MRI assessment of early response to certolizumab pegol in rheumatoid arthritis: a randomised, double-blind, placebo-controlled phase IIIb study applying MRI at weeks 0, 1, 2, 4, 8 and 16.

OBJECTIVES: To identify the first time point of an MRI-verified response to certolizumab pegol (CZP) therapy in patients with rheumatoid arthritis (RA). METHODS: Forty-one patients with active RA despite disease-modifying antirheumatic drug therapy were randomised 2:1 to CZP (CZP loading dose 400 mg every 2 weeks at weeks 0-4; CZP 200 mg every 2 weeks at weeks 6-16) or placebo→CZP (placebo at weeks 0-2; CZP loading dose at weeks 2-6; CZP 200 mg every 2 weeks at weeks 8-16). Contrast-enhanced MRI of one hand and wrist was acquired at baseline (week 0) and weeks 1, 2, 4, 8 and 16. All six time points were read simultaneously, blinded to time, using the Outcome Measures in Rheumatology Clinical Trials RA MRI scoring system. Primary outcome was change in synovitis score in the CZP group; secondary outcomes were change in bone oedema (osteitis) and erosion scores and clinical outcome measures. RESULTS: Forty patients were treated (27 CZP, 13 placebo→CZP), and 36 (24 CZP, 12 placebo→CZP) completed week 16. In the CZP group, there were significant reductions from baseline synovitis (Hodges-Lehmann estimate of median change, -1.5, p=0.049) and osteitis scores (-2.5, p=0.031) at week 16. Numerical, but statistically insignificant, MRI inflammation reductions were observed at weeks 1-2 in the CZP group. No significant change was seen in bone erosion score. Improvements across all clinical outcomes were seen in the CZP group. CONCLUSIONS: CZP reduced MRI synovitis and osteitis scores at week 16, despite small sample size and the technical challenge of reading six time points simultaneously. This study provides essential information on optimal MRI timing for subsequent trials. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov, NCT01235598.

P-Glycoprotein inhibitor valspodar (PSC 833) increases the intracellular concentrations of daunorubicin in vivo in patients with P-glycoprotein-positive acute myeloid leukemia.

PURPOSE: The aim of the present study was to evaluate the effect of the cyclosporine derivative valspodar (PSC 833; Amdray, Novartis Pharma, Basel, Switzerland) on the concentration of daunorubicin (dnr) in leukemic blast cells in vivo during treatment. PATIENTS AND METHODS: Ten patients with acute myeloid leukemia (AML) were included. Leukemic cells from seven of the patients were P-glycoprotein (Pgp)-positive. dnr 100 mg/m(2) was given as a continuous infusion over 72 hours. After 24 hours, a loading dose of valspodar was given, followed by a 36-hour infusion of 10 mg/kg per 24 hours. Blood samples were drawn at regular intervals, and concentrations of dnr and its main metabolite, daunorubicinol, in plasma and isolated leukemic cells were determined by high-pressure liquid chromatography. RESULTS: The mean dnr concentrations in leukemic cells 24 hours after the start of infusion (before valspodar) were 18.8 micromol/L in Pgp-negative samples and 13.5 micromol/L in Pgp-positive samples. After 8 hours of valspodar infusion, these values were 25.8 and 24.0 micromol/L, respectively. The effect of valspodar was evaluated from the ratio of the area under the curve (AUC) for dnr concentration versus time in leukemic cells to the AUC for dnr concentration against time in the plasma. For the seven patients with Pgp-positive leukemia, the mean ratio increased by 52%, from 545 on day 1 to 830 on day 2 (P<.05) when valspodar was given. In the three patients with Pgp-negative leukemia, no significant difference was observed. CONCLUSION: These results strongly suggest that valspodar, by interacting with Pgp, can increase the cellular uptake of dnr in leukemic blasts in vivo.

Comparison of the intracellular pharmacokinetics of daunorubicin and idarubicin in patients with acute leukemia.

