Continuous Drug Exposure Research Articles

Side Effects on the Skeletal System Exerted by Continuous and by Interval Treatment with Tyrosine Kinase Inhibitors in An Animal Model of Juvenile Rodents,

Abstract 3741 Background:Chronic myeloid leukemia (CML) is a rare malignancy in children. The oncogenic transformation of hematopoietic stem cells results from the dysregulated tyrosine kinase (TK) activity of the BCR-ABL fusion protein. As front line treatment the TK inhibitor (TKI) imatinib (IMA; Gleevec®, Novartis) has radically changed the hematological, cytogenetic and molecular response rates in adults as well as in children with CML. IMA blocks the BCR-ABL oncogene, however, as side effect also related TKs (c-FMS, PDGF-R) which are important for bone (re)modeling are inhibited. This may result in impaired development and activity of osteoclasts and -blasts with consecutive disturbances in bone and mineral metabolism (Vandyke K, Blood 2010). In children with CML impaired longitudinal growth while on IMA treatment has been described (Millot F, ASH 2009 Abstr. #863; Shima H, J Pediatr 2011). Preliminary data in adults with CML treated intermittently with IMA (one month “on”, one month “off”) showed no negative impact on maintaining a complete cytogenetic remission (Rosti G, ASH 2010 Abstr. #3412). Aim:We hypothesized that i) in comparison to regular daily drug intake intermittent IMA treatment may cause less impairment on bone metabolism and ii) that the TKI bosutinib (BOSU, Pfizer) which does not inhibit PDGF-R might exert less bone damage. Therefore the impact of continuous and intermittent IMA and continuous BOSU treatment on the growing skeleton was investigated in juvenile rodents. Methods:Prepubertal (prep) 4 weeks (w) old, male Wistar rats were exposed via the drinking water to IMA over a 10 w period and to BOSU by micro-osmotic pumps (ALZET®, Charles River) subcutaneously over 4 w. Compared to controls independent groups were offered regular (500 mg/l IMA; 2.5 mg/kg BOSU) and high (1000 mg/l IMA; 5 mg/kg BOSU) doses daily, while a another group obtained high dose IMA intermittently only every half week, respectively. Parameters of bone morphology (epiphyseal cartilage width, bone length, bone mass density (BMD)) were analyzed using pQCT (XCT Research SA+, Stratec Medizintechnik) at defined time points (prep; pubertal (pub); postpubertal; (postp)). Results:Compared to controls, animals from all IMA-groups showed significantly shortened femoral and tibial lengths and a decrease in trabecular, but not cortical BMD (Fig. 1A). The alterations caused by intermittent IMA treatment corresponded strictly to the cumulative drug intake. Thus, alterations in rats receiving high dose IMA intermittently were identical to animals receiving a regular dose daily. Interestingly, rats receiving a high dose daily revealed a shortened epiphyseal line of the femoral cartilage during the whole period of TKI treatment while this effect could not be observed in the tibia (Tab. 1). If the high dose was applied intermittently also minor effects on the epiphyseal cartilage were observed. All effects were more pronounced in prep animals. Effects with BOSU on bone length were less pronounced in the high dosage group and no alterations could be observed with regular dosing (Fig. 1B). [Display omitted] Table 1:Width of the femoral and tibial epiphyseal cartilage [mm ± SD]agecontrolsregular dosehigh dosehigh dose; intermittentlypreppubpostppreppubpostppreppubpostppreppubpostpIMAfemur0.937 ± 0.0751.038 ± 0.11090.820 ± 0.0750.875 ± 0.051.033 ± 0.1150.716 ± 0.0290.850 ± 0.0410.866 ± 0.0760.883 ± 0.0761.075 ± 0.0640.983 ± 0.1530.900 ± 0.086tibia0.762 ± 0.0620.787 ± 0.0750.600 ± 0.0500.787 ± 0.0940.900 ± 0.000.600 ± 0.000.725 ± 0.0640.700 ± 0.050.583 ± 0.11550.675 ± 0.0640.816 ± 0.0570.600 ± 0.100BOSUfemur–1.075 ± 0.156––0.983 ± 0.126––0.983 ± 0.161––––tibia–0.700 ± 0.071––0.733 ± 0.029––0.683 ± 0.029–––– Conclusion:The rodent model presented is useful to study, predict, and mimic side effects of TKI treatment on bone metabolism. Impairments of the growing skeleton were comparable to the effects of IMA treatment as reported in children with CML. Preliminary data on BOSU exhibit minor bone side effects. As anticipated, intermittent IMA treatment revealed ∼50% less side effects on the growing bone than continuous drug exposure. However, whether this approach is also appropriate to sufficiently control CML in children has still to be proven in controlled clinical trials. Disclosures:Suttorp:Novartis: Research Funding; Pfizer: Research Funding.

Abstract B144: Elesclomol, an inducer of oxidative stress, targets oxidative phosphorylation (oxphos) in melanoma cells.

