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Background: The generally poor prognosis and poor quality of life for lung cancer patients have highlighted the need for a conceptual model of integrative practice. Although the philosophy of integrative oncology is well described, conceptual models that could guide the implementation and scientific evaluation of integrative practice are lacking. Purpose: The present paper describes a conceptual model of integrative practice in which the philosophical underpinnings derive mainly from integrative oncology, with important contributions from Traditional Chinese Medicine (tcm) and the discipline of nursing. The conceptual model is described in terms of its purpose, values, concepts, dynamic components, scientific evidence, clinical approach, and theoretical underpinnings. The model argues that these components delineate the initial scope and orientation of integrative practice. They serve as the needed context for evaluating and interpreting the effectiveness of clinical interventions in enhancing patient outcomes in lung cancer at various phases of the illness. Furthermore, the development of relevant and effective integrative clinical interventions requires new research methods based on whole-systems research. An initial focus would be the identification of interrelationship patterns among variables that influence clinical interventions and their targeted patient outcomes.

There is ongoing debate about the criteria that are required for a new treatment to be approved for prescription and reimbursement and for clinicians to be convinced of that treatment’s true clinical utility [...]

Summary Improving the therapeutic index of anthracycline-based regimens in the management of metastatic breast cancer is a goal that physicians strive for in orderto improve quality of life and increase the small fraction of long-term disease-free survivors that have been observed in studies using anthracycline-based regimens. There are supporters and opponents of continuous and dose-intensive therapy for improving quality of life and providing a survival advantage, but the risk of cumulative toxicity associated with anthracycline-based regimens, especially, doxorubicin, is limiting in these approaches. Myocet™, has equivalent antitumour efficacy to doxorubicin, but significantly less cardiotoxicity. Consequently, the recommended cumulative lifetime dose of Myocet is 780 mg/m 2 , compared with 450 mg/m 2 for doxorubicin. By using Myocet in the metastatic setting, and perhaps eventually in the adjuvant treatment for primary breast cancer, an increased number of cycles can be givenwithin the cardiotoxicity risk threshold, improving quality of life by delaying relapse. This improvement in therapeutic index may contribute to the fraction of long-term disease-free survivors and fulfils a previously unmet need in the overall management of breast cancer.

Purpose. To describe the pharmacokinetics of R- andS-Ifosfamide (IFF), and their respective 2 and 3 N-dechloroethylated (DCE)metabolites (R2-, R3-, S2, S3-DCE-IFF) in cancer patients.

PURPOSE To compare the efficacy and toxicity profiles of a combination of fluorouracil (5-FU) with recombinant human interferon alfa-2a (Roferon-A; Hoffman La-Roche AG, Basel, Switzerland) versus the combination of 5-FU with leucovorin (LV) in the treatment of advanced colorectal cancer. PATIENTS AND METHODS A total of 496 previously untreated colorectal cancer patients were randomized to receive either Roferon-A (9 MIU) subcutaneously three times per week, with 5-FU (750 mg/m2/d) by continuous intravenous (i.v.) infusion (CIV) on days 1 to 5, then, after a 9-day hiatus, as a weekly i.v. bolus at the same dose (IFN/5-FU); or LV (200 mg/m2/d) by i.v. infusion plus 5-FU (370 mg/m2/d) by i.v. bolus on days 1 to 5, repeated every 4 weeks (LV/5-FU). RESULTS There were no significant differences between IFN/5-FU and LV/5-FU in the overall response rate (21% v 18%), duration of response (7.3 v 6.2 months), or survival time (median, 11.0 v 11.3 months). Toxicity profiles differed; constitutional symptoms and myelosuppression were more frequent and more severe with IFN/5-FU, and gastrointestinal symptoms with LV/5-FU. More patients interrupted treatment for adverse events (AEs) with IFN/5-FU than with LV/5-FU. Five treatment-related deaths occurred with each regimen. CONCLUSION The combination IFN/5-FU produced response rates, response durations, and survival times similar to those with LV/5-FU. Biochemical modulation of 5-FU by either IFN or LV appears to result in equivalent efficacy; however, fewer patients were able to tolerate the specified IFN/5-FU combination used in this study.

Summary A number of 1-aryl-4-methyl-1-penten-3-ones 1 were converted to the corresponding Mannich bases 2 and analogues 3 . Attempts to form the azines 4 from several members in series 1 led to the isolation of the corresponding pyrazolines 5 or aryl aldehyde azines 6 . Replacement of the isopropyl group of a compound in series 1 by methyl and ethyl functions led to ketones that reacted with hydrazine producing the corresponding azines. The Mannich bases displayed greater activity than the precursor ketones towards murine P388 and L1210 leukemia cells as well as to a panel of human tumour cell lines. Certain of the Mannich bases had selective toxicity towards some human tumour cell lines and others to L1210 cells (in contrast to human T lymphocytes). Several drug-resistant cell lines were shown to be free from cross resistance to a number of the Mannich bases.

