Market and patient access to new oncology products in Europe: a current, multidisciplinary perspective.

To air challenging issues related to patient and market access to new anticancer agents, the Biotherapy Development Association--an international group focused on developing targeted cancer therapies using biological agents--convened a meeting on 29 November 2007 in Brussels, Belgium. The meeting provided a forum for representatives of pharmaceutical companies and academia to interact with European regulatory and postregulatory agencies. The goal was to increase all parties' understanding of their counterparts' roles in the development, licensure, and appraisal of new agents for cancer treatment, events guided by an understanding that cancer patients should have rapid and equitable access to life-prolonging treatments. Among the outcomes of the meeting were a greater understanding of the barriers facing drug developers in an evolving postregulatory world, clarity about what regulatory and postregulatory bodies expect to see in dossiers of new anticancer agents as they contemplate licensure and reimbursement, and several sets of recommendations to optimize patients' access to innovative, safe, effective, and fairly priced cancer treatments.

Chemopotentiating effects of a novel NAD biosynthesis inhibitor, FK866, in combination with antineoplastic agents.

FK866 is a novel anticancer agent that was previously shown to interfere with NAD superset+ biosynthesis by inhibition of nicotinamide phosphoribosyltransferase and to initiate apoptosis in cancer cells. As NAD superset+ is involved in cellular DNA repair processes, the present in vitro study on THP-1 and K562 leukemia cells was conducted to investigate the cytotoxicity of FK866 combination treatment with various cytotoxic agents: the antimetabolite Ara-C, the DNA-intercalating agent daunorubicin and the alkylating compounds 1-methyl-3-nitro-1-nitrosoguanidinium (MNNG) and melphalan. Cell viability after drug exposure was assessed by propidium iodide (PI) staining. Non-cytotoxic concentrations of FK866 (10 superset-9 M or less), applied simultaneously or 24 hours before adding cytotoxic agents, caused a depletion in the intracellular NAD superset+ and--to a lesser extent-- NADH levels in THP-1 cells. After 48 and 72 hours treatment with daunorubicin and Ara-C, respectively, increased cell death was observed in THP-1 cells that were pretreated with FK866, as compared to cells exposed to antineoplastic drugs alone. However, this effect was transient, and there was no difference in cell survival after 72 hours incubation with daunorubicin or 96 hours with Ara-C. - Non-toxic concentrations of FK866 added 8, 16, or 24 hours before starting treatment with the PARP-activating agent MNNG synergistically decreased intracellular NAD superset+ contents, and increased MNNG-induced cytotoxicity both in THP-1 and K562 cells for at least 72 hours. This effect was less pronounced when FK866 was used in combination with another alkylating agent, melphalan. The PARP inhibitor 3-aminobenzamide delayed MNNG-induced cytotoxicity by 24 hours both in cells that were pretreated with FK866 and in non-pretreated cells. 48 hours later, the protective effect of 3-aminobenzamide could no longer be observed, but FK866-pretreated cells retained increased sensitivity to MNNG. - In conclusion, the chemosensitizing effect of FK866 on cell death induced by antineoplastic drugs was particularly obvious in combination with substances like MNNG that cause NAD superset+ depletion per se. It was less pronounced and only transiently measurable in combination with daunorubicin, Ara-C, and melphalan, respectively. These results may indicate different levels of DNA damage implicated in the action of the cytotoxic agents used.

Poly ADP-ribose polymerase (PARP) inhibitors transiently protect leukemia cells from alkylating agent induced cell death by three different effects.