Abstract Mitochondria are important gatekeepers of cell survival in melanoma cells and their role as oxygen sensors, ATP producers, or inducers of reactive oxygen species are established. However, the role(s) of metabolic processes other than glycolysis are not well characterized in melanoma development and progression. Here we show that melanoma cells exhibit higher levels of oxidative phosphorylation (Oxphos) compared to human epidermal melanocytes. Furthermore, we present several independent lines of evidence (bioenergetics analysis using Seahorse XF24, stable isotope labeling of melanoma cells in culture, immunoblot analysis of isolated mitochondria, electron microscopy, and rho zero cells) that elesclomol, an inducer of oxidative stress, suppresses Oxphos in melanoma cells without a significant effect on glycolysis in vitro. Melanoma cells with intrinsically high levels of glycolysis are less sensitive to elesclomol. In addition, following chronic continuous drug exposure, resistant cells manifest a high glycolytic phenotype. Finally hypoxia inducible factor 1 alpha, a master metabolic switch of cell metabolism towards glycolysis, is upregulated following elesclomol treatment in a number of melanoma cell lines examined. Our results suggest that Oxphos is an important therapeutic target and that elesclomol specifically impinges on this process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B144.

An Overview of Drug-induced Liver Injury

Drug-induced liver injury (DILI) represents liver damage from various therapeutic drugs, herbal medicines, plants, or nutritional supplements. DILI is the most frequent reason for drug withdrawal and may lead to acute liver failure, liver transplant, or fatality. Its pathophysiology includes direct hepatotoxicity and idiosyncratic immune reactions. The diagnosis requires serum alanine aminotransferase or alkaline phosphatase elevation > 2-fold above the upper limit of normal, a causality assessment score ≥ 3, and exclusion of other possible causes of liver disease. While early diagnosis and drug discontinuation are imperative to recovery, continued drug exposure may lead to serious undesirable outcomes.

Paclitaxel-Eluting Polymer Film Reduces Locoregional Recurrence and Improves Survival in a Recurrent Sarcoma Model: A Novel Investigational Therapy

Locoregional recurrences occur in up to 50% of patients after macroscopically complete (R0/R1) resections of abdominal, pelvic, and retroperitoneal sarcomas. Efficacy of a drug-eluting polymer film in reducing locoregional recurrence rates was assessed in a murine recurrent sarcoma model. Poly(glycerol monostearate-co-caprolactone) polymer films were synthesized with and without 300μg paclitaxel (Pax-film and unloaded film). Cytotoxicity was assessed against CS-1 (human chondrosarcoma) cells in vitro and in vivo in nude mice. Following R0/R1 resection of primary subcutaneous tumors, mice were blindly randomized to: (1) Pax-film implant, (2) unloaded film implant, (3) paclitaxel 300μg IV (Pax IV), or (4) no other therapy ("untreated"). Locoregional recurrence, overall survival (OS), and tumor mitotic index were evaluated. Pax-films, but not unloaded films, reduced CS-1 viability in vitro for >50days (P<0.001). In vivo, locoregional recurrence was observed in 2 of 12 Pax-film mice (17%), 9 of 13 unloaded film mice (69%), 8 of 9 Pax IV mice (89%), and 7 of 8 untreated mice (88%) (P<0.01). Median OS was 81, 64, 48, and 56days, respectively. Paclitaxel levels in local tissues were 50- to 300-fold greater in Pax-film mice compared with Pax IV mice. Tumor mitotic index adjacent to Pax-films was significantly lower than adjacent to unloaded films. Tumor bed implantation of Pax-films after R0/R1 resection is superior to Pax IV as evidenced by reduced locoregional recurrence and improved OS in a murine recurrent sarcoma model. Continuous local drug exposure via polymer films represents a potentially novel approach for treatment of locally aggressive sarcomas.

Episcleritis Related to Drug-Induced Lupus Erythematosus following Infliximab Therapy: A Case Report

Drug-induced lupus erythematosus is defined as a lupus-like syndrome temporally related to continuous drug exposure which resolves after discontinuation of the offending drug. Herein, we describe a patient with distinct clinical manifestations of anti-TNF-associated DILE related to infliximab therapy. The patient exhibited clinical and laboratory findings of lupus-like illnesses as well as ocular disorders, such as episcleritis. The main message is that the symptoms of DILE should not be overlooked, although sometimes other systematic conditions may underlie them. As a result, it is very important for the clinicians to evaluate the symptoms of DILE and manage appropriately these cases.