En premiere partie, nous presentons brievement les facteurs gui influencent la radioreponse intrinseque, incluant les modificateurs de radiosensibilite gui interviennent par competition avec des radicaux libres, la reparation chimique et enzymatique de l'ADN, la conformation de la chromatine, la modulation du cycle cellulaire et le processus de transduction de signaux. En deuxieme partie, nous decrivons un groupe de lignees de cellules radioresistantes gui sont egalement resistantes aux agents alcoylants. La radioresistance est associee α la reduction de la valeur du coefficient α dans le modele lineaire-quadratique. Ces lignees de cellules sont sensibilisees aux radiations par depletion de GSH en conditions aerees avec augmentation de la valeur du coefficient α

Troxacitabine (Troxatyl) is a novel L-enantiomer nucleoside analog with activity in pancreatic cancer xenograft models.Troxacitabine 1.5 mg/m(2) was administered by 30-min infusions daily x5 every 4 weeks to 54 patients with advanced pancreatic cancer. Patients were evaluated for objective tumor response, time to tumor progression (TTP), changes in tumor marker CA 19-9, survival, safety, pain, analgesic consumption, Karnofsky performance status and weight change.Median TTP was 3.5 months (95% CI 2.0-3.8), median survival 5.6 months (95% CI 4.9-7.4), and the 1 year survival rate 19%. Best responses were stable disease in 24 patients with eight patients having stable disease for at least 6 months (15%). A 50% or greater decrease in CA 19-9 was seen in seven of 44 assessed patients (16%). Grade 3 and 4 neutropenia were observed in 37% and 30% of patients with one episode of febrile neutropenia. The most common drug-related non-hematological toxic effects reported were cutaneous, with 22% and 6% of patients reporting grade 2 and 3 skin rash, respectively and 4% grade 2 hand-foot syndrome.Troxacitabine administered by a bolus daily x5 monthly regimen has modest activity in advanced pancreatic adenocarcinoma.

Gap junctional intercellular communication (GJIC) is a form of cell-cell communication mediating the exchange of small molecules between neighboring cells. Gap junctions (GJs) are formed by connexins (Cxs), and are subject to tight and dynamic regulation. They are involved in the cell cycle, differentiation, and cell signaling. The loss of Cxs and GJs is a hallmark of carcinogenesis, while their induction in cancer cells leads to a reversal of the cancer phenotype, induction of differentiation, and regulation of cell growth. On the basis of the observations about Cx loss in breast cancer, this review examines Cxs' involvement in breast cancer metastasis. Previous work indicates that Cx expression is inversely correlated to metastatic potential. This is probably because of the loss of cooperation between neighboring cells, leading to cell heterogeneity and cell dissociation in the tumor. The possible involvement of Cx activity during metastasis will be discussed.

By flexibly docking 9593 compounds of the NCI-3D database against the refined structure of beta-tubulin using DOCK 4.0, a long-forgotten synthetic steroid derivative, NSC12983, has been identified as a microtubule-stabilizing agent. The 32 top scorers includes NSC12983 and the three added references: paclitaxel, docetaxel and IDN5109. That is, 12.5% of the 0.33% top scorers are active. In addition, NSC12983 is active on Mycobacterium tuberculosis in vitro and in vivo, which might be due to its ability to promote the assembly of essential cell division protein.

A homology model for human Bcl-2 was built. Paclitaxel and baccatin III were individually docked into the Bcl-2 loop domain. The conformational space of the loop region on ligand binding was explored by molecular dynamics. The ligand-Bcl-2 interaction energy was calculated by minimization in the binding site. It was found that both paclitaxel's core skeleton and its C-13 side chain contribute significantly to its interaction with Bcl-2. A portion of the Bcl-2 loop domain was found to be locked by the bound paclitaxel and baccatin III in a similar conformation.

Cultured rat liver epithelial cells (RLE) transformed with repeated treatments of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) demonstrate many features of the common biochemical phenotype of multidrug resistance (MDR) seen in vivo in 'resistant hepatocytes'. The cells have increased glutathione-S-transferase placental subunit (GST-Yp), gamma-glutamyltranspeptidase (GGT), glutathione (GSH) and glutathione peroxidase and are resistant to MNNG. Phenotypically identical RLE cells spontaneously transformed by selective culture conditions showed low levels of GGT and GST and were not resistant to MNNG. Both chemical and spontaneous transformants are cross resistant to doxorubicin although resistance is consistently greater in chemical transformants. No direct correlation was found between the degree of resistance to doxorubicin and MDR gene expression in either of the chemically or spontaneously transformed RLE cells. These observations suggest that in chemical carcinogenesis, other mechanisms of drug detoxification are involved and that MDR expression is not a consistent feature.

To describe the pharmacokinetics of R- and S-Ifosfamide (IFF), and their respective 2 and 3 N-dechloroethylated (DCE) metabolites (R2-, R3-, S2, S3-DCE-IFF) in cancer patients.(R,S)-IFF was administered (1.5 g/m2) daily for 5 days in 13 cancer patients. Plasma and urine samples were collected and analyzed using an enantioselective GC-MS method. An average of 97 observations per patient were simultaneously fitted using a pharmacokinetic-metabolism (PK-MB) model. A population PK analysis was performed using an iterative 2-stage method (IT2S).Auto-induction of IFF metabolism was observed over the 5 day period. Increases were seen in IFF clearance (R: 4 vs. 7 L/h; S: 5 vs. 10 L/h), and in the formation of DCE (R: 7 vs. 9%; S: 14 vs. 19%) and active metabolites (4-OHM-IFF; R: 71 vs. 77%; S: 67 vs. 71%). A novel finding of this analysis was that the renal excretion of the DCE metabolites was also induced.This population PK-MB model for (R,S)-IFF may be useful in the optimization of patient care, and gives new insight into the metabolism of (R,S)-IFF.