Polyadenosylation of nuclear enzymes is well known to regulate the cellular repair capacity after DNA damage. PARP mediates the transfer of poly-ADP-ribose moieties on itself and other nuclear proteins by the breakdown of NAD+. The present study investigated how modulation of PARP activity interferes with cell death induced by two different alkylating agents used in cancer chemotherapy. 1-methyl-3-nitro-1-nitrosoguanidinium (MNNG) decreased cellular reduction capacity (WST-1 assay) in HL60 and CCRF-CEM cells, accompanied by increased activity of PARP and depletion of intracellular NAD+ and ATP. Pretreatment with the PARP inhibitors 3-AB or 4-AN resulted in transient cell protection, which was associated with a switch from necrosis to apoptosis in CCRF-CEM cells and enhanced apoptosis in HL60 cells. Both PARP inhibitors delayed the drop in WST-1 reduction and retained NAD+ and ATP levels required for apoptosis. Furthermore, 3-AB or 4-AN prevented progressive DNA degradation in MNNG-treated CCRF-CEM cells. In contrast to MNNG, we did not observe early activation of PARP, decrease in WST-1 reduction, or wasteful consumption of NAD+ and ATP after treatment with melphalan. However, preincubation with 3-AB or 4-AN resulted in decreased HL60 cell membrane blebbing and reduced formation of apoptotic bodies. In conclusion, the cell death preventing effects of PARP inhibitors are mediated by their ability to maintain cellular energy metabolism, to inhibit the activation of endonucleolytic DNA degradation and to prevent cell blebbing. Surprisingly, these protective effects of PARP inhibitors on different cell functions seem to be independent of each other and are rather determined by the respective cytotoxic mechanisms implicated by different drugs. Our results support the hypothesis, that PARP activation and/or cleavage plays a regulatory role in the induction of apoptosis.

4-1 BB ligand--just another costimulating molecule?

Initially, scientific interest in the 4-1BB/4-1BB Ligand system focused on the role of the 4-1BB (CD137) receptor in the costimulation of T cells. More recently, evidence is accumulating that 4-1BBL is more than "just" the ligand for a costimulatory molecule. In this review we discuss the functional properties of 4-1BB Ligand such as its preference for CD8 positive T cells and the differences to costimulation via the B7/CD28 system. Furthermore, the available data regarding its ability to transduce signals bidirectionally, i.e. also back into the ligand bearing cell, its release as a soluble form following shedding from the cell surface, and its role in the interaction of tumor cells with the immune system are reviewed.

Commentary: Immune escape versus tumor tolerance: how do tumors evade immune surveillance?

Despite an increasing understanding of immunological mechanisms it is still not clear how tumors evade immune-surveillance of the host and how tumors interact with the immune-system. In particular, the question whether tumors arise because of an insufficient immune response or whether tumor cells employ active strategies to escape the control of the immune system is still open. Data from a great number of in vitro studies and animal models offer arguments for both the theory of an immune escape as well as that of tumor tolerance. In this article the available results regarding the mechanisms of host-tumor-interaction are discussed with focus on these two contrary hypotheses.

Drug-induced apoptosis in chronic lymphocytic leukemia.

Glucocorticoids and fludarabine are able to induce typical features of apoptosis in CLL lymphocytes. Cysteinyl aspartate specific proteases (caspases) play a key biochemical role in the apoptotic pathway. Caspase activation following cytotoxic stimuli leads to highly specific proteolytic cleavage of functionally important cellular enzymes. One of them is poly ADP-ribose) polymerase (PARP). To some extent caspase activation seems to be under the control of the Bcl-2 family of interacting proteins. We determined the role of Bcl-2-family proteins Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic), activation of caspase-3 (CPP32/Yama) and activation of PARP in CLL apoptosis. All 21 analyzed CLL samples expressed Bcl-2 and Bax. Four of 13 (31%) samples with a low Bcl-2/Bax ratio exhibited in vitro prednisolone resistance, whereas eight of nine (88%) samples with a high Bcl-2/Bax ratio were in vitro resistant (

Colonic mucosal proliferation is related to serum deoxycholic acid levels.

BACKGROUND: Hyperproliferation of the colorectal mucosa is regarded as an early step in colorectal carcinogenesis. Deoxycholic acid, a secondary bile acid, stimulates colorectal epithelial proliferation in animals and is considered a tumor promoter in human colorectal carcinogenesis. The aim of this study was to investigate the correlation between colorectal mucosal proliferation and the serum deoxycholic acid level. METHODS: From each of 19 patients (10 men and 9 women) with (n = 3) or without (n = 16) colorectal adenoma, 18 biopsy specimens were obtained by colonoscopy, 3 from each of the 6 colonic segments. A crude nuclei fraction was prepared, and DNA was stained by propidium iodide to determine the proliferative index (the percentage of cells in the S and G2/M phases of the cell cycle) by flow cytometry. Serum levels of deoxycholic acid were determined by gas-liquid chromatography. RESULTS: The colonic proliferation rates (median of the values obtained in all segments, 14.1%; range, 10.0-18.7%) and the fasting serum deoxycholic acid levels (median, 0.86 micromol/L; range, 0.28-1.58 micromol/L) showed a significant correlation (r = 0.51, P = 0.03). Serum lithocholic, cholic, chenodeoxycholic, and ursodeoxycholic acid levels were not correlated with the proliferation rates. CONCLUSIONS: Levels of deoxycholic acid in serum are correlated with the rates of the colorectal mucosa. These results are consistent with the concept that deoxycholic acid promotes colorectal carcinogenesis.