A Case of Angiotensin Converting Enzyme Inhibitor-Induced Systemic Lupus Erythematosus

Drug-induced lupus erythematosus is defined as a lupus-like syndrome related to continuous drug exposure which resolves after discontinuation of the offending drug. Here we report a case of a 70-year-old man who developed drug-induced lupus erythematosus after receiving angiotensin converting enzyme inhibitor medication for unstable angina pectoris, for 5 years. He was hospitalized with arthralgia, edema, and newly developed pleural effusion. The serum analysis revealed an elevated level of antinuclear

Amrubicin Targets Nuclear Matrix-Bound Topoisomerase II In Human Myeloid Leukemia Cells

AbstractAbstract 1830Amrubicin, a synthetic anthracycline which is approved in Japan for the treatment of lung cancer, is currently being evaluated in a global phase III trial for second line treatment of SCLC. Anthracyclines can exert cytotoxicity through inhibiting topoiosomerase II (Topo II), resulting in the blockade of DNA transcription and replication. Similar to etoposide, Amrubicin also is thought to induce covalent DNA protein complexes (stabilized DNA-topo II complexes) and double strand DNA breaks. In this study, we compared the cytotoxic and DNA damage-inducing effects of amrubicin to two other Topo II inhibitors and evaluated their effects on purified human Topo II as well as on the MV4-11 leukemia cell line. Cellular cytotoxicity assays demonstrated that amrubicin, etoposide and daunorubicin induced MV4-11 leukemia cell death at EC50's of 0.7 μM, 1.8 μM and 0.2 μM, respectively. In addition, DNA damage as represented by H2AX phosphorylation, was observed following drug treatment but was less extensive with amrubicin than with daunorubicin. One level of DNA organization consists of the folding of chromatin into 50–100 kb loops, which are attached at their bases to the nuclear matrix. The matrix attachment regions (MARS) of DNA contain strong Topo II binding sites and mediate the attachment of the chromatin loops to the nuclear matrix. We examined the effects of amrubicin on purified human Topo II, nuclear matrix-bound Topo II, and Topo II activity in MV4-11 leukemia cells in order to gain insights into the mechanism of amrubicin-induced apoptosis. The results were compared to those obtained with the “classical” Topo II-active drugs, etoposide and daunorubicin. Amrubicin, etoposide and daunorubicin inhibited the DNA strand passage reaction of purified human Topo II by stabilizing the formation of Topo II-DNA cleavable complexes (at IC50 values of 20 μM, 750 μM and 4 mM, respectively). In MV4-11 leukemia cells, cleavable complex stabilization by IC50 concentrations of etoposide and daunorubicin resulted in extensive DNA fragmentation from 1–50 kb. In contrast, DNA cleavage by amrubicin was limited to the formation of >50 kb fragments even after 24 hrs of continuous drug exposure. The >50 kb fragments were similar in size to the 50–100 kb chromatin loops that are attached to the nuclear matrix. The hypothesis that amrubicin inhibits selectively nuclear matrix-bound Topo II resulting in detachment of the chromatin loops from the matrix was examined directly by using isolated nuclear matrix preparations from MV4-11 cells. Amrubicin was much more potent in inducing chromatin loop release from isolated nuclear matrix preparations than either etoposide or daunorubicin despite the fact that all three drugs stabilized Topo II-DNA cleavable complex formation. Chromatin loop release by amrubicin was Mg++ dependent and reversible by 4 mM ATP indicating that it was Topo II mediated. Taken together, our results suggest that in MV4-11 cells amrubicin selectively targets nuclear matrix-bound Topo II, while etoposide and daunorubicin are less selective and induce generalized and extensive DNA damage. Exposure to etoposide and daunorubicin is associated with risk of myelodysplastic syndrome and secondary acute myeloid leukemia, which may be related to their ability to induce generalized DNA damage. As a more selective DNA damaging agent, further clinical development of amrubicin is warranted. Disclosures:Fernandes:celgene corp: Consultancy, Research Funding. Sung:celgene corp: Employment.

Assessment of genotoxicity associated with hydroxyurea therapy in children with sickle cell anemia

Hydroxyurea induces fetal hemoglobin, improves laboratory parameters, and ameliorates clinical complications of sickle cell anemia (SCA), but its long-term efficacy and safety in this patient population remain incompletely defined. Although generally considered non-DNA reactive, an important safety concern is that hydroxyurea may indirectly cause genotoxic damage. To better address this safety issue of hydroxyurea in patients with SCA, we measured the production of micronuclei (MN) in red blood cells (RBCs) as a marker of genotoxicity. Blood samples were collected from children with SCA enrolled in the Hydroxyurea Study of Long-term Effects (ClinicalTrials.gov NCT00305175). Flow cytometry quantified circulating MN-containing erythrocyte sub-populations before and during hydroxyurea exposure. The frequency of micronucleated reticulocytes (MN-CD71(+)) and micronucleated mature erythrocytes (MN-RBC) was then tested for associations with laboratory and clinical data. In cross-sectional analysis of 293 blood samples from 105 children with SCA and a median of 2 years of hydroxyurea therapy, exposure to hydroxyurea was associated with significantly increased frequencies of MN-CD71(+) and MN-RBC compared to baseline. The increases were evident by 3 months of therapy, and did not escalate further with up to 12 years of continuous drug exposure. In prospective longitudinal analysis, substantial inter-individual variation in the effect of hydroxyurea on %MN-CD71(+) was observed that was associated with the expected laboratory effects of hydroxyurea. In conclusion, clinically relevant exposure to hydroxyurea is associated with increased MN production consistent with erythroblast genotoxicity but with substantial inter-patient variability. Associations between increased %MN-CD71(+) and laboratory benefits suggest that hydroxyurea effects on MN production may be related to individual patient sensitivity to hydroxyurea within the bone marrow.

