerve. Clinical and radiologic deterioration occurred more commonly with leptomeningeal and enhancing brain parenchymal lesions. CONCLUSION: MR imaging can be used to confirm clinical suspicion and to show subclinical disease and the response of pathologic lesions to treatment.

Measurement of P-glycoprotein expression in human neuroblastoma xenografts using in vitro quantitative autoradiography.

P-glycoprotein (P-gp) has a role in multidrug resistance (MDR) encountered in human cancers. In this study, we used the colchicine-resistant cell line BE(2)-C/CHCb(0.2), a strain of neuroblastoma cell line BE(2)-C, as a model to measure variations of P-gp expression in cells grown in vitro and in vivo. Cells were cultured in the medium supplemented with colchicine. At the beginning of the study the drug was withdrawn and, after 22 days, added back to the culture medium. Cells were harvested at various time points and xenografted in nude mice. P-gp content in cells was measured by self-competitive binding assay and in tumors, by quantitative autoradiography (QAR). Both assays were carried out using 125I-labeled monoclonal antibody MRK16, reactive with P-gp. Concentration of P-gp in cells varied from a maximum of 1,361 pmol/g in the presence of colchicine to a minimum of 374 pmol/g in the absence of colchicine in the culture medium. P-gp concentration in the tumors ranged from 929 to 188 pmol/g, which correlated with P-gp content in the cells at the time of their injection in the mice. QAR is an accurate and reliable method to quantify P-gp expression in tumors. Changes in colchicine concentration in the ambient medium of BE(2)-C/CHCb(0.2) cells growing in vitro resulted in a change in phenotype of P-gp expression, which was stable under conditions of in vivo growth over approximately 9 cell divisions in nude mice xenografts. Therefore, P-gp content in xenografts depends only on the level of resistance of the cells at the time of their injection in the mice.

Measurement of P-glycoprotein expression in multidrug-resistant human neuroblastoma cell lines using self-competitive binding assay.

To date, all reported measurements of multidrug resistance have been semiquantitative. The purpose of the present study is to establish and validate the self-competitive binding assay technique utilizing monoclonal antibody for quantitative estimation of multidrug resistance in tumor cells. This technique is used for P-glycoprotein concentration measurement in BE(2)-C human neuroblastoma cell line and its sublines with primary resistance to colchicine and actinomycin D. Monoclonal antibody MRK-16 was used in this study. It was labeled with iodine-125 (125I) to trace the concentration of antibody-antigen complexes. The binding data were obtained by varying the concentration of the unlabeled antibody. The results were fitted to a model equation to estimate the number of binding sites and antibody-antigen dissociation constant. The P-glycoprotein concentration was significantly higher in the resistant sublines than in the sensitive line. The highest levels were achieved in actinomycin D-resistant cells: 2.1 x 10(6) binding sites/cell versus 5.4 x 10(4) binding sites/cell in the sensitive cells. The consistency of the results was verified by repeating the study three times for each cell line. The binding assay results were confirmed by Western blot experiments performed on the same cell lines.

Evaluation of carbon-14-colchicine biodistribution with whole-body quantitative autoradiography in colchicine-sensitive and -resistant xenografts.

UNLABELLED: Quantitative autoradiography (QAR) with radiolabeled monoclonal antibodies in xenografted animals has been extensively described in the past, either on individual tissues or on the whole body. We applied whole-body QAR to identify multidrug resistant tumors using 14C-colchicine (14C-CHC). METHODS: Two groups of five animals each were xenografted with CHC-sensitive and CHC-resistant human neuroblastoma cells. Animals were injected intravenously with 4 microCi/0.11 mumole 14C-CHC per gram of body weight and sacrificed after 60 min. Whole-body QAR was carried out using 25-microns thick sections. RESULTS: Fusion images allowed direct comparison of 14C-CHC uptake in tumor and nontumor tissues. Mean 14C-CHC distribution in sensitive and resistant tumors was 882.0 +/- 43.6 and 399.6 +/- 157.7 nCi/g corresponding to 24.5 +/- 1.21 and 11.1 +/- 4.38 nmole/g, respectively (p < 0.001), with normal tissue distribution in both groups being similar. Three-dimensional QAR showed that the uptake of 14C-CHC was in the cellular zones of the tumor. This method has potential in biodistribution studies of novel radiopharmaceuticals such as 14C-CHC. CONCLUSION: These studies further suggest that PET imaging of 11C-CHC is feasible to distinguish between sensitive and resistant tumor deposits in vivo.