Five patients with acute non-lymphoblastic leukemia were treated with a mixture of daunorubicin 50 mg/m2 and idarubicin 10 mg/m2 given as a short-time infusion. Daunorubicin, idarubicin and the main metabolites daunorubicinol and idarubicinol were separated and the concentrations in plasma and leukemic cells were determined by HPLC. Although idarubicin was given in one-fifth of the dose, the intracellular peak concentration was 70% of that of daunorubicin. The initial elimination of idarubicin from leukemic cells was somewhat faster but in the terminal phase the drug was retained longer than daunorubicin. Intracellular concentrations of both metabolites were low and probably of little importance for the activity of the drug. We conclude that the intracellular pharmacokinetics of idarubicin, with higher peak concentration and longer terminal retention, is a possible explanation for the higher toxicity and suggested better antileukemic effect of this drug.

Intracellular concentrations of mitoxantrone in leukemic cells in vitro vs in vivo.

The aim of this study was to determine the intracellular pharmacokinetics of mitoxantrone in vivo and to use these results to establish how leukemic cells should be incubated to perform clinically relevant in vitro studies of this drug. Blood samples were obtained from 11 patients with acute nonlymphoblastic leukemia at certain intervals up to 20 h after the infusion of mitoxantrone 12 mg/m2. Plasma and leukemic cells were separated and the drug concentrations were determined with HPLC. Before treatment, leukemic cells from 12 patients were incubated with 0.02, 0.05, 0.1, 0.2 and 1.0 microM mitoxantrone for 1-4 h and thereafter cultured in suspension culture for 20 h; during this time cell samples were taken at certain intervals for drug determination. In cells incubated with 0.05 and 0.2 microM mitoxantrone the cytotoxic effect was measured with the DiSC assay after cultivation for 4-5 days. In vivo, the intracellular levels exceeded the plasma concentrations already at the end of infusion and after 2 h the intracellular concentrations were 200-300 times higher than in plasma. In vitro, the intracellular steady state level of mitoxantrone was reached after 1-2 h and there was a pronounced intracellular retention even after 20 h culture in drug-free medium. Incubation with 0.05 microM during 1 h gave intracellular concentrations of mitoxantrone similar to those achieved in vivo. This incubation concentration gave a mean cytotoxic effect of 53% living cells measured with the DiSC assay, which gives good possibilities to discriminate between mitoxantrone-sensitive and unsensitive cells. We believe that exposing leukemic cells in vitro for in vivo mimicking mitoxantrone concentrations could increase the clinical relevance of predictive assays.

In vitro chemosensitivity testing in acute non lymphocytic leukemia using the bioluminescence ATP assay.

The ATP assay is a short term in vitro chemosensitivity assay where the amount of viable cells are determined by their content of ATP. The aim of the study was to compare the in vitro results of six cytostatic drugs to the clinical outcome in 83 acute non-lymphocytic leukemia (ANLL) patients. The secondary ANLL at diagnosis showed an in vitro resistance to daunorubicin that was significantly higher compared to de novo ANLL at diagnosis (P<0.003). De novo ANLL at diagnosis that achieved complete remission (CR) were significantly more sensitive to daunorubicin compared to those who didn't achieve CR (P<0.05). There was an vitro correlation between topoisomerase II active drugs but not between these drugs and ara-C. In vitro ara-C sensitivity (< or = the median of the de novo ANLL at diagnosis) was correlated to poor overall survival (P = 0.02). In vitro sensitivity to daunorubicin and mitoxantrone was associated with prolonged disease free survival (P = 0.03 and P = 0.04). We conclude that despite significant correlation to clinical parameters for daunorubicin and mitoxantrone the predictive value of the ATP assay in this material was insufficient for directing therapy.

Intracellular uptake and cytotoxic effect in vitro of doxorubicin and epirubicin in human leukemic and normal hematopoietic cells.