Gap junctions are intercellular channels that are formed from members of a family of proteins, the connexins (Cxs). Gap junctions play an important role in vital functions, including the regulation of cell growth and cell differentiation. Here, we examined the expression of Cx43, a major Cx in breast tissue, in 32 surgical specimens obtained from breast cancer patients who underwent a primary surgical resection prior to chemotherapy or radiotherapy treatments. The expression of Cx43 gap junctions was compared to the levels of estrogen, progesterone, and erbB2 tyrosine kinase receptors. In addition, a panel of breast cancer cell lines and a series of normal rat mammary tissues and rat mammary tumors induced in vivo by dimethylbenz(a)anthracene were studied. We demonstrated that the lack of Cx43 gap junctions is a common feature of human mammary cancer tissues compared to nonneoplastic breast tissues surrounding primary tumors. Cx43 gap junctions were not observed in ductal carcinomas in situ, infiltrating ductal carcinomas, and infiltrating lobular carcinomas, and they seem to be independent of estrogen, progesterone, and erbB2 receptor status. In breast cancer cell lines and rodent mammary carcinoma tissues, down-regulation of Cx43 occurs at the mRNA level, suggesting a transcriptional mechanism for the decrease of Cx43 protein in breast cancer. In summary, this study provides evidence of decreased expression of Cx43 gap junctions in breast cancer at various stages of progression as well as breast cancer cell lines and raises the possibility that Cx43 may be a useful marker for detecting early oncogenesis in the breast. Because Cx43 gap junctions are lacking in breast cancer and restoration of Cx43 has been shown to reverse the malignant phenotype in vitro, pharmacological up-regulation of Cx43 may prove beneficial in cancer therapeutics.

The phenomenon of the "bystander effect" (BE) observed in suicide gene therapy studies leads to the intriguing possibility that cytotoxicity can be achieved even in tumor cells that have not themselves been targeted with novel genetic material. There is considerable data suggesting the role of gap junction-mediated intercellular communication (GJIC) in the BE. Transfer of connexin (Cx)-encoding genes, the building blocks of GJIC, has been shown both in vitro and in vivo to increase the BE. Since the loss of GJIC is a common feature of cancer cells, we examined the consequence of GJIC up-regulation on the BE in suicide gene therapy. We used 8-bromo-cyclic-AMP to induce Cx43 and GJIC. In mixing assays, using various proportions of cells containing viral thymidine kinase delivered by an adenoviral delivery system or stably transduced by a retrovirus vector, 8-bromo-cyclic-AMP enhanced the BE of cell killing using ganciclovir. The induction in cell killing was more significant when a low percentage of the cell population was infected, which is the relevant clinical situation. We have demonstrated that this is not due to an effect on infectivity or suicide gene expression. Since decreased GJIC is part of the transformed phenotype, induction of Cxs provides an element of selectivity to suicide gene therapy. Our study adds strength to the rationale to develop clinically tolerable GJ inducers to potentiate the effect of suicide gene therapy via the BE.

Notwithstanding ongoing progress in anticancer therapeutics development, the persistent problem remains to selectively target tumors while sparing normal tissues. This is confounding largely because the differences between normal and tumor cells are often subtle and part of a gradient, where a gene product may be more or less expressed in tumor compared with the host normal tissue, but seldom expressed (or turned off) in tumors. The role of glutathione (GSH) and related enzymes in cellular resistance to xenobiotics, including chemotherapy is well established. This study is among those attempting to modulate GSH to therapeutic advantage. The authors briefly describe the experience with the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine, and then in greater detail outline recent evidence for a potentially more selective approach using the cysteine prodrug L-2-oxothiazolidine-4-carboxylate. This has led to a detailed study of the activating enzyme 5-oxo-L-prolinase, including enzymatic and immunocharacterization, as well as in vitro study of the effect of its modulators on anticancer drug toxicity. Using high affinity antibodies the authors have generated interesting information on the distribution of this enzyme in tumor versus normal human tissues. Finally, the authors have been studying the potential for modulating gap junctions as a part of anti-cancer therapeutics, since they transport GSH between cells and are generally deficient in tumor cells. Preliminary studies suggest that gap junction induction may dramatically deplete GSH concentration in tumor cells and sensitize them to a variety of treatments.