In vivo imaging and specific targeting of P-glycoprotein expression in multidrug resistant nude mice xenografts with [125I]MRK-16 monoclonal antibody.

Multidrug resistance (MDR) in tumors is associated with P-glycoprotein (Pgp) expression. In vivo quantitation of Pgp may allow MDR to be evaluated noninvasively prior to treatment planning. The purpose of this study was to radiolabel MRK-16, a monoclonal antibody that targets an external epitope of P-glycoprotein, and perform in vivo quantitation of P-glycoprotein in a MDR xenograft nude mouse model. MRK-16 was labeled with 125I by the iodogen method, with subsequent purification by size exclusion chromatography. Groups of 10 Balb c mice were each xenografted with colchicine-resistant or sensitive neuroblastoma cell lines, respectively. Whole body clearance and tumor uptake over time was quantitated by gamma camera imaging, and biodistribution studies were performed with [125I]MRK-16 and an isotype matched control antibody, A33. Quantitative autoradiography and immunohistochemistry analysis of tumors was also evaluated to confirm specific targetting of [125I]MRK-16. Peak tumor uptake was at 2-3 days post-injection, and was significantly greater in resistance compared to sensitive tumors (mean % injected dose/g +/- SD) (18.76 +/- 2.94 vs 10.93 +/- 0.96; p < 0.05). Quantitative autoradiography verified these findings (19.13 +/- 0.622 vs 12.08 +/- 0.38, p < 0.05). Specific binding of [125I]MRK-16 was confirmed by comparison to [131I]A33 in biodistribution studies, and localized to cellular components of tissue stroma by comparison of histologic and autoradiographic sections of sensitive and resistant tumors. Immunoblot analysis demonstrated a 4.5-fold difference in P-glycoprotein expression between sensitive and resistant cell lines without colchicine selective pressure. We conclude that in vivo quantitation of P-glycoprotein in MDR tumors can be performed with [125I]MRK-16.(ABSTRACT TRUNCATED AT 250 WORDS)

Calculated and TLD-based absorbed dose estimates for I-131-labeled 3F8 monoclonal antibody in a human neuroblastoma xenograft nude mouse model.

Preclinical evaluation of the therapeutic potential of radiolabeled antibodies is commonly performed in a xenografted nude mouse model. To assess therapeutic efficacy it is important to estimate the absorbed dose to the tumor and normal tissues of the nude mouse. The current study was designed to accurately measure radiation does to human neuroblastoma xenografts and normal organs in nude mice treated with I-131-labeled 3F8 monoclonal antibody (MoAb) against disialoganglioside GD2 antigen. Absorbed dose estimates were obtained using two different approaches: (1) measurement with teflon-imbedded CaSO4:Dy mini-thermoluminescent dosimeters (TLDs) and (2) calculations using mouse S-factors. The calculated total dose to tumor one week after i.v. injection of the 50 microCi I-131-3F8 MoAb was 604 cGy. The corresponding decay corrected and not corrected TLD measurements were 109 +/- 9 and 48.7 +/- 3.4 cGy respectively. The calculated to TLD-derived dose ratios for tumor ranged from 6.1 at 24 h to 5.5 at 1 week. The light output fading rate was found to depend upon the tissue type within which the TLDs were implanted. The decay rate in tumor, muscle, subcutaneous tissue and in vitro, were 9.5, 5.0, 3.7 and 0.67% per day, respectively. We have demonstrated that the type of tissue in which the TLD was implanted strongly influenced the in vivo decay of light output. Even with decay correction, a significant discrepancy was observed between MIRD-based calculated and CaSO4:Dy mini-TLD measured absorbed doses. Batch dependence, pH of the tumor or other variables associated with TLDs which are not as yet well known may account for this discrepancy.