Leukemic cells from patients presenting with acute nonlymphoblastic leukemia and normal hematopoietic bone marrow cells from healthy donors for allogeneic bone marrow transplantation were incubated for 3 h with doxorubicin and epirubicin at different concentrations. The intracellular uptake at the end of the incubation was determined by photofluorometry in leukemic cells from 15 patients and in normal cells from 9 donors for bone marrow transplantation. Cytotoxicity in vitro against granulocyte/macrophage colony-forming units (CFU-GM) was determined in normal cells from 7 donors, and in vitro toxicity against leukemic cells was determined by a clonogenic technique in cells from 6 patients and by vital dye staining (DiSC) following 4 days' culture in cells from 15 patients. Epirubicin was significantly less toxic than doxorubicin to normal hematopoetic cells (72% +/- 20% survival of cells for epirubicin vs 45% +/- 13% for doxorubicin at a concentration of 0.2 microM; P less than or equal to 0.005). As analyzed by the DiSC assay, 0.2 microM epirubicin was slightly more toxic to leukemic cells than was the same concentration of doxorubicin (47% vs 61% survival, P less than or equal to 0.01), but the clonogenic assay revealed no difference in toxicity to leukemic cells. At a concentration of 0.2 microM, the mean intracellular uptake of epirubicin in leukemic cells was 0.43 +/- 0.26 nmol/mg protein as compared with 0.33 +/- 0.14 nmol/mg protein for doxorubicin (not significant). In normal cells, the uptake of epirubicin at a concentration of 0.2 microM was 0.47 +/- 0.25 nmol/mg protein as compared with 0.31 +/- 0.21 nmol/mg protein for doxorubicin (not significant). The reduced myelotoxicity observed in vitro together with the retained toxicity to leukemic cells indicates that the therapeutic index of epirubicin is better than that of doxorubicin.

Comparison of the intracellular pharmacokinetics of doxorubicin and 4'-epi-doxorubicin in patients with acute leukemia.

A direct comparison of the intracellular pharmacokinetics of 4'-epi-doxorubicin and doxorubicin was carried out in five patients with leukemia who were given weekly low doses of a combination of these drugs at 20 mg each in an i.v. injection. Blood samples were collected for 48 h after administration and the drug concentrations in leukemic cells were determined by high-performance liquid chromatography (HPLC). The intracellular peak concentrations of 4'-epi-doxorubicin were higher than those of doxorubicin in all patients. The AUC for the intracellular drug concentration vs time curve was significantly higher for 4'-epi-doxorubicin. The intracellular uptake and retention were also studied in vitro after incubation of isolated leukemic blast cells with the two drugs; they showed the same pattern observed in vivo. We conclude that 4'-epi-doxorubicin and doxorubicin exhibit different pharmacokinetics in malignant cells. The therapeutic significance of this finding requires further evaluation.

Toxicity of cytostatic drugs to normal bone marrow cells in vitro.

In this study we compared how different concentrations and periods of incubation of anthracyclines, amsacrine, and cytosine arabinoside would affect normal hematopoietic bone marrow cells in terms of interindividual differences in toxicity, the age of the donor, and the proliferative capacity of the bone marrow. Bone marrow was obtained from 36 donors in connection with bone marrow transplantation. After separation the mononuclear cell fraction was incubated with doxorubicin, 4-epidoxorubicin, daunorubicin, idarubicin, aclarubicin, mitroxantrone, amsacrine, and cytosine arabinoside for 1 h, for 3 h, or continuously. The cells were thereafter cultured in soft agar and CFU-GM were counted after 10-12 days. The results showed a large interindividual variation in toxicity for all drugs tested. Daunorubicin, idarubicin, aclarubicin, and mitoxantrone had a pronounced cytotoxic effect after 1 h of incubation. Doxorubicin and 4-epi-doxorubicin showed the greatest cytotoxic effect after 3 h and were also more toxic to normal bone marrow cells from donors over 40 years of age. Ara-C had a low cytotoxic effect after 1 and 3 h of incubation, even at high concentrations, but exerted a pronounced degree of toxicity during continuous incubation. Daunorubicin, idarubicin, and ara-C also showed increased toxicity to cell samples with a low proliferating capacity in the control. The conclusions drawn from these results are that interindividual variation, proliferation capacity, incubation conditions, and the age of the donors are factors of importance in the toxicity of drugs to normal hematopoietic bone marrow cells.

Effect of cytokines on the toxicity of cytostatic drugs to normal bone marrow cells in vitro.