Cellular resistance mechanisms with impact on the therapy of multiple myeloma.

Multiple myeloma (MM) is characterized by bone marrow infiltration with abnormal plasma cells which synthesize monoclonal immunoglobulins (Ig) or Ig fragments. Regularly, MM cells exhibit a high intrinsic resistance to available chemotherapeutic strategies. A number of cellular alterations including the cellular membrane, such as mutations of the glucocorticoid receptor or expression of membrane transport proteins, detoxification mechanisms and altered expression of topoisomerases, have been described. In addition to anti-apoptotic survival mechanisms, involving abnormalities of several oncogenes and suppressor genes (ras, c-myc, p53, Rh and bcl-2), the broad resistance spectrum might be explained but clinical studies which include the evaluation of resistance factors are missing. On the other hand, risk factor evaluation is important as a number of therapeutical strategies with different intensities from corticosteroid monotherapy up to high-dose chemotherapy with tandem autologous bone marrow transplantation exist.

Intracellular pharmacokinetics of anthracyclines in human leukemia cells: correlation of DNA-binding with apoptotic cell death.

Anthracyclines are major components of the most therapeutical strategies in hematological oncology. These drugs are not directly cytotoxic but induce apoptosis. Due to their lipophilicity, anthracyclines are rapidly distributed in myeloid and lymphatic leukemia cells. Within 20 min after treatment, daunorubicin and idarubicin are found to intercalate into DNA. Shortly after treatment, DNA damage occurs and increases within 3 hours. Apoptosis can be monitored 12-24 hours after treatment, at this timepoint the majority of DNA strand breaks have already been repaired. A statistically highly significant linear correlation could be established between the DNA binding rate of anthracyclines and the resulting cell death. This indicates that DNA binding is a prerequisite for the induction of apoptosis. With respect to cellular resistance mechanisms, 2 different pharmacodynamic phases can be distinguished: intracellular distribution and cellular reaction. The endpoint of the "distribution phase" is marked by the DNA intercalation of the anthracyclines. Cellular resistance mechanisms which decrease the DNA binding include membrane transport mechanisms and vesicular trapping of the drugs. The "reaction phase" might be disturbed by complex antiapoptotic mechanisms. The assessment of DNA binding in malignant cells during or shortly after treatment with anthracyclines might be a useful tool to distinguish cellular resistance mechanisms.

In vitro efficacy of known P-glycoprotein modulators compared to droloxifene E and Z: studies on a human T-cell leukemia cell line and their resistant variants.

P-glycoprotein(P-gp)- related resistance is one of the major obstacles in treating leukemia patients. Therefore, it is of clinical interest to find new potential modulators and compare their P-gp-modulating efficacy. The present analysis investigated the influence of P-gp modulators, such as verapamil, tamoxifen, droloxifene E, droloxifene Z, SDZ PSC 833 (PSC 833) and dexniguldipine in a leukemic T-cell line (CCRF-CEM) and its P-gp-resistant counterparts (CCRF-CEM/ACT400 and CCRF-CEM/VCR1000). P-gp expression was assessed with an immunocytological technique using the monoclonal antibody 4E3.16. It was characterized as the percentage of P-gp positive cells and also expressed as a D value by using the Kolmogorov Smirnov statistic. The efficacy of P-gp modulators was determined with the rhodamine-123 accumulation test and the MTT test. An in vitro modulator concentration between 0.1 microM and 3 microM was determined, where no genuine antiproliferative effect was apparent. The modulators PSC 833 and dexniguldipine were the significant (p

Serum thymidine kinase in non-Hodgkin lymphomas with special regard to multiple myeloma.