In vivo uptake of carbon-14-colchicine for identification of tumor multidrug resistance.

UNLABELLED: A major limitation in the treatment of cancer with natural product chemotherapeutic agents is the development of multidrug resistance (MDR). Multidrug resistance is attributed to enhanced expression of the multidrug resistance gene MDR1. Colchicine (CHC) is known to be one of the MDR drugs. We have previously demonstrated that it is possible to distinguish multidrug-resistant tumors from multidrug-sensitive tumors in vivo on the basis of tritium (3H) uptake following injection of 3H-CHC. METHODS: The present studies were carried out in xenografted animals using 14C-CHC which may be more indicative of 11C-labeled CHC distribution with regard to circulating metabolites, since metabolic processes following injection of (ring C, methoxy-11C)-CHC may produce significant amounts of circulating 1-carbon fragments (i.e., methanol and/or formaldehyde). Experiments were carried out at a dose of 2 mg/kg. RESULTS: Activity concentration per injected dose was approximately twice as great in sensitive as in resistant tumors (p < 0.05) at 60 min following intravenous injection of 14C-CHS. About 75% of total activity was CHC in the sensitive tumors. The findings are further confirmed by the quantitative autoradiographic evaluation of resistant and sensitive tumors. CONCLUSIONS: These studies confirm our previous observations that it is possible to noninvasively distinguish multidrug-resistant tumors from sensitive tumors in vivo based on uptake of an injected MDR drug using a 14C-labeled CHC at the same position and of comparable specific activity to a 11C-CHC tracer used for PET imaging.

BCNU-resistant human glioma cells with over-representation of chromosomes 7 and 22 demonstrate increased copy number and expression of platelet-derived growth factor genes.

We used standard karyotypic analyses of first-division cells to identify a subpopulation of cells in primary malignant gliomas with over-representation of chromosomes 7 and 22. These cells are a minor subpopulation in the primary tumor but become the dominant population after treatment in vitro of the cells with the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The selection for a cell with this specific karyotypic abnormality suggests that these chromosomes contain genes important to the growth of BCNU-resistant cells. Southern blot hybridization analyses demonstrate an increased copy number of the genes encoding platelet-derived growth factor (PDGF) A-chain and B-chain, which have been mapped to chromosomes 7 and 22, respectively. Reverse transcription followed by polymerase chain reaction (RT-PCR) analysis demonstrates increased expression of these genes. In addition, these cells secrete a mitogenic factor that stimulates 3H-thymidine uptake in NIH 3T3 cells. This factor is sensitive to anti-PDGF antibodies and beta-mercaptoethanol, but not to anti-EGF antibodies. These data suggest that autocrine and/or paracrine mechanisms occur in human malignant gliomas, and that over-expression of PDGF may play a role in the growth of BCNU-resistant cells in these tumors.

In-vivo identification of tumor multidrug resistance with 3H-colchicine [corrected].

Multidrug resistance (MDR) is a major obstacle in the clinical treatment of cancer with natural-product anticancer agents. Identification of MDR in vivo could be important in the design of chemotherapeutic regimens. As a first step in developing radiolabeled drugs to detect MDR, we measured the in vivo distribution of radiolabel from [ring C, methoxy-3H]-colchicine ([3H]-CHC) in immunosuppressed mice bearing xenografts of colchicine-resistant and sensitive tumor cell lines. Experiments were done at trace (1 microgram/kg) and LD50 (4 mg/kg) dose levels. Activity concentration/injected dose was more than twice as great in sensitive as in resistant tumors (p less than 0.01) at 60 min following retroorbital injection of [3H]-CHC. There was no significant difference in activity distribution between trace- and high-dose injections for any of the tissues sampled. Chromatographic analysis of plasma and tumor extracts demonstrated extensive extravascular metabolic degradation of [3H]-CHC. The ratio of [3H]-CHC concentration of injected dose between sensitive and resistant tumors was 3:1 (p less than 0.05), due primarily to protein-bound [3H]-CHC. This preliminary study demonstrates that it is possible to distinguish multidrug resistant from sensitive tumors in vivo on the basis of radiolabel uptake from an injected MDR drug. Colchicine, labeled with 11C at the [ring C]-methoxy group, may be useful as a radiopharmaceutical for quantitative identification of MDR in human tumors using PET.