We evaluated in vitro how growth factors influenced the effect of cytostatic drugs on normal hematopoietic progenitor cells. Bone marrow was obtained from 15 donors for bone marrow transplantation. After separation the mononuclear fraction was incubated with granulocyte colony-stimulating factor (G-CSF) at 5 and 50 ng/ml, with granulocyte/macrophage colony-stimulating factor (GM-CSF) at 1 and 10 ng/ml, and with interleukin 3 (IL-3) at 0.5 and 5 ng/ml for 24 h prior to incubation with cytostatic drugs. These incubations were performed with 0.05 microM mitoxantrone and 0.2 microM daunorubicin for 1 h, and cells were thereafter cultured for colony-forming units--granulocyte/macrophage (CFU-GM) in soft agar for 10-12 days. Incubation with 0.05 microM cytosine arabinoside was performed continuously throughout the culture period. The proliferation of normal hematopoietic progenitor cells stimulated with GM-CSF at 10 ng/ml and with IL-3 at 5 ng/ml was significantly increased to 218% and 215% colonies, respectively, as compared with the control stimulated with conditioned medium only. Stimulation with G-CSF, on the other hand, did not induce any significantly enhanced proliferation relative to the control. Daunorubicin applied in combination with G-CSF at 5 ng/ml or with IL-3 at 0.5 ng/ml exerted a significantly higher degree of cytotoxicity on normal hematopoietic progenitor cells, resulting in 21% and 30% surviving colonies as compared with the 38% recorded for daunorubicin alone (P < 0.05). Neither G-CSF nor IL-3 at a higher concentration nor GM-CSF exerted a significantly altered degree of toxicity relative to cells incubated with daunorubicin alone. The cytotoxic effect exerted on normal hematopoietic cells by mitoxantrone or ara-C was unchanged or significantly decreased after stimulation with growth factors as compared with the effect on cells incubated with cytostatic drugs alone. We conclude that G-CSF and IL-3 augment the effect of daunorubicin on normal hematopoietic progenitor cells.

Intracellular concentrations of anti cancer drugs in leukemic cells in vitro vs in vivo.

A comparison of intracellular concentrations of daunorubicin, doxorubicin and ara-C in myeloid blast cells was carried out in vivo and in vitro. In vivo, blood samples were obtained from 27 patients with acute nonlymphoblastic leukemia during and up to 4 days after drug infusion. Leukemic cells were isolated and drug concentrations were determined by HPLC. Before treatment, leukemic cells from 21 patients were isolated from blood and bone marrow, and in vitro incubations were done with anthracyclines for 1-3 h at concentrations of 0.1-1.0 microM and with ara-C for 1 h to 5 days at concentrations of 0.5-5.0 microM. The cells were cultured for 5 days, during which cell samples were taken for drug determination. The results showed that incubation with 0.2 microM daunorubicin for 1 h and 0.2 microM doxorubicin for 3 h and continuous exposure to 0.5 microM ara-C gave intracellular concentration curves similar to those obtained in vivo. After 5 days' culture, the cytotoxic effect was determined by vital dye staining with fast green, the addition of an internal standard of fixed goose erythrocytes, cytospin centrifugation and counter-staining of living cells with haematoxylin/eosin (DiSC). Incubations at the above-mentioned concentrations exerted a cytotoxic effect of approximately 50%. We conclude that in mimicking the in vivo situation, it is important to consider differences in intracellular pharmacokinetics.

Effects of verapamil on uptake and in vitro toxicity of anthracyclines in human leukemic blast cells.

The effect of a calcium channel blocker, verapamil, on intracellular uptake and cytotoxicity of anthracyclines in vitro was studied on leukemic cells from 32 patients with acute non-lymphoblastic leukemia. Cells were isolated from peripheral blood or bone-marrow and incubated with a concentration of 0.2 mumol/l, 0.5 mumol/l and/or 1.0 mumol/l of doxorubicin or daunorubicin in the absence and presence of verapamil at a concentration of 2 mumol/l and/or 10 mumol/l. Intracellular uptake was determined at the end of the incubations by photofluorometer and the in vitro cytotoxicity was determined after 5 days culturing in liquid medium by dye exclusion according to Weisenthal. Verapamil significantly increased the intracellular uptake of anthracyclines 0.5 mumol/l and 1.0 mumol/l and the cytotoxic effect of anthracyclines 0.2 mumol/l and 0.5 mumol/l and affected doxorubicin and daunorubicin equally. There were no significant differences between the two concentrations of verapamil. Cells from different FAB-groups were equally affected by verapamil. The effect on intracellular uptake was higher in cells from patients who were resistant to therapy compared to those who achieved a complete remission. We conclude that verapamil has an effect on intracellular uptake and cytotoxicity of anthracyclines on tumor cells from patients with acute non-lymphoblastic leukemia. The prognostic and therapeutic relevance of this has to be further evaluated.