S-TK (Serum thymidine kinase) levels were determined by means of a radioenzyme assay (REA). In 95% of healthy controls (n = 97), S-TK values were below 8.5 U/L. In patients with monoclonal gammopathies of undetermined significance (MGUS) (n = 27) or polyclonal gammopathies (n = 45) the cut off was 10.3 U/L respectively 25 U/L. Patients with viral disease (n = 16), especially infections with Epstein-Barr virus, Hepatitis-virus and HIV, had elevated S-TK values of up to 215 U/L. In 95 patients with multiple myeloma (MM) and 103 patients with other various non-Hodgkin lymphomas (NHL) S-TK levels were investigated. With regard to monoclonal gammopathies, MGUS had lower S-TK than MM patients (p < 0.05) and patients with stage I MM according to Durie and Salmon had S-TK levels significantly lower than those with more advanced stages (p < 0.01). There was a correlation between S-TK and plasma cell labeling index (r = 0.56, p < 0.001). Patients with chronic lymphocytic leukemia showed significantly higher S-TK levels in the RAI stages 3 and 4 than in stages 1 and 2 (p < 0.01). In cases of other malignant NHL in progression sensitivities of S-TK were found to be: immunocytoma 36%, centrocytic/centroblastic-centrocytic lymphoma 54% and high-grade NHL 40% (cut off defined on lymphomas in remission). S-TK levels varied in MM according to the course of disease and response to therapy decreasing at remission and increasing again at relapse. Analogous variations were found in the other NHL. After two years, 83% of patients with a pretreatment S-TK of < 10 U/L and 47% of the patients with a S-TK of > or = 10 U/L were still alive. S-TK proved to be a highly significant prognostic indicator for MM patients (log-rank and Wilcoxon: p < 0.0001). In the other NHL patients with a S-TK level greater than 10 U/L had a median follow-up of only 7 months. NHL patients with lower S-TK levels did not yet reach the median survival time (log-rank and Wilcoxon. p < 0.005). Our results suggest that the determination of S-TK may help to monitor the clinical course of NHL during therapy and predict the prognosis of NHL.

Evaluation of serum neural cell adhesion molecule as a prognostic marker in multiple myeloma.

Serum neural cell adhesion molecule (NCAM), a possible prognostic marker for multiple myeloma (MM), was determined by means of an enzyme immunoassay, which showed good linearity and high precision. In 95% of healthy controls (n = 70), NCAM values were below 18.7 U/mL. In patients with monoclorlal gammopathies of undetermined significance (MGUS) (n = 31) or polyclonal gammopathies (n = 53) the cut off was 23.1 U/mL. MM in active stage (n = 52) showed significantly higher NCAM levels (p < 0.001) than in asymptomatic stage (n = 44). In active myeloma the sensitivity of serum markers were found to be: NCAM 40%, beta 2-microglobulin beta 2-M) 52% and serum thymidine-kinase (S-TK) 41% (cut off defined on MGUS). The combined sensitivities ranged between 55 and 60% (NCAM+ beta 2-M, beta 2-M+S-TK, NCAM+S-TK). No correlation with beta 2-M or S-TK could be demonstrated. However, NCAM values were correlated with the concentration of monoclonal immunoglobulin (IgG-paraprotein: r = 0.45; IgA-paraprotein: r = 0.58). In the follow-up of patients with myeloma, NCAM values decreased in response to chemotherapy and were low in smouldering myeloma. But in three patients with progression NCAM did not reflect the tumor activity. At the time of censor, 80% of patients (n = 80) with a pre-treatment NCAM of < 18.5 U/mL and 61% of patients with a NCAM of > 18.5 U/mL were still alive. NCAM showed a low prognostic significance (log-rank: p < 0.07). Seven of ten myeloma patients with CD56 expression on plasma cell surface, which was examined by flow cytometry, displayed a high concentration of NCAM in serum. All other non-Hodgkin's lymphomas (21 immunocytoma, 27 chronic lymphocytic leukemia, 16 centrocytic/centroblastic-centrocytic lymphoma, 24 high-grade lymphoma) had low NCAM concentrations in serum and did not significantly vary in follow-up. In conclusion, serum NCAM could be a marker for the staging and monitoring of MM. However, it seems, that NCAM did not provide additional prognostic information relating to beta 2-M, S-TK or paraprotein.

Idarubicin monotherapy in multiply pretreated leukemia patients: response in relation to P-glycoprotein expression.