5-Oxo-L-prolinase (5-OPase) is an enzyme of the gamma-glutamyl cycle involved in the synthesis and metabolism of glutathione (GSH), which is known to protect cells from the cytotoxic effects of chemotherapy and radiation. Previous studies on rats have shown that administration of the cysteine prodrug L-2-oxothiazolidine-4-carboxylate, a 5-oxo-L-proline analogue that is metabolized by 5-OPase, preferentially increases the GSH content of normal tissues while paradoxically decreasing it in the tumor and results in an enhanced in vivo tumor response to the anticancer drug melphalan. These observations initiated the present study of 5-OPase in experimental models and clinical specimens to investigate the potential role of this enzyme in the selective modulation of GSH in normal and tumor tissues. First, 5-OPase activity was measured in tissues of tumor-bearing rats, in the peripheral mononuclear cells of normal human subjects, and in surgically resected tumor and the adjacent normal tissues from patients. We found that the activity of 5-OPase in human kidney, liver, and lung is significantly lower than that found in rats. Second, we have raised a polyclonal IgG anti-5-OPase antibody by immunizing rabbits with purified 5-OPase from rat kidney. This antibody has very high affinity (shown by immunoprecipitation) and specificity (shown by Western blot) and cross-reacts with human 5-OPase (shown by Western blot and immunohistochemistry). It was then used to examine the distribution of 5-OPase in paired normal and neoplastic human specimens using Western blot and immunohistochemistry. Examination of paired normal and neoplastic tissues of stomach and lung revealed a significantly lower level of 5-OPase in tumor tissues than in the paired normal tissues. In colon tissues, there is no significant difference in 5-OPase level between the normal and tumor tissues. These findings could have implications for both carcinogenesis and therapy.

Glutathione transferase (GSTs) have been shown to be overexpressed in a number of tumor cell lines selected for resistance to chemotherapeutic drugs and have been implicated in some studies of clinical specimens. In tumor cell lines selected for resistance to chemicals that alkylate DNA, the isoform most frequently overexpressed is GST-Yc, a member of the alpha class GSTs. To date, two variations of the cDNA designated Yc1 with subtle differences have been described, and Yc2 is shown to be clearly distinct. Transfection of a Yc1 cDNA constitutively expressed in rat liver into rat mammary cancer cells confers resistance to alkylators, however, to a lesser extent than is observed in the cells selected for resistance. It has therefore been widely suggested that the GST that is overexpressed in selected resistant cells represents a distinct and novel isoform. We have previously described a rat mammary carcinoma cell line (MLNr) that is resistant to alkylating agents, and overexpresses a GST with characteristics similar to GST-Yc1 and not Yc2. It has many features common to the several other GST-Yc overexpressing alkylator resistant cell lines. We have cloned the specific Yc cDNA overexpressed in MLNr and analyzed it in detail and found that it is identical to one of the previously reported Yc1 cDNAs, suggesting that there is no additional Yc gene specifically induced by nitrogen mustards. Another hypothesis to explain the difference in the level of resistance in selected versus GST-Yc transfected cells is the lack of concurrent increased glutathione (GSH) in the transfectants, which is a common feature in the selected resistant cells. Experiments in which we modulated GSH levels suggest that this is not likely. These studies add to our speculation that other mechanisms may be involved in alkylator resistance.

Glutathione S-transferase (GST) represents a multifunctional enzyme family consisting of four known cytosolic isoforms (alpha, mu, pi, and Phi) that detoxify a variety of xenobiotic chemicals and may confer resistance to both chemotherapeutic drugs and carcinogens in various experimental models. GST-pi has already been extensively studied in clinical specimens, including breast cancer. We studied the immuno-histochemical distribution and relative immunopositivity of GST-alpha and GST-mu, based on a grading system for immunointensity, in samples of 51 neoplastic and 46 normal breast samples and 12 lymph node metastases from patients treated with intensive chemotherapy and bone marrow transplant. In normal breast tissue, GST-alpha localized predominantly to the cytoplasm of scattered cells lining the luminal aspects of the ducts. Occasional cells showed both cytoplasmic and nuclear GST-alpha immunoreactivity. GST-mu was stained in myoepithelial cells preferentially as well as in occasional ductal cells (including apocrine epithelium), vascular smooth muscle, and plasma cells. GST-alpha and GST-mu were detected in 22 of 51 (43%) and 24 of 48 (50%) invasive cancers, respectively. In paired samples of normal and malignant tissue from the same patient, GST-alpha immunostaining in cancers was significantly less intense compared to that of normal breast tissue in 13 of 41 (32%) cases. No such trend was found for GST-mu in paired samples. Neither GST-alpha nor GST-mu immunopositivity in tumor or nonneoplastic breast was found to correlate with relapse-free or overall survival in this clinical context; however, the apparent decreased expression of GST-alpha in malignant versus normal breast epithelial cells could have important implications in breast carcinogenesis.

Given the potential for improved outcomes, a phase I trial was initiated to develop a paclitaxel/cisplatin regimen that could be delivered every two weeks to women with newly diagnosed advanced ovarian cancer.From 1992 to 1994, 29 (28 eligible) patients were enrolled in a dose-seeking trial. All received 60 mg/m2 of cisplatin preceded by paclitaxel infused over three hours. The paclitaxel dose was excalated from an initial level of 90 mg/m2 by 10 mg/m2 increments in successive cohorts of patients.At 120 mg/m2 of paclitaxel, the dose-limiting toxicity was granulocytopenia which prevented retreatment on time. The recommended dose level was therefore paclitaxel 110 mg/m2 infused over three hours with cisplatin 60 mg/m2, repeated bi-weekly for eight cycles.This bi-weekly schedule of paclitaxel/cisplatin provides no advantage in terms of dose-intensity nor total dose of paclitaxel in comparison to more common regimens given tri-weekly.