A novel size-tunable nanocarrier system for targeted anticancer drug delivery

We have developed a nanocarrier drug-delivery system based on micelles formed by a new class of well-defined linear PEGylated two-arm oligomer of cholic acids in aqueous solution. By varying the length of the linear PEG chains and the configuration of cholic acid oligomer, one can easily fine-tune the physicochemical properties of the amphiphilic polymers and the resulting micelles. These include particle size, critical micelle concentration, and drug-loading capacity. High level of hydrophobic anticancer drugs such as PTX, etoposide and SN-38 can be readily loaded into such nanocarriers. The loading capacity of the nanocarrier for PTX (PTX) is extremely high (12.0 mg/mL), which is equivalent to 37.5% (w/w) of the total mass of the micelle. PTX-loaded nanocarriers are much more stable than Abraxane ® (PTX/human serum albumin nanoaggregate) when stored in bovine serum albumin solution or dog plasma. PTX release profile from the micelles is burst-free and sustained over a period of seven days. The anti-tumor activity of PTX-loaded nanocarriers against ovarian cancer cell line in vitro, with continuous drug exposure, is similar to Taxol ® (formulation of PTX dissolved in Cremophor EL and ethanol) or Abraxane ®. Targeted drug delivery to tumor site with these novel micelles was demonstrated by near infrared fluorescence (NIRF) imaging in nude mice bearing ovarian cancer xenograft. Furthermore, PTX-loaded nanocarriers demonstrated superior anti-tumor efficacy compared to Taxol ® at equivalent PTX dose in ovarian cancer xenograft model.

Radiation- and Chemoinduced Multidrug Resistance in Colon Carcinoma Cells

Radiation can induce multidrug resistance (MDR) and thus interfere with simultaneous or subsequent chemotherapy. In SW620 colon carcinoma cells, the interrelation of various biological endpoints of MDR was analyzed and the potential of fractionated irradiation and chemoselection to evoke MDR was compared. To induce/select an MDR phenotype, SW620 were exposed to either 27 Gy in 1.8-Gy daily fractions or to 50% inhibiting concentrations of doxorubicin or cisplatin, given over 6-15 weeks. Expression of genes involved in MDR, including glutathione metabolism, was determined by semiquantitative RT-PCR (reverse transcription-polymerase chain reaction). Efflux was analyzed by flow cytometry after staining with rhodamine-123 or 5-chloromethyl fluorescein diacetate. Apoptosis was monitored after pulse exposure to doxorubicin or cisplatin. Colony-forming assays were performed under continuous drug exposure. A pronounced gene induction was found in MRP2 after cisplatin selection and up to 3 weeks after radiation. LRP was activated only shortly after radiation. Radiation enhanced rhodamine-123 efflux to a similar extent as short-term chemoselection but not as much as long-term drug exposure. Drug-induced apoptosis was slightly delayed in preirradiated cells. Clonogenic growth in the progeny of irradiated cells was less sensitive to cisplatin but not to doxorubicin. Fractionated radiation can induce an MDR phenotype in SW620. However, long-term drug exposure establishes a more efficient selection. Various endpoints are not fully concordant regarding the extent of MDR. Posttranscriptional modifications, pleiotropic regulation, and alternative pathways may cause these discrepancies.

Assessment of Genotoxicity Associated with Hydroxyurea Therapy in Children with Sickle Cell Anemia.