The aim of the study was to test whether fractionated (weekly) idarubicin administration to multiply pretreated leukemia patients is effective and tolerable for outpatient treatment, and whether idarubicin alone can overcome P-glycoprotein (P-gp)-related resistance. P-gp was assessed with an immunocytological technique using the monoclonal antibody 4E3.16. P-gp. expression was characterized as a percentage of P-gp-positive blasts. Additionally, the function of P-gp was determined with the rhodamine-123 (R-123) accumulation test in combination with or without verapamil and expressed as the R123 accumulation ratio. Fractionated idarubicin (12 mg/m2/week) was given to 36 acute myelogenous leukemia (AML) patients, 12 acute lymphoblastic leukemia (ALL) patients, and eight chronic myelogenous leukemia (CML) patients in blast crisis. Furthermore, 11 AML and four ALL patients were treated with fractionated daunorubicin at a dose of 50 mg/m2/week. All patients had been pretreated with drugs inducing P-gp-related resistance including daunorubicin and/or doxorubicin or vindesine (CML patients). Of 71 pretreated patients, 51 (72%) had a P-gp value between 25 and 98%. Six of these patients with increased P-gp expression had a nonpumping P-gp; four of them were CD34 positive. Of 51 patients with increased P-gp expression, 30 (59%) were CD34 positive. With regard to idarubicin monotherapy, overall response was 33/56 (59%) patients, and 23/33 (70%) responding patients showed a P-gp expression between 25 and 95%. All idarubicin-responding patients with high P-gp expression before treatment showed a clear reduction of P-gp-positive blasts. No patients with P-gp expression between 34 and 85% treated with fractionated daunorubicin showed response or reduction of P-gp-positive blasts in bone marrow. This study demonstrates that P-gp-related resistance can be overcome in multiply pretreated leukemia patients with idarubicin alone, and that the protocol used here is tolerable for outpatient treatment.

Cellular resistance mechanisms with impact on the therapy of multiple myeloma.

Multiple myeloma (MM) is characterized by bone marrow infiltration with abnormal plasma cells which synthesize monoclonal immunoglobulins (Ig) or Ig fragments. Regularly, MM cells exhibit a high intrinsic resistance to available chemotherapeutic strategies. A number of cellular alterations including the cellular membrane, such as mutations of the glucocorticoid receptor or expression of membrane transport proteins, detoxification mechanisms and altered expression of topoisomerases, have been described. In addition to anti-apoptotic survival mechanisms, involving abnormalities of several oncogenes and suppressor genes (ras, c-myc, p53, Rb and bcl-2), the broad resistance spectrum might be explained but clinical studies which include the evaluation of resistance factors are missing. On the other hand, risk factor evaluation would be important as a number of therapeutical strategies with different intensities from corticosteroid monotherapy up to high-dose chemotherapy with tandem autologous bone marrow transplantation exist.

Association of bcl-2, bax, bcl-xL and interleukin-1 beta-converting enzyme expression with initial response to chemotherapy in acute myeloid leukemia.

Bcl-2 expression is able to confer drug resistance to chemotherapy-induced programmed cell death. Bax, a partner protein of bcl-2 with extensive aminoacid homology, is a promoter of apoptosis. Apparently the equilibrium of bcl-2 and bax hetero- and homodimers is important for the susceptibility of cells for stimuli inducing apoptosis. In this study we determined the role of bcl-2 to bax expression ratio, bcl-xL and ICE expression level for predicting clinical response to chemotherapy in acute myelold leukemia (AML). Bone marrow samples from 14 patients with AML were examined using an immunophosphatase staining method. Initial bone marrow blast portion was over 80% in all cases. Clinical response was defined by bone marrow aspiration 4 weeks after treatment initiation. There was a significant correlation between bcl-2 to bax expression ratio and clinical response (P < 0.005). No patients with a bcl-2/bax ratio >1.0 achieved complete remission after induction therapy. No significant correlation between bcl-2- and p-glycoprotein-expression was observed in this group. Conversely a high expression of ICE indicated a good clinical response (P < 0.01), whereas expression of bcl-xL had no influence on therapeutic success in this group.

P-glycoprotein expression in patients with acute leukemia-clinical relevance.