Experimental changes in pain threshold and severe pain threshold for electrically induced pain.

Pain and severe pain thresholds were measured in groups of 15 (Experiment 1) and 20 (Experiment 2) normal volunteers. In Experiment 1, the subjects underwent a series of ten constant level, painful stimuli each of 10 sec duration at an arbitrarily chosen level between the thresholds. This level was recorded. It ranged from 14% to 81% of the difference between thresholds. The thresholds were then remeasured. For the second experiment the experimental stimulus commenced at a non-painful level and increased over a period of 5 sec to a level midway between thresholds for a series of three stimuli and again thresholds were remeasured. An overall effect was demonstrable only in Experiment 2, where the severe pain threshold was significantly reduced (p less than 0.001). There was a marked individual variation with increases and decreases in both thresholds occurring in different individuals at significance levels varying from p less than 0.05 to p less than 0.001. No subject changed the two thresholds in opposite directions. Examination of the responses in Experiment 1 suggested that for any increase in either threshold to occur it was necessary that the repeated painful stimulus should be at a level below the mid-point between the two thresholds, but that this was not a sufficient condition.

Reversibility of elemental liquid diet-enhanced methotrexate toxicity by refeeding with chow.

The toxic effects of methotrexate administration [20 mg/kg, bolus intraperitoneally (ip)] to rats fed a regular chow diet (n = 10) was compared with results in animals fed an elemental, chemically defined, liquid diet (n = 10) for 7 days. All animals receiving an elemental diet became anorectic and lethargic within 60 hr of methotrexate injection. All animals in this group subsequently developed enteritis and died within 150 hr. There was no clinical evidence of enteritis in rats fed a regular chow diet and mortality as zero in this group (p less than 0.001). In a second study one group of rats (n = 9) was fed a regular chow diet for 7 days; four groups were fed an elemental, chemically defined, liquid diet (n = 9 per group) for 7 days. At 24 hr and 8 hr prior to, or 24 hr after methotrexate administration, one group was refed a regular chow diet; the fourth group was maintained on an elemental liquid diet throughout the study period. All rats fed a regular chow diet survived following methotrexate injection (20 mg/kg, ip). All rats fed an elemental diet throughout the study period died. Those rats refed a regular diet 24 or 8 hr prior to methotrexate injection demonstrated a significant improvement in survival (100% in the 24-hr group, 55% in the 8-hr group). However, those animals refed a regular diet 24 hr after methotrexate injection demonstrated a 100% mortality.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of methotrexate metabolism in rats by administration of an elemental liquid diet. I. Changes in drug enterohepatic circulation.