Acquired drug resistance is a major factor in the failure of doxorubicin-based chemotherapy in breast cancer. We determined the ability of megestrol acetate and/or tamoxifen to reverse doxorubicin drug resistance in a doxorubicin-resistant breast cancer line (the human MCF-7/ADR). The cytotoxicity of doxorubicin, megestrol acetate, and/or tamoxifen was determined in the sensitive and resistant cell lines utilizing the sulphorhodamine B assay. Tamoxifen alone produced an IC50 (concentration resulting in 50% inhibition of control growth) of 10.6 microM, whereas megestrol acetate alone resulted in an IC50 of 48.7 microM in the MCF-7/ADR cell line. The IC50 of doxorubicin in MCF-7/ADR was 1.9 microM. Neither megestrol acetate alone nor tamoxifen alone at 1 or 5 microM altered the IC50 of doxorubicin. However, the combination of tamoxifen (1 or 5 microM) and megestrol acetate (1 or 5 microM) synergistically sensitized MCF-7/ADR cells. Additionally, megestrol acetate and tamoxifen inhibited iodoarylazidoprazosin binding to P-glycoprotein, and, in their presence, there was an increased doxorubicin accumulation in the MCF-7/ADR cells. Furthermore, the combination of tamoxifen and megestrol acetate had much less effect on the cytotoxicity of doxorubicin in MCF-7 wild-type cells. Clinically achievable concentrations of tamoxifen and megestrol acetate can largely sensitize MCF-7/ADR to doxorubicin. The combination of these three drugs in a clinical trial may be informative.

AGG to AGT mutations in codon 249 of the p53 tumor-suppressor gene are frequently observed in hepatocellular carcinomas (HCC) from areas where exposure to aflatoxin B1 (AFB) occurs. We developed a sensitive allele-specific polymerase chain reaction (AS-PCR) assay to detect this point mutation in non-neoplastic human liver tissues. Three oligonucleotide primers, 1 specific for the mutant allele and 2 specific for the wild-type allele were used. The mutant allele primer differed from the wild-type allele due to a G-to-T transversion in its terminal 3' nucleotide. The first stage involved amplification of exon 7 of p53 followed by a selective amplification of mutant codon 249 sequences. This method allowed for the detection of a mutant codon 249 allele in the presence of as many as 105 copies of the wild-type allele and was 100-fold more sensitive than the restriction fragment length polymorphism-PCR technique. We have applied this AS-PCR protocol to examine codon 249 AGT transversion in tumor and matched non-tumor liver samples from North American patients with hepatitis and from Mozambiquan patients exposed to AFB. Mutations were detected in 5 of 6 samples of non-neoplastic liver from Mozambiquan patients, all of whom were HBsAg- or HBcAg-positive and AFB-exposed. In contrast, no mutations were detected in non-neoplastic liver from North American patients with either HBV- or HCV-derived hepatitis and cirrhosis. This procedure is a simple and powerful approach for screening p53 codon 249 AGT mutation in heterogeneous non-neoplastic hepatocyte populations.

Glutathione (GSH) is known to play a role in cellular sensitivity to some chemotherapeutic agents and to radiation. Depletion of cellular glutathione increases toxicity of these drugs, and this approach is being explored in the clinic as a form of biochemical modulation using the drug buthionine sulfoximine. The fact that some drug-resistant cell lines have increased GSH levels, and that enhancing glutathione concentrations in animal tissues protects against a variety of xenobiotic agents, suggests a different potential approach to improve anticancer therapy. We previously showed a selective enhancement by the cysteine "pro-drug," L-2-oxothiazolidine-4-carboxylate (OTZ), of GSH concentration in some normal tissues of tumor-bearing rats, whereas there is a paradoxic GSH depletion in tumor. OTZ has been shown to protect animals from a variety of toxins, and in vitro studies showed a selective increase in GSH in normal cells that results in reduced sensitivity to some chemotherapy drugs. This report describes evidence that OTZ provides this effect in an in vivo rat mammary tumor model. We have examined the OTZ "activating" enzyme, 5-oxoprolinase, in these tumors and found it to be 4-fold lower than that of normal rat liver. This may explain at least the lack of increased GSH in tumor in response to OTZ. A limited number of human breast cancer samples show similar activity.

An assay for the diastereoisomers of the biochemical modifier L-buthionine-(R,S)-sulfoximine (BSO) in human plasma has been developed using capillary electrophoresis (CE). Separation of the diastereoisomers is achieved by the micellar electrokinetic chromatography (MEKC) mode of CE. Plasma is injected directly onto the separation capillary without any extraction step, and BSO is detected directly by ultraviolet absorbance measurements at 190 nm without prior derivatization. The whole assay, including capillary conditioning, takes approximately 30 min. Intra- and inter-day R.S.D. values are approximately 7% at sample concentrations around 25 micrograms ml-1, and approximately 3% at sample concentrations around 500 micrograms ml-1. The limit of detection in plasma is 3.9 micrograms ml-1 (S/N = 2). The assay has been used to quantitate the diastereoisomers of BSO in patient samples in a pharmacokinetic study.