CCNU toxicity after an overdose in a patient with Hodgkin's disease. Effects on colony-forming cells (CFU-C) and colony-stimulating activity (CSA).

An overdose of CCNU (600 mg over a 15-d period) was unintentionally ingested by a patient with advanced Hodgkin's disease subjected to combination chemotherapy. A severe bone marrow depression occurred 3 weeks after the start of the CCNU treatment. The nadir of the platelet count was reached after 4 weeks and that of the granulocyte count after 5 weeks. At the nadir of the white blood cell count, colony-forming cells (CFU-C) were found in significantly reduced numbers in the bone marrow, and were not found at all in the peripheral blood; the amount of colony-stimulating activity (CSA) produced by peripheral blood cells was reduced. However, the cells producing CSA recovered earlier than the CFU-C, and the CSA peak value was reached about 1 week before the peak value for CFU-C in the bone marrow. Thus, in vivo CSA-producing cells appeared to be more resistant to CCNU than were CFU-C, and their recovery appeared to be a prerequisite for the recovery of CFU-C and myelopoietic cells.

A controlled comparison of two different clinical grade devices for CD34+ cell selection of autologous blood stem cell grafts.

Six patients who were to undergo autologous PBSC transplantation with positively selected CD34+ cells were included in this study to compare the efficiency of two devices for clinical grade stem cell selection, the Isolex 300i (Baxter, Munich, Germany) and CEPRATE SC (CellPro, Bothell, WA). PBSC were mobilized by chemotherapy and G-CSF and were collected by leukapheresis on a CS3000 cell separator on 2 consecutive days. The two apheresis products were pooled for CD34 selection. The pooled apheresis products from each patient were divided into two equal portions to be separated on each of the two devices. Cell selection was performed according to the manufacturers' instructions. Enumeration of CD34+ cells was performed by flow cytometry using the HPCA-2 MAb. Purity and yield were significantly better with Isolex than with CEPRATE. Median purity was 93.0% (range 80%-98%) for Isolex and 61.5% (range 27%-72%) for CEPRATE (p = 0.03); median yields for Isolex and for CEPRATE were 48.0% (range 18%-73%) and 23.0% (range 17%-29%), respectively (p = 0.03). The number of CD34+ cells/kg body weight was also significantly higher with Isolex (median 3.8x10(6), range 1.7-5.2) compared with CEPRATE (median 2.35x10(6), range 0.7-4.3) (p = 0.03). Thus, the Isolex 300i device gave products of higher purity and recovered a higher proportion of the CD34+ cells in the harvest before separation. The yield was still poor with both devices, however, and further optimization of the technique for clinical grade stem cell selection is warranted.

Effect of cytokines on the toxicity of cytostatic drugs on leukemic cells in vitro and in vivo.

Most cytostatic drugs exert their effect on cells in active cell cycle. To improve the effect of cytostatic drugs we have tried, prior to treatment in vitro, to recruit tumor cells from G0 with growth factors. Leukemic cells from the bone marrows of 26 patients with AML and CML in blast crisis were incubated with G-CSF, GM-CSF and IL-3 for 24 h prior to incubation with cytostatic drugs. The cells were incubated with mitoxantrone, etoposide or daunorubicin for 1 h, or with Ara-C continuously. Prior to treatment, 4 patients with AML received GM-CSF for 24 h, after which blast cells from bone marrow were incubated with cytostatic drugs. After incubation with the cytostatic drugs, cells were cultured in a suspension culture for 4 d. The drug effect was determined with a bioluminescence ATP method. Leukemic cells were significantly stimulated by all three cytokines compared to an untreated control. GM-CSF and IL-3 increased the amount of cells 3- to 4-fold and G-CSF increased the amount 3 times compared to untreated cells. G-CSF significantly enhanced the cytotoxic effect of daunorubicin, mitoxantrone, etoposide and Ara-C by 20-40%, which GM-CSF and IL-3 showed a significantly increased toxicity for Ara-C only. Although the cytokines induced a higher percentage of cells killed with the cytostatic drugs, proliferation of the remaining cells resulted in an increased total number of cells from 1.5 to 3 times compared to the unstimulated incubations. We conclude that cytokines induce a higher level of toxicity of cytostatic drugs on leukemic cells, but the increased proliferation of the remaining cells may offset the clinical benefit.