Abstract 2554Poster Board II-531 Introduction:Hydroxyurea induces fetal hemoglobin (HbF), improves laboratory parameters, and reduces acute clinical complications of sickle cell anemia (SCA), but its long-term efficacy and safety remain incompletely defined. One long-term safety concern is that hydroxyurea may elicit DNA alterations via genotoxic damage. During normal erythropoiesis, red blood cells (RBC) extrude their nucleus as they develop into functional reticulocytes. Occasionally, membrane bound DNA remains in the cell after erythrocyte maturation and these inclusion bodies are known as micronuclei (MN) or Howell-Jolly Bodies. MN-containing reticulocytes are formed at higher frequency upon exposure to genotoxic agents. Patients with SCA have increased basal MN production while also having decreased MN clearance due to diminished splenic filtrative function. In a previous small cross sectional study, we showed that hydroxyurea exposure further increases MN production in SCA patients. To better address this long-term safety issue of hydroxyurea, we evaluated MN production and clearance both in a large cross-sectional and prospective study of children with SCA on hydroxyurea therapy. Patients and Methods:A high-throughput flow cytometric technique was used to detect and quantitate MN within circulating erythrocyte subpopulations. After written informed consent, venous blood samples were collected from children with SCA enrolled in the Hydroxyurea Study of Long-term Effects (HUSTLE, ClinicalTrials.gov NCT00305175). A total of 105 subjects had at least 1 MN measurement, including 37 subjects with serial measurements at baseline and at follow-up time points up to 24 months of hydroxyurea exposure. MN were quantified in both reticulocytes (MN-CD71(+)) and mature RBC (MN-RBC), and then tested for associations with individual subject laboratory and clinical data. Results:In cross-sectional analysis of 293 samples from 105 children with SCA and a median of 2 years hydroxyurea exposure (range 3 months – 12 years), hydroxyurea therapy significantly lowered %CD71(+) reticulocytes (mean fold reduction 0.53 ± 0.44, p < 0.001). Compared to baseline values, hydroxyurea treatment increased levels of MN in both reticulocytes (%MN-CD71(+), mean fold increase 1.80 ± 0.91, p < 0.05) and mature RBC (%MN-RBC, mean fold increase 1.89 ± 1.39, p<0.01). The increase in MN-CD71(+) was evident by 6 months of hydroxyurea treatment, but did not significantly escalate further with up to 12 years of continued drug exposure (Fig. 1). [Display omitted] To prospectively determine the genotoxic effect of hydroxyurea, serial measurements over 2 years were performed on 37 patients. After 9 months on hydroxyurea therapy all subjects were on a stable maximum tolerated dose (MTD, average 25.1 mg/kg/day); 15 of 37 children had > 3.0 fold increase in %MN-CD71(+) while 22 of 37 had < 3.0 fold increase (Mean 3.68 ± 0.65 vs. 1.52 ± 0.52, p < 0.001). The observed inter-individual variation was associated with the predicted laboratory effects of hydroxyurea; increases in %MN-CD71(+) were positively correlated with MTD values of HbF (r2=0.22, p=0.005), mean corpuscular volume (r2=0.35, p=0.002), and mean corpuscular hemoglobin (r2=0.29, p=0.006) but negatively correlated with absolute neutrophil count (r2=0.14, p=0.02) and bilirubin levels (r2=0.20, p=0.008). There were no significant associations between %MN-CD71(+) and gender, age, or hydroxyurea dosage although %MN-RBC clearance decreased with age. Conclusions:A highly sensitive and quantitative flow cytometric technique can detect circulating MN-containing erythrocytes and this technique may be used to assess the in vivo genotoxic effect of any drug. Children with SCA have high basal MN production that is probably related to the degree of erythropoiesis in these patients. Hydroxyurea therapy was associated with genotoxicity but with substantial inter-patient variability in hydroxyurea induced %MN-CD71(+) levels. The increases in %MN-CD71(+) are observed within 6 months of starting hydroxyurea therapy but persist at the same level in patients with up to 12 years of continued drug exposure. Correlations between increased %MN-CD71(+) and predicted hydroxyurea effects on other laboratory parameters suggest that hydroxyurea induces measurable genotoxicity that may be related to individual patient sensitivity and efficacy of hydroxyurea within the bone marrow. These patients will be monitored further to confirm that hydroxyurea does not pose any long term safety issues. Disclosures:Off Label Use: Off label use of hydroxyurea in children with sickle cell anemia. Avlasevich:Litron Laboratories: Employment. Dertinger:Litron Laboratories: Employment.

Downregulation of the Common Cytokine Receptor Subunit Beta c by Omacetaxine in CML: A Potential Molecular Mechanism to Overcome Cytokine-Mediated Resistance against BCR-ABL-Inhibitors.