The objective of this study was to determine the pharmacokinetics of pamidronate disodium in plasma and urine after a single intravenous infusion of the drug to cancer patients at risk for developing bone metastases. Thirty-six patients were randomized into six treatment groups to receive 30-, 60- or 90-mg doses of the drug by 4- or 24-h intravenous infusions. Plasma and urine samples were collected at intervals for up to 144 h after drug administration and were assayed for pamidronate disodium using validated reversed-phase HPLC methods. The percentage of the administered dose excreted in urine following a 4- or 24-h infusion of 30-, 60- or 90-mg pamidronate disodium ranged from 30% to 60% except for one individual who excreted 96% by this route of elimination. There was a linear relationship between amount of drug excreted in urine and dose. Curve fitting of ARE (amount of drug to be excreted in urine) data indicated that the disposition kinetics of the drug was consistent with a biexponential process with overall mean plus minus S.D. half-life values of 2.1 plus minus 1.8 and 26.9 plus minus 8.7 h for the alpha and beta phases, respectively. The results of this study showed that the drug exhibited dose proportionality in its pharmacokinetic behavior over the 30--90-mg range regardless of whether it was infused over a 4- or 24-h interval.

Ifosfamide (IFF) is a nitrogen mustard with significant activity against a number of tumors. Since it is a chiral molecule, it has been suggested that enantioselective metabolism could result in different efficacy and toxicity profiles for (R)- and (S)-ifosfamide. Both experimental animal and clinical data suggest that N-dechloroethyl metabolites of (S)-IFF are more significantly associated with neurological toxicity, which may limit therapeutic use of IFF. We have used purified ifosfamide enantiomers to examine the pharmacokinetics; spectrum of toxicity including lethality, weight loss, and myelosuppression; and antitumor effects of the mixture compared to each of the purified enantiomers. In the MatB mammary carcinoma grown in female Fischer rats we demonstrated that the antitumor efficacy appears to be the same for (R)-IFF and (S)-IFF, while the (R)-IFF has greater myelotoxicity. Pharmacokinetic analysis of plasma concentration-time confirms that the (R)-IFF is metabolized to a greater extent than (S)-IFF via the activation pathway. These data suggest that purified (R)-IFF may be an effective way to delivery active cytotoxic drug while limiting the generation of neurotoxic metabolites.

A major limitation to successful cancer treatment is the existence of drug resistance. While several mechanisms of drug resistance have now been well characterized, mechanisms of resistance to alkylating drugs have remained less well defined. Several experimental models of alkylator resistance have implicated isoforms of glutathione S-transferase (GST) but transfection experiments using cloned isoforms of GST have yielded conflicting results. While there are several plausible explanations for these apparently contradictory findings, the issue that clonal variability might potentially confound the results of conventional transfection experiments has been raised. To address this issue properly, we have studied rat GST-Yc expression and drug sensitivity to alkylating drugs in populations of mouse NIH 3T3 fibroblasts following either transfection or transduction with an N2-based retrovirus vector. In comparison with cells treated with an antisense vector, Yc-transfected and Yc-transduced populations of NIH 3T3 cells expressed increased levels of GST-Yc mRNA (Northern blot), increased levels of immunodetectable GST-Yc (Western blot), and, respectively, 1.4- and 1.9-fold increases in total GST activity and 6.1- and 8.3-fold increases in glutathione peroxidase activity (associated with the Yc subunit). Yc-transfected and Yc-transduced cell populations were, respectively, 5.8- (P0.001) and 2.4-fold (P0.05) resistant to chlorambucil and 10.8- (P0.01) and 5.4-fold (P0.001) resistant to mechlorethamine. The range of resistance of clonal isolates from either population was 1.8-6.0-fold for chlorambucil and 4.6-6.1-fold for mechlorethamine (P0.05). In contrast, these cells showed unaltered sensitivity to the antimetabolite methotrexate, a nonalkylating drug. These results clearly demonstrate that the rat GTS-Yc is able to confer alkylating drug resistance in mouse fibroblasts. The ability to confer alkylating drug resistance following retrovirus-mediated gene transfer also raises the possibility of using GST-Yc somatic gene transfer to confer protection to the hematopoietic system in a gene therapy strategy applicable to cancer.

The role of glutathione-S-transferase (GST) in alkylator drug resistance has been studied in MatB rat mammary carcinoma cells. A series of GST transfectant cell lines was established by using an expression vector containing the complementary DNA for the rat GST Yc gene under regulation of the SV40 early region promoter and the antibiotic resistance plasmid pSV2neo. Transfectant cell lines expressing up to 4-fold higher total GST activity than in the parental wild type cell line were identified. Southern blot analysis confirmed a DNA fragment corresponding in size to the transfected GST Yc complementary DNA. Wild type MatB cells contain very low levels of Yc protein, whereas the Yc+ clones showed greatly increased amounts of the Yc subunit. The effect of increased GST Yc activity on the sensitivity of the transfected clones to various cytotoxic agents was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell survival assay. The clones expressing recombinant GST Yc were more resistant to melphalan (6- to 12-fold), mechlorethamine (10- to 16-fold), and chlorambucil (7- to 30-fold). In late passage populations of the GST Yc+ clones that had been grown over a period of 14 months under continuous selection in G418, GST activity was decreased and it was paralleled by a decrease in Yc protein. These late passage clones with diminished GST Yc content also demonstrate a partial reversion toward the wild type phenotype as determined by cytotoxicity assays using melphalan, mustargen, and chlorambucil. Interstrand DNA cross-links induced by mechlorethamine were significantly lower at 0, 2, and 20 h posttreatment in one of the GST Yc+ clones when compared to wild type MatB cells. These studies indicate that GST Yc overexpression can confer resistance to alkylating agents and that this correlates with inhibition of DNA cross-link formation.