Severe enteritis and death consistently occur within 150 hours of a single intraperitoneal dose of 25 mg/kg methotrexate given to rats fed an elemental diet. Rats fed a regular chow diet show no clinical evidence of gastrointestinal morbidity or mortality at this dose. The etiology of this enhanced toxicity is not clear, however. It this study rats were randomized to receive an elemental diet (n = 45) or a regular chow diet (n = 45) for 7 days. They were then given a bolus dose of 3H methotrexate (25 mg/kg) intraperitoneally. At 20 and 40 minutes and at 1, 2, 12, 24, 48, 72, and 96 hours after injection, five animals from each group were anesthetized, and mean methotrexate levels in bile and serum, and tissue methotrexate concentrations in proximal, mid, and distal small intestine and in liver were obtained. Mean methotrexate levels in bile were significantly elevated in rats fed an elemental, chemically defined diet at 12, 24, 48, and 72 hours compared with regular diet fed rats. Mean serum methotrexate levels were elevated in both the inferior vena cava (IVC) and portal vein (PV) at 12 (IVC), 24 (PV), and 48 hours (IVC) in elemental, liquid diet fed rats. There were no statistical differences between methotrexate levels in rats fed an elemental diet or a regular chow diet in proximal or mid small intestine at any of the time points measured. In the distal small intestine, methotrexate levels were significantly elevated at 24 hours in rats fed an elemental, chemically defined liquid diet (P less than 0.03) compared with regular diet fed rats. Methotrexate levels in liver of rats fed an elemental, chemically defined liquid diet were significantly elevated at 24 and 48 (P less than 0.05) hours. Administration of an elemental, chemically defined liquid diet prolongs the enterohepatic circulation of methotrexate in rats and delays clearance of the drug from the systemic circulation. Thus, prolonged exposure of methotrexate to the cells of the intestinal mucosa explains the increased gastrointestinal side effects of the drug in rats fed an elemental, chemically defined liquid diet. If these results are applicable to man, elemental, chemically defined liquid diets are contraindicated as the sole nutritional source in patients receiving methotrexate. Such qualitative alteration of dietary intake may be a factor in the unpredictability of gastrointestinal side effects associated with high dose methotrexate administration.

Systemic absorption of a leucovorin mouth wash: a pharmacologic study.

Mucositis is one of the major problems encountered after the administration of systemic chemotherapy. Leucovorin, routinely used as a rescue agent for methotrexate may reduce toxicity, but may also reduce the effectiveness of the chemotherapeutic agent. If leucovorin is administered as a mouth wash, local toxicity may be reduced without loss of methotrexate efficacy. In order to study this, 15 normal human volunteers were given leucovorin mouth wash and then had plasma determinations of 5-methyl-tetrahydrofolate and citrovorum factor. Small but statistically significant increases in plasma levels of 5-methyl-tetrahydrofolate were observed with no increase in levels of plasma citrovorum factor. It is concluded therefore that a small amount of leucovorin is absorbed systemically when administered as a mouth wash, but such an amount would most likely not be significant enough to reduce the effect of methotrexate therapy, but may reduce mucositis.

Comparison of gas chromatographic/mass spectrometric and microbiological assays for 5-fluorouracil in plasma.

The concentrations of 5-fluorouracil in 99 plasma samples were determined by both microbiological and gas chromatographic/mass spectrometric assays. The values determined by the two methods were similar (correlation coefficient = 0.90). A regression of the natural logarithms of the concentrations (0.01-94 micrograms ml-1) determined by the two methods gave a line whose slope and intercept were not statistically different (p greater than 0.05) from 1 and 0 respectively. Thus, the microbiological assay has specificity over a sufficient concentration range to serve as a practical routine laboratory method for the determination of 5-fluorouracil.

Mutagenic and chemotherapeutic activity in L1210 leukemia of several monofunctional alkylating agents.

The mutagenic and chemotherapeutic activities of the following monofunctional alkylating agents were compared in vivo: beta-chloroethylamine, dimethyl- and diethylaminoethyl chloride; methyl- and ethylmethanesulfonate; methyl- and ethylnitrosourea; and procarbazine. The bifunctional alkylating agent diethylamine 2,2'-dichloro-N-methyl-hydrochloride was used as reference. The alkylating activity was assessed by reacting with 4-(p-nitrobenzyl)pyridine, and antitumor activity was determined against L1210 in vitro and in vivo. The L1210 response, which is consistent with useful alkylating reactivity, was very marked with the two nitrosoureas and procarbazine. The nitrosoureas and, to some extent, procarbazine decreased the tumorigenicity of L1210 leukemia as evidenced by the increase in survival times with increasing numbers of treatment generations. After treatment for about five transfers (10(6) cells i.p.) with methylnitrosourea (40 mg/kg i.p. on Days 1, 3, and 5), the untreated control mice consistently survived free of tumor, whereas the treated mice died before Day 30. After treatment with ethylnitrosourea (80 mg/kg), the survival times also increased but more in the treated than in the corresponding control groups. Methylnitrosourea was most efficient in increasing the survival times and abolishing tumor transplantability. Antigenic change and loss of growth potential presumably were the reason for this increase in survival time, as indicated by tests in X-irradiated and nude mice. The fact that nitrosoureas and triazenes, besides reducing tumorigenicity, have similarities in their chemistry in that they decompose or are metabolically converted into diazohydroxides and then to carbonium ions is possibly of significance.