Abstract 3256Poster Board III-1▪▪This icon denotes an abstract that is clinically relevant. Introduction:Inhibition of BCR-ABL by imatinib is standard treatment for patients with chronic myeloid leukemia (CML). Despite favourable response rates, overcoming primary and secondary resistance against BCR-ABL inhibitors is of upmost clinical importance. A proposed mechanism of CML stem-cell survival is cytokine-dependent activation of anti-apoptotic and promitogenic signalling cascades despite continued treatment with tyrosine kinase inhibitors (TKI). Additionally, resistance inducing point mutations of the BCR-ABL kinase domain besides lowering the drug-target affinity of TKIs also seem to interfere with cytokine signalling by not yet fully understood mechanisms. Omacetaxine mepesuccinate (OM, formerly known as homoharringtonine) is showing promising results in phase II clinical trials even in patients with the highly resistant mutation T315I. Preliminary results suggest deselection of the aggressive T315I-clone by OM. We therefore went back to pre-clinical resistance models to study mechanisms of OM-dependent antileukemic activity. Methods:Two murine cell lines with induced expression of unmutated and T315I-mutant BCR-ABL (32Dp210, Baf3p210), and a human CML cell line with induced imatinib-resistance due to continuous drug exposure (KBM5s, KBM5r-T315I) were used in addition to primary CD34+ enriched stem cell cultures. Results:Dye exclusion proliferation assays confirmed BCR-ABL-dependent sensitization of cell lines to OM in the low nanomolar dose range. Expression of BCR-ABL-mutant T315I does not confer cross-resistance to OM in myeloid cell lines. However, it slightly reduces OM-sensitivity in lymphoid Baf3p210-T315I cells with a 2.5-fold increased IC50-value. OM induced suppression of cell viability is mediated by Caspase-3-dependent apoptosis as detected by flow cytometry. Addition of Interleukin-3 (IL3), shown to revert BCR-ABL-inhibitor dependent cytotoxicity in the murine 32Dp210 and Baf3p210 cell lines, does not affect OM-induced antiproliferative activity. In addition, cytotoxicity of OM against CD34-enriched CML stem cells grown in the presence of a high vs low physiological growth factor mix (GF) is unaffected by the respective GF-condition. Looking at potential mechanisms, we found marked OM-induced downregulation of the beta-subunit of the IL3-receptor (IL3-R) in both, cell lines, and primary stem cell cultures as detected by Western blot, irrespective of the mutational status, at the clinically relevant concentration of 40 nM OM. Due to the significantly reduced IL3-R-expression in BCR-ABL-T315I-expressing cells compared to unmutated BCR-ABL-expressing cells, OM-treatment leads to near complete eradication of the IL3-R in BCR-ABL-T315I positive cells. Combined treatment of Baf3p210 cells with Nilotinib and OM reveals that OM overrides cytokine mediated rescue of TKI treatment. Conclusions:The observed cytokine-independent in-vitro cytotoxicity of OM may be explained by OM-induced suppression of IL3-R-expression. Loss of IL3-R-expression in BCR-ABL-T315I expressing cells could be one mechanism governing the clinically observed deselection of the resistant clone in patients treated with OM. Cytokine receptor directed action of OM in CML needs to be explored as a potential strategy to overcome cytokine dependent resistance development against TKI-treatment, and to further explore OM-activity against disease maintaining stem cells. Disclosures:Craig:ChemGenex: Employment.

Multidrug Resistance in a Urothelial Cancer Cell Line After 1-Hour Mitomycin C Exposure

A factor pertinent to the design of cancer chemotherapy is multidrug resistance. Research in this area conventionally involves in vitro models using resistant cell lines generated by continuous low dose drug exposure for many months, unlike the exposure experienced by residual superficial bladder cancer cells during chemotherapy adjuvant to resection. Recently we noted a measure of multidrug resistance induced by 3 short exposures to mitomycin C during 10 weeks. We currently report detectable functional resistance after a single 1-hour insult. RT112 bladder cancer cells (Catalog No. ACC 418, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) were exposed to a range of mitomycin C concentrations for 1 hour. Cells regrew in 3 of 24 cultures at 15.6, 3.91 and 0.98 mg/ml exposure. These cells were subjected to 3 functional tests of cross resistance to epirubicin, including MTT cytotoxicity assay, quantitative accumulation by flow cytometry and nuclear uptake or exclusion by live cell fluorescence microscopy. MTT assay and flow cytometry revealed clear indications of resistance. Intracellular distribution, in which nuclear exclusion indicates resistance, was distinctively resistant in 1 subline and another 2 were equivocal. Results indicate that some multidrug resistance potential exists even in a cloned cell line that is capable of surviving 1 short drug exposure and expanding after that insult. The exposures used are consistent with those probably experienced by many superficial transitional cell carcinoma cells during an intravesical chemotherapy application. The result gives added weight to considering multidrug resistance induction in dose scheduling or drug combinations for topical chemotherapy.

Drug-induced lupus: an update on its dermatologic aspects

Drug-induced lupus erythematosus (DILE) is defined as an entity characterized by clinical manifestations and immunopathological serum findings similar to those of idiopathic lupus but which is temporally related to continuous drug exposure and resolves after discontinuation of the offending drug. Similar to idiopathic lupus, DILE can be divided into systemic lupus erythematosus (SLE), subacute cutaneous lupus erythematosus (SCLE) and chronic cutaneous lupus erythematosus (CCLE). Based on the literature review and retrospective analysis of our case series, we focused on the dermatological aspects of DILE. The cutaneous features of drug-induced SLE are protean, including particularly purpura, erythema nodosum and photosensitivity as well as the skin lesions characterizing both urticarial and necrotizing vasculitis. The typical laboratory profile of systemic DILE consists of positive antinuclear antibodies (ANA) and antihistone antibodies, the latter being regarded as the serum marker of this subset. The drugs most frequently implicated in the development of systemic DILE are hydralazine, procainamide, isoniazid and minocycline. Drug-induced SCLE usually presents with annular polycyclic or papulosquamous cutaneous manifestations as in the idiopathic form, but blisters or targetoid lesions mimicking erythema multiforme cannot rarely be associated. The clinical presentation is often generalized, with involvement of the lower legs that are usually spared in idiopathic SCLE. ANA and anti-Ro/SSA antibodies are usually present, whereas antihistone antibodies are uncommonly found. Drugs associated with SCLE include particularly calcium channel blockers, angiotensin-converting enzyme inhibitors, thiazide diuretics, terbinafine and the recently reported tumour necrosis factor (TNF)-alpha antagonists. Drug-induced CCLE is very rarely described in the literature and usually refers to fluorouracile agents or TNF-alpha antagonists. The picture is characterized by the occurrence of classic discoid lesions, but aspects of lupus tumidus can occasionally develop. ANA are demonstrated in around two-thirds of the cases. Management of DILE is based on the withdrawal of the offending drug. Topical and/or systemic corticosteroids and other immunosuppressive agents should be reserved for resistant cases.