The effectiveness of a calcium antagonist analog Ro11-2933 to modulate doxorubicin (DOX) response in DOX-sensitive (WT) and -resistant (DOXr, 200-fold) cell lines was investigated and compared to verapamil (VP) in vitro and in vivo in rats bearing mammary carcinoma using equivalent nontoxic doses. In vitro exposure to a nontoxic concentration of Ro11-2933 (2 microM) normalizes the DOX accumulation defect observed in DOXr cells, increases DOX-induced DNA single-strand breaks and effectively sensitizes DOXr cells to DOX. Ten microM VP was required to obtain an effect equivalent to that seen with 2 microM Ro11-2933. Intravenous administration of DOX at 5 mg/kg to the rat bearing the DOXr tumors has no significant therapeutic effect on tumor growth (P.5), whereas it was found effective in inhibiting the growth of WT tumors (P.05). Ro11-2933 or VP administered alone has no significant effect on tumor growth as compared to a saline-treated group (P.1). Combination of Ro11-2933 with DOX effectively inhibits DOXr tumor growth as compared to DOX alone. Combination of DOX with VP was found less effective than Ro11-2933 and the results were not statistically significant from DOX treatment alone (P.5). Our data demonstrate that Ro11-2933 is well tolerated after i.v. administration and an effective modulator of DOX resistance in a solid tumor model.

We have investigated the effect of a combined chemoimmunotherapy protocol with liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE), 5-fluorouracil (5-FU), and 5-formyltetrahydrofolate (leucovorin) on the growth of hepatic metastases using carcinoma H-59, a liver-homing subline of the Lewis lung carcinoma (P. Brodt, Cancer Res., 46: 2442-2448, 1986). C57BL/6 mice inoculated with the tumor cells via the intrasplenic route received three i.v. injections of liposomal MTP-PE, the first of which was administered 3 days prior to tumor cell inoculation. Chemotherapy with 5-FU and leucovorin at the maximal tolerated doses (30 mg/kg per injection) was initiated immediately after tumor inoculation and continued on alternate days for a total of 4 injections. The incidence of liver metastases in animals which received the combined therapy was compared to that in animals treated with chemotherapy or immunotherapy alone. We found that while the number of liver metastases was reduced in all of the treatment groups as compared to control untreated or placebo-treated animals, the combined effect of 5-FU leucovorin and liposomal MTP-PE was significantly better than that of chemotherapy or immunotherapy alone. This was reflected in a reduced incidence (70% as compared to 100% in all other groups) and in a significant reduction in the number and size of the liver nodules. Our results suggest that the efficacy of 5-FU and leucovorin in the treatment of hepatic metastases could be significantly augmented by the addition of the liposome-encapsulated immunoadjuvant MTP-PE.

A specific biochemical phenotype is expressed during chemical hepatocarcinogenesis, which includes increased activity of the various isoenzyme forms of glutathione S-transferase (GST) composed of class alpha (Ya/Yc), class mu (Yb) and class pi (Yp) gene products. In vitro cell lines of normal and chemically transformed rat liver epithelial cells provide an opportunity to examine the regulation of expression of GST isoenzymes. We have studied the effect of the state of proliferation in culture on both the enzymic activity and the isoenzyme-specific mRNA expression. In normal rat liver epithelial cells (WB-F344), basal expression of the Yp subunit decreases, and of the Yb subunit increases, in cells at confluence compared with those in logarithmic-phase growth. In a subline of WB-F344 cells that has been chemically treated in vitro (GN6), there was greater Yp expression; however, the effect of growth status on both subunits was the same as in the nontransformed WB-344 cells, and the Ya subunit was not expressed. Inhibition of RNA synthesis with actinomycin D was limited, demonstrating pronged half-lives of the GST mRNAs, and shows a slightly greater decrease in GST-Yp specific mRNA levels in the confluent cells. Also nuclear run-off experiments demonstrate identical transcription rate in confluent and pre-confluent cells. These data suggest that the increase in Yp steady-state RNA in preconfluent cells is due to increased stability of the GST-Yp mRNA. In tumor cells derived from GN6 cells, the regulatory effects of growth status on the Yb and Yp expressions were absent. However, in these cells Ya subunit was expressed and this was subject to the effects of cell proliferation. We conclude that the growth status of cells in culture exerts a significant regulatory control on the GST isoenzyme gene expression. The effects are probably mediated at independent regulatory sites in each gene. The state of transformation of rat liver epithelial cells may determine responsiveness to this effect.