P-glycoprotein (P-gp) is a crucial factor in the development of chemotherapy resistance in malignant disorders. Between 1989 and 1995, P-gp expression was studied in bone marrow blast cells of 322 (239 AML; 83 ALL) acute leukemia patients. 166 AML patients with the AML-6 protocol (EORTC), containing daunorubicin, vincristine and conventional-dose cytarabine (ara-C), and 63 AML patients treated with intermediate-does Ara-C plus amsacrine. Further 71 ALL patients were treated according to a German standard polychemotherapy protocol (BMFT04/1989). P-gp was determined by using monoclonal antibodies C219 and 4E3, and the cutoff point for P-gp overexpression was set at >/= 10%. A significant (P < 0.06) difference in P-gp overexpression was demonstrated between AML (21.6%) and ALL (10.2%) patients at primary diagnosis and between primary diagnosis and relapse/refractoriness in AML (21.6%; 51.0%) and ALL (10.2%; 27.2%) patients. According to FAB classification P-gp overexpression was detected in AML patients significantly (P < 0.05) more frequently in classes M4, M5a and M5b and less frequently in M3, as compared to other types. For AML patients with P-gp overexpression at primary diagnosis or early relapse/refractoriness, the predictive value for nonresponse to the AML-6 protocol was 91 and 95%, respectively, while late-relapsed AML patients with P-gp overexpression had a significantly (P < 0.05) lower predictive value of 73% for nonresponse. Additionally, in refractory and late-relapsed P-gp--overexpressing AML patients treated with intermediate-dose ara-C plus amsacrine the predictive values for nonresponse were 44 and 39%, respectively, significantly (P < 0.05) lower as compared to AML-6 protocol-treated refractory or late-relapsed AML patients. In P-gp-overexpressing treated ALL patients the predictive values of 50 and 55% for non-response were calculated at primary diagnosis and late relapse, respectively. We conclude that P-gp overexpression is a common phenomenon in AML patients at primary diagnosis or relapse, has an inverse influence on AML-6 treatment outcome and should be taken into consideration in the development of new therapy strategies.

In vitro stimulation of erythropoiesis by stem cell factor alone in myelodysplastic syndrome patients with elevated endogenous erythropoietin serum levels.

Anemia remains a therapeutic problem in patients with myelodysplastic syndrome (MDS). In view of the recently reported potential of stem cell factor (SCF) in restoring erythropoiesis in combination with erythropoietin (Epo), we first aimed to define a correlation between SCF serum levels and anemia in MDS. Endogenous SCF levels in 50 MDS patients were determined by using a quantitative sandwich enzyme immunoassay. Broad interindividual variations were observed, but SCF serum levels were in the normal range with no correlation to peripheral blood count. A soft agar culture system was used to further define the role of SCF for stimulation of erythroid growth. Bone marrow mononucleated cells of 20 MDS patients (4 refractory anemia, 5 refractory anemia with excess of blasts, 7 refractory anemia with excess of blasts in transition, and 4 chronic myelomonocytic leukemia) were investigated, and SCF plus Epo was able to stimulate burst-forming unit-erythroid significantly more than SCF or Epo alone independent of French-American-British group. When mononucleated cells from six MDS patients (two refractory anemia, two refractory anemia with excess of blasts, and 2 refractory anemia with excess of blasts in transition) with elevated serum Epo levels were incubated in the presence of SCF and autologous serum, a significant dose-dependent stimulation of burst-forming unit-erythroid number and cells per colony was detected. Erythroid differentiation was further enhanced by adding serum with high colony-stimulating activity obtained from patients with severe aplastic anemia. Our data suggest that in MDS patients with high endogenous Epo serum levels SCF alone might be effective in stimulating erythropoiesis in vivo.

Cytotoxicity of anthracyclines: correlation with cellular uptake, intracellular distribution and DNA binding.

In order to interact with topoisomerase II and induce genotoxicity, anthracyclines have to cross the outer cell membrane and the cytoplasm, enter the nucleus, and bind to the DNA. We incubated sensitive and resistant hematopoietic cells from cell lines and patient cells with daunorubicin, idarubicin, and its active derivative idarubicinol, extracted the anthracyclines from whole cells and nuclei, and determined their concentration fluorimetrically. Additionally, the DNA binding of the drugs was evaluated in the same cells by determining fluorescence resonance energy transfer between the anthracyclines and DNA-bound Hoechst dye 33342. We found a several thousand-fold accumulation of anthracyclines in sensitive and resistant hematopoietic cells; 30-60% of the drugs are found in the nucleus, resulting in 200- to 300-fold differences in concentration between the nucleus and outer fluids. A small proportion of the intracellular or intranuclear anthracyclines is bound to the DNA. The amount of DNA-bound anthracyclines correlates directly to cell death. It takes an additional 10 min for idarubicin and 30 min for daunorubicin to satisfy DNA binding sites after the drugs have arrived in the nucleus. The described methods provide the means to perform ex vivo studies on clinical material.