Vorinostat increases carboplatin and paclitaxel activity in non‐small cell lung cancer cells

We observed a 53% response rate in non-small cell lung cancer (NSCLC) patients treated with vorinostat plus paclitaxel/carboplatin in a Phase I trial. Studies were undertaken to investigate the mechanism (s) underlying this activity. Growth inhibition was assessed in NSCLC cells by MTT assay after 72 hr of continuous drug exposure. Vorinostat (1 microM) inhibited growth by: 17% +/- 7% in A549, 28% +/- 6% in 128-88T, 39% +/- 8% in Calu1 and 41% +/- 7% in 201T cells. Vorinostat addition to carboplatin or paclitaxel led to significantly greater growth inhibition than chemotherapy alone in all 4 cell lines. Vorinostat (1 microM) synergistically increased the growth inhibitory effects of carboplatin/paclitaxel in 128-88T cells. When colony formation was measured after drug withdrawal, vorinostat significantly increased the effects of carboplatin but not paclitaxel. The % colony formation was control 100%; 1 microM vorinostat, 83% +/- 10%; 5 microM carboplatin, 41% +/- 11%; carboplatin/vorinostat, 8% +/- 4%; 2 nM paclitaxel, 53% +/- 11%; paclitaxel/vorinostat, 46% +/- 21%. In A549 and 128-88T, vorinostat potentiated carboplatin induction of gamma-H2AX (a DNA damage marker) and increased alpha-tubulin acetylation (a marker for stabilized mictrotubules). In A549, combination of vorinostat with paclitaxel resulted in a synergistic increase in alpha-tubulin acetylation, which reversed upon drug washout. We conclude that vorinostat interacts favorably with carboplatin and paclitaxel in NSCLC cells, which may explain the provocative response observed in our clinical trial. This likely involves a vorinostat-mediated irreversible increase in DNA damage in the case of carboplatin and a reversible increase in microtubule stability in the case of paclitaxel.

Pegylated interferon alfa-2B induced lupus in a patient with chronic hepatitis B virus infection: case report

Drug-induced lupus is defined as a lupus-like syndrome temporally related to continuous drug exposure which resolves after discontinuation of the offending drug. Newer biological agents such as interferons (IFN) and anti-tumor necrosis factor-alpha therapies have been involved in the induction of lupus-like syndromes. Prompt recognition and discontinuation of the suspected drug are associated with excellent outcomes in most cases. In this article, we present the case who developed lupus during therapy with pegylated-IFN alpha-2b for chronic hepatitis B virus infection after 8 months.

Metronomic Chemotherapy

Chemotherapy drugs are usually administered at doses that are high enough to result in an obligatory break period to allow for the observation of potential side effects and institution of supportive care, if required. In recent years, efforts to administer chemotherapy on a more continuous basis, with a much shorter break period, or none at all, have received increased interest, and the practice has come to be known as metronomic chemotherapy. The basis for success with this currently investigational approach may be rooted in continuous drug exposure to susceptible cancer cells, inhibition of tumor blood vessel growth-a process known as tumor angiogenesis, and/or alterations in tumor immunology. Increased benefit also appears to occur when metronomic chemotherapy is used in combination with newer, targeted antiangiogenic agents, and therefore represents a promising approach to combination therapy, particularly as targeted oncology drugs make their way into veterinary oncology applications. There is still much to be learned in this field, especially with regard to optimization of the proper drugs, dose, schedule, and tumor applications. However, the low cost, ease of administration, and acceptable toxicity profiles potentially associated with this therapeutic strategy make metronomic chemotherapy protocols attractive and suitable to veterinary applications. Preliminary clinical trial results have now been reported in both human and veterinary medicine, including adjuvant treatment of canine splenic hemangiosarcoma and incompletely resected soft tissue sarcoma, and, further, more powerful studies are currently ongoing.