Serum and cerebrospinal fluid distribution of 5-methyltetrahydrofolate after intravenous calcium leucovorin and intra-Ommaya methotrexate administration in patients with meningeal carcinomatosis.

Serum and cerebrospinal fluid (CSF) concentrations of citrovorum factor (CF) and 5-methyltetrahydrofolic acid (5-MTHFA) were measured after i.v. infusion of leucovorin (50 or 100 mg/sq m) in seven patients undergoing treatment for meningeal carcinomatosis by intra-Ommaya reservoir injection of methotrexate (MTX). Serum CF levels rapidly rose after leucovorin administration as did 5-MTHFA, its conversion product. A small amount of CF entered the CSF, but peak CSF 5-MTHFA increased about 10-fold. The concentration X time (C X t) of additional 5-MTHFA in the CSF was greater [114.4 +/- 36.1 (S.E.) microgram/ml X min] after 100-mg/sq m doses of leucovorin than after 50 mg/sq m [14.2 +/- 4.3 micrograms/ml X min] (p less than 0.05). The CSF MTX concentration exceeded CSF 5-MTHFA by 2 to 3 logs throughout the 48 hr of observation, while serum 5-MTHFA and CF exceeded serum MTX by 0.5 to 2 logs. This study demonstrates that leucovorin administered i.v. to patients receiving intra-Ommaya MTX does not increase CSF concentrations of "rescue" folate above those of CSF MTX and are unlikely to interfere with MTX action against meningeal tumor. On the other hand, i.v. leucovorin does permit serum "rescue" folate to operate, thus reducing the systemic toxicity that may follow intraventricular administration of MTX.

Prognostic factors in the response of primary osteogenic sarcoma to preoperative chemotherapy (high-dose methotrexate with citrovorum factor).

Forty-three patients, ranging in age from 7 to 30 years (median age, 17 yr), with primary osteogenic sarcoma (OS), confirmed by biopsies and with no evidence of metastatic disease at the time of diagnosis, received T-7 chemotherapy for an average of 4 months before surgery, including high-dose methotrexate (HDMTX) and citrovorum factor rescue (CFR) (median, 7 courses), and 1 course each of bleomycin, cyclophosphamide, and dactinomycin, and adriamycin. At the time of definitive surgery, the surgical specimen showed a good histologic response to chemotherapy (grade III or IV response) in 29 (67%) of 43 patients and a poor histologic response (grade I or II response) in 14 (33%) of 43 patients. Among those who responded well, no patient relapsed, as all received a complete course of preoperative and postoperative chemotherapy for more than 5 to over 28 months after the initiation of treatment (medium, 13 mo). Among those who responded poorly, 6 of 14 patients relapsed with pulmonary metastases (a thoracotomy was beneficial to 1), 4 of 6 patients are alive with disease, and 1 patient died of progressive disease. On retrospective analysis, we observed that good and poor responders did not differ in the distribution of sex, age, race, primary site of disease, or histologic subtype of OS. An elevated alkaline phosphatase level that returned to normal under preoperative chemotherapy indicated a good response. Neither the 24-, 48-, and 72-hour serum MTX levels nor the fluid intake and urinary output during 3 days that followed HDMTX with CFR correlated significantly with tumor response. Based on our studies with this form of therapy, we concluded that the response of OS to preoperative chemotherapy is of prognostic value.