Clinical importance of P-glycoprotein-related resistance in leukemia and myelodysplastic syndromes--first experience with their reversal.

P-glycoprotein (P-gp) expression in mononuclear bone marrow cells was analyzed in 119 patients, including 60 with chronic myelogenous leukemia (CML), 48 with myelodysplastic syndromes (MDS), and 11 with acute myelogenous leukemia (AML). For P-gp measurement an immunocytological method using monoclonal antibodies C219, 4E3, and MRK 16 and the reverse transcription-polymerase chain reaction technique were applied. According to our results obtained in healthy volunteers using the immunocytological method, the limit for P-gp overexpression was set at > or = 10% P-gp-positive mononuclear bone marrow cells and at > or = 30% P-gp-positive mononuclear peripheral blood cells. All 42 CML patients in chronic phase had normal P-gp expression. P-gp overexpression was demonstrated in four of six patients in accelerated myelogenous blast cell phase and in four of 12 CML-BC patients. Of eight CML patients in blast crisis (BC) with normal P-gp expression, partial remission was achieved in three and minor response in five after prednisone/vindesine therapy. All four of the 12 CML-BC patients with P-gp overexpression did not respond to this therapy. Normal P-gp expression was seen in 41 (85.4%) of 48 untreated MDS patients. While P-gp overexpression did not develop during therapy in any of the myelodysplastic syndrome patients treated with low-dose ara-C alone, four of eight treated with low-dose ara-C plus GM-CSF and four of 11 treated with low-dose ara-C and IL-3 developed P-gp overexpression after therapy. Furthermore, 11 AML patients at primary diagnosis, including five AML patients with P-gp overexpression, who were treated with idarubicin, vepesid, and cytarabine V (ara-C) showed a complete remission. Additionally, one daunorubicin-cytarabine-pretreated refractory AML patient was treated with the oral form of the P-gp modulator drug dexniguldipine and achieved complete remission for a duration of 7 months. Our results suggest that in CML patients in BC, P-gp expression influences outcome after therapy. Further more, studies in a larger series of patients are necessary to prove the efficacy and toxicity of idarubicin/vepesid and cytardbine--or dexniguldipine-containing--therapy in relation to P-gp expression of AML patients.

Mdr1/P-glycoprotein expression in natural killer (NK) cells enriched from peripheral or umbilical cord blood.

The sensitivity to antineoplastic agents of subpopulations of haematopoietic cells during cancer chemotherapy is an open question. The performance of natural killer (NK) cells, possibly assisting the elimination of tumour cells under drug treatment might be of particular interest. We examined the expression of the transmembrane multidrug transporter mdr1/P-glycoprotein in NK-cells (CD56+) enriched from the peripheral blood or the umbilical cord blood from healthy donors by indirect immunocytofluorescence using the monoclonal P-glycoprotein antibody C219 and a polymerase chain reaction (PCR) approach with amplimers specific for the human mdr1 cDNA. As the antibody C219 apparently cross-reacts with the human mdr3 gene product whose functions are as yet unclear we also checked expression of this gene by PCR using mdr3 specific amplimers. Distinct, but rather inhomogeneous mdr1/P-glycoprotein expression was found in NK-cells enriched from the peripheral blood. NK-cells enriched from the umbilical cord blood showed quite strong mdr1 expression levels throughout, exceeding the values found in the moderately multidrug-resistant cell line CCRF VCR 100 which is permanently cultivated in the presence of 100 ng/ml vincristine. Mdr1/P-glycoprotein expression was mirrored by lowered sensitivities of the cultivated NK-cells towards actinomycin D or adriamycin. The drug sensitivity could be modulated by treatment of the cells with the immunosuppressive drug cyclosporin A. Expression of the mdr3 gene was low or absent in all NK-cell samples examined so far.