Many in vitro tumor models have been examined to help understand the precise mechanisms responsible for drug resistance. The importance of these results in vivo remains uncertain. MatB 13762 is a rat mammary adenocarcinoma cell line that can be grown both in vitro and as a solid tumor in Fischer 344 rats, thus permitting the examination of tumor cell drug resistance under both conditions. Two cell lines have been selected in vitro for resistance to Adriamycin (AdrR) and melphalan (MlnR), respectively. Each subline has the following features: AdrR, increased mdr-1 messenger RNA, a high level of cross-resistance to vincristine and atypical low level resistance to melphalan and 1,3-bis(2-chloroethyl)-1-nitrosourea, decreased cellular glutathione content, and increased expression of Yc and Yp glutathione S-transferase isozymes; MlnR, low level drug resistance to melphalan and cross-resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea, Adriamycin, and vincristine; increased cellular concentration of glutathione; elevated glutathione S-transferase activity; and greatly increased messenger RNA specific to the Yc and Yp glutathione-S-transferase subunits. Most of the biochemical and molecular features described above are present but significantly less prominent in tumors grown in vivo. This model provides the opportunity to examine the magnitude of expression and the clinical significance of in vitro resistance in an in vivo model.

The effects of GSH depletion in a human breast cancer cell line and a multi-drug resistant subline (ADRr) were determined in a number of experimental conditions. The ADRr cells contained lower GSH concentration which cannot be explained solely on the basis of differences in cell kinetics, and yet the rate-limiting synthetic enzyme gamma-glutamylcysteine synthetase was increased 2-fold. Inhibition of GSH synthesis by BSO resulted in more rapid and more pronounced GSH depletion in ADRr compared to the wild-type cells, suggesting that enhanced GSH utilization and efflux in the resistant cells account for the lowered basal concentration. In addition, the gamma-glutamyl moiety salvage enzyme gamma-glutamyltranspeptidase was reduced markedly in the ADRr cell line. Since these cells have overexpression of the efflux pump protein P-glycoprotein, we examined the effects on cellular GSH of inhibition of the pump's function by verapamil. We found that verapamil significantly depleted cellular GSH. In a rat mammary carcinoma cell line selected in Adriamycin for multi-drug resistance, a similar molecular phenotype has been described including diminished cellular GSH concentration. Verapamil treatment of these cells also resulted in significant depletion of cellular GSH. These results are consistent with the recent report that combined treatment of BSO and verapamil has an additive effect on cytotoxicity. It is likely that decreased basal GSH concentration is due to oxidation and conjugation of it in reactions catalyzed by the enhanced peroxidase and GST found in these cells.

Resistance to (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU), an experimental antitumor compound, was investigated in the sensitive SK-MG-1 cells and the 20-fold more resistant SKI-1 human glioma cells [which are 3-fold more resistant to 1,3,bis(2-chloroethyl)-1-nitrosourea (BCNU)]. The transport of SarCNU was examined by utilizing tritiated sarcosinamide. Sarcosinamide uptake into SK-MG-1 cells is via the catecholamine carrier that accommodates epinephrine. Dixon plot analysis of SarCNU inhibition of sarcosinamide uptake reveals that SarCNU is also accommodated by this carrier. The uptake of 0.5 mM [3H]sarcosinamide was temperature dependent, with similar levels of intracellular sarcosinamide accumulating at steady state in both cell lines. The uptake of sarcosinamide in SKI-1 cells obeyed Michaelis-Menten kinetics over a 200-fold range of concentrations with a Km of 1.52 +/- 0.151 mM and Vmax of 0.659 +/- 0.066 nmol/10(6) cells/min. This represents a more than 5-fold decrease in the uptake affinity and a more than 4-fold increase in the transport capacity compared with SK-MG-1 cells (Km = 0.282 +/- 0.041 mM; Vmax = 0.154 +/- 0.024 nmol/10(6) cells/min). The initial rate of sarcosinamide uptake is similar in both cell lines. Dixon plot analysis confirmed that SarCNU is a competitive inhibitor of sarcosinamide transport in SKI-1 cells with a Ki of 17.5 mM, which is more than 5-fold greater than the Ki obtained in SK-MG-1 cells. The steady state accumulation of SarCNU is significantly reduced by 47% in SKI-1 cells compared with the SK-MG-1 cells (cell to medium ratios of 0.65 +/- 0.11 and 1.22 +/- 0.08, respectively) (p less than 0.005). The accumulation of BCNU was comparable in the two cell lines. Since the Vmax of sarcosinamide (SarCNU) uptake is increased in the SKI-1 cells, the decrease in intracellular SarCNU is not related to decreased drug influx via the catecholamine carrier in SKI-1 cells. The efflux of tritiated sarcosinamide was temperature dependent and similar in both cell lines, with 54 and 58% of sarcosinamide being freely exchangeable in SKI-1 and SK-MG-1 cells, respectively. SarCNU efflux may or may not be altered. Since the expression of mdr is higher in the sensitive cells, it is unlikely that increased efflux of SarCNU mediated by the P-glycoprotein is responsible for drug resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

4710 Background: DCs are antigen presenting cells stimulating cell-mediated immunity by effects on both CD4+ and CD8+ T-cells. We report results of a phase I/II study of a vaccine comprised of matu...

The use of glycine as nitrogen source by Escherichia coli K12 involves L-serine as obligatory intermediate. The pathway includes glycine cleavage enzymes, serine transhydroxymethylase and probably L-serine deaminase. This conversion of glycine to serine occurs in prototrophic strains only when glycine is used as nitrogen source. The growth rate with glycine as sole nitrogen source is slow; apparent doubling time 360 min.