Behavioral adaptation in C. elegans produced by antipsychotic drugs requires serotonin and is associated with calcium signaling and calcineurin inhibition

Chronic administration of antipsychotic drugs produces adaptive responses at the cellular and molecular levels that may be responsible for both the main therapeutic effects and rebound psychosis, which is often observed upon discontinuation of these drugs. Here we show that some antipsychotic drugs produce significant functional changes in serotonergic neurons that directly impact feeding behavior in the model organism, Caenorhabditis elegans. In particular, antipsychotic drugs acutely suppress pharyngeal pumping, which is regulated by serotonin from the NSM neurons. By contrast, withdrawal from food and drug is accompanied by a striking recovery and overshoot in the rate of pharyngeal pumping. This rebound response is absent or diminished in mutant strains that lack tryptophan hydroxylase (TPH-1) or the serotonin receptors SER-7 and SER-1, and is blocked by serotonin antagonists, which implicates serotonergic mechanisms in this adaptive response. Consistent with this, continuous drug exposure stimulates an increase in serotonin and the number of varicosities along the NSM processes. Cyclosporin A and calcineurin mutant strains mimic the effects of the antipsychotic drugs and reveal a potential role for the calmodulin–calcineurin signaling pathway in the response of serotonergic neurons. Similar molecular and cellular changes may contribute to the long-term adaptive response to antipsychotic drugs in patients.

Clozapine-Induced ERK1 and ERK2 Signaling in Prefrontal Cortex Is Mediated by the EGF Receptor

The atypical antipsychotic drug clozapine is effective in treatment-refractory schizophrenia. The intracellular signaling pathways that mediate clozapine action remain unknown. A potential candidate is the mitogen-activated protein kinase extracellular signal-regulated kinase (MAPK-ERK) cascade that links G-protein-coupled receptor and ErbB growth factor signaling systems, thereby regulating synaptic plasticity and connectivity, processes impaired in schizophrenia. Here, we examined how clozapine differentially modulated phosphorylation of the MAPK isoforms, ERK1/ERK2 in primary murine prefrontal cortical neurons compared to the typical antipsychotic drug haloperidol. While clozapine and haloperidol acutely decreased cortical pERK1 activation, only clozapine but not haloperidol stimulated pERK1 and pERK2 with continued drug exposure. This delayed ERK increase however, did not occur via the canonical dopamine D(2)-Gi/o-PKA or serotonin 5HT(2A)-Gq-phospholipase-C-linked signaling pathways. Rather, epidermal growth factor (EGF) receptor signaling mediated clozapine-induced ERK activation, given dose-dependent reduction of pERK1 and pERK2 stimulation with the EGF receptor inhibitor, AG1478. Immunocytochemical studies indicated that clozapine treatment increased EGF receptor (Tyr1068) phosphorylation. In vivo mouse treatment studies supported the in vitro findings with initial blockade, subsequent activation, and normalization of the cortical ERK response over 24 h. Furthermore, in vivo clozapine-induced ERK activation was significantly reduced by AG1478. This is the first report that clozapine action on prefrontal cortical neurons involves the EGF signaling system. Since EGF receptor signaling has not been previously linked to antipsychotic drug action, our findings may implicate the EGF system as a molecular substrate in treatment-resistant schizophrenia.

Drug-induced lupus erythematosus

Drug-induced lupus erythematosus (DILE) is defined as a lupus-like syndrome temporally related to continuous drug exposure which resolves after discontinuation of the offending drug. There are currently no standard diagnostic criteria for DILE and the pathomechanisms are still unclear. Similarly to idiopathic lupus, DILE can be diveded into systemic (SLE), subacute cutaneous (SCLE) and chronic cutaneous lupus (CCLE). Systemic DILE is characterized by typical lupus-like symptoms including skin signs, usually mild systemic involvement and a typical laboratory profile with positive antinuclear and anti-histone antibodies, while anti-double strand (ds) DNA and anti-extractable nuclear antigens antibodies are rare. High risk drugs include hydralazine, procainamide and isoniazid. Drug-induced SCLE is very similar to idiopathic SCLE in terms of clinical and serologic characteristic, and it is more common than the systemic form of DILE. Drugs associated with SCLE include calcium channel blockers, angiotensin-converting enzyme inhibitors, interferons, thiazide diuretics and terbinafine. Drug-induced CCLE is very rarely reported in the literature and usually refers to fluorouracile agents or non steroidal anti-inflammatory drugs. Recently, cases of DILE have been reported with anti-TNFalpha agents. These cases present with disparate clinical features including arthritis/arthralgia, skin rash, serositis, cytopenia and variable laboratory abnormalities. DILE to anti-TNFalpha agents differs in several ways to classic DILE. The incidence of rashes is higher compared to classical systemic DILE. In most cases of classic DILE visceral involvement is rare, whereas several cases of anti-TNFalpha DILE with evidence of renal disease have been reported. Low serum complement levels as well as anti-extractable nuclear antigen antibodies and anti-dsDNA antibodies are rarely present in classic DILE, whereas they are reported in half the cases of anti-TNFalpha DILE; in contrast, anti-histone antibodies are described in classic DILE more often than in anti-TNFalpha DILE. Recognition of DILE in patients receiving anti-TNFalpha therapy can be difficult due to the symptoms of their underlying disease. A temporal association (months to years) of the offending drug with characteristic or suggestive symptoms, and resolution of symptoms on drug withdrawal is the best evidence for this diagnosis of DILE.