In vitro drug testing in patients with acute leukemia with incubations mimicking in vivo intracellular drug concentrations.

The differential staining cytotoxicity (DiSC) assay was evaluated as a predictive test for response to therapy in patients with acute non-lymphoblastic leukemia. Incubations were designed in such a way that the intracellular concentrations of cytostatic drugs in vitro paralleled those in vivo. Leukemic cells were isolated from 53 patients with acute non-lymphocytic leukemia. 13 of these patients died early due to supportive care failure and were not evaluable for the predictive drug testing. Of the remaining 40 patients, 25 entered a complete remission (CR) and 15 had a resistant disease (RD). According to the patients randomization to therapy the cells were incubated with anthracyclines and Ara-C separately and in combination. After 4 days of culturing in liquid medium the in vitro cytotoxicity was determined by dye exclusion according to Weisenthal. The cytotoxic effect in vitro was significantly higher on cells from patients who achieved a CR compared to patients with RD after incubations with anthracyclines 0.2 mumol/l (p less than or equal to 0.005), Ara-C 0.5 mumol/l (p less than or equal to 0.05) and with the combination of anthracyclines with Ara-C (p less than or equal to 0.0005). The best predictive value was achieved when incubations with 0.2 mumol/l anthracyclines and 0.5 mumol/l Ara-C were analyzed together. With these incubations cells from 20 out of 21 patients who achieved CR showed either less than or equal to 60% surviving cells after the anthracycline incubation or less than or equal to 35% surviving cells after the Ara-C incubation. Cells from 11 out of 13 patients with RD did not fulfill either of these criteria. In vitro drug sensitivity was significantly correlated to a prolonged survival (p less than 0.01). We conclude that, when performed with incubations that mimic in vivo tumor cell exposure to cytostatic drugs, the DiSC assay shows a high correlation to clinical outcome for patients with acute non-lymphocytic leukemia.

High single dose of mitoxantrone and cytarabine in acute non-lymphocytic leukemia: a pharmacokinetic and clinical study.

In a phase I-II study, the authors evaluated the intracellular pharmacokinetics, toxicity, and efficiency of a high dose of mitoxantrone given as first induction in acute non-lymphocytic leukemia. Twenty-two patients with previously untreated de novo ANLL were included and received 30 or 40 mg/m2 mitoxantrone on day 1 by intravenous infusion over 1 hour and 500 mg/m2 ara-C twice a day for 5 days. If there was no complete remission (CR), a second induction with ara-C, etoposide, and amsacrine was given. The CR rate after two courses with this regimen was 77%. Median duration of severe neutropenia was 18 days in the 30-mg/m2 group and 25 days in the 40-mg/m2 group. Two patients had fatal lung complications probably unrelated to mitoxantrone. A third patient had a possible mitoxantrone-induced reversible lung complication. In the leukemic cells, we found a high accumulation of mitoxantrone which, in contrast to the plasma concentration, remained stable during the 48 hours studied. Compared with previous results with 12 mg/m2 mitoxantrone, the AUC for intracellular concentrations versus time for the first 20 hours studied was increased by 150% to 0.638 nmol/mg cell protein x hour with 30 mg/m2 mitoxantrone and by 260% to 1.103 nmol/mg cell protein x hour with 40 mg/m2 mitoxantrone. In conclusion, a high dose of mitoxantrone results in a high intracellular exposure of the leukemic cells, which may be an advantage in improving survival of these patients.