Continuous Drug Exposure Research Articles

Risk-Adjusted Therapy of Acute Lymphoblastic Leukemia Can Decrease Treatment Burden and Improve Survival: Treatment Results of 2169 Unselected Pediatric and Adolescent Patients Enrolled in the Trial ALL-BFM 95.

An unselected patient population of 2169 children with acute lymphoblastic leukemia (ALL) was enrolled in the multicenter trial ALL-BFM 95 of the Berlin-Frankfurt-Münster (BFM) study group. The study was designed to minimize acute and long-term toxicity through well-directed treatment reduction in a large patient subset with expectedly good prognosis. On the other hand extensive therapy intensification was implemented in a subgroup with very poor prognosis. The precondition was the clear definition of distinct risk groups enabled by a new risk stratification: patients (pts) with translocation t(9;22) and t(4;11), poor response to the cytoreductive prednisone prephase and/or to induction treatment were allocated to the high-risk (HR) group. Non-HR pts were stratified by age, white blood cell count (WBC) and immunophenotype to standard risk (SR, age 1–5 years, WBC <20 Gp/L, and no T-immunology) or medium risk (MR, all other pts). Compared to the previous trial ALL-BFM 90, the following treatment modifications were made in ALL-BFM 95: 1. reduction of the daunorubicin dose by 50% in induction in SR pts; 2. extension of maintenance by 12 months in SR boys; 3. in the MR group, randomized evaluation of intermediate-dose (ID) cytarabine in addition to high-dose methotrexate in the extracompartment/consolidation phase; 4. omission of preventive cranial radiotherapy (pCRT) in all MR pts with Non-T-ALL; 5. intensification of consolidation/reinduction in HR pts by dose increase in the block elements and reintroduction of protocol II as element providing a continuous drug exposure. Except for the randomized question, event-free survival was analyzed in comparison to the matching subsets of the historical control group treated in ALL-BFM 90. Estimated 6-year event-free survival (6y-pEFS) for the total group was 80±1% (median follow-up 7.3 years). The large SR group (35% of pts) achieved excellent 6y-pEFS of 89±1% despite significant reduction of the daunorubicin dose. The elongation of maintenance in boys yielded no significant relapse reduction. In MR (53% of pts), 6y-pEFS was 80±1%; no improvement was accomplished by the use of additional ID-cytarabine. Omission of pCRT in non-T-ALL MR pts was possible without reduction of EFS though the 6-year cumulative incidence of CNS relapses increased from 2.0±0.5% to 4.5±0.8%%. In the small HR group (12% of pts), treatment intensification led to considerable improvement over the previous ALL-BFM trials yielding a 6y-pEFS of 49±3%. Compared with the previous trial ALL-BFM 90, favorable results in SR and MR were firmly established despite significant treatment reduction in the majority of these patients.

Microfluidic Tissue Model for Live Cell Screening

We have developed a microfluidic platform modeled after the physiologic microcirculation for multiplexed tissue-like culture and high-throughput analysis. Each microfabricated culture unit consisted of three functional components: a 50 microm wide cell culture pocket, an artificial endothelial barrier with 2 microm pores, and a nutrient transport channel. This configuration enabled a high density of cancer cells to be maintained for over 1 week in a solid tumor-like morphology when fed with continuous flow. The microfluidic chip contained 16 parallel units for "flow cell" based experiments where live cells were exposed to a soluble factor and analyzed via fluorescence microscopy or flow-through biochemistry. Each fluidically independent tissue unit contained approximately 500 cells fed with a continuous flow of 10 nL/min. As a demonstration, the toxicity profile of the anti-cancer drug paclitaxel was collected on HeLa cells cultured in the microfluidic format and compared with a 384-well dish for up to 5 days of continuous drug exposure.

Induction of tachyzoite egress from cells infected with the protozoan Neospora caninum by nitro- and bromo-thiazolides, a class of broad-spectrum anti-parasitic drugs

Neospora caninum represents an important pathogen causing stillbirth and abortion in cattle and neuromuscular disease in dogs. Nitazoxanide (NTZ) and its deacetylated metabolite tizoxanide (TIZ) are nitro-thiazolyl-salicylamide drugs with a broad-spectrum anti-parasitic activity in vitro and in vivo. In order to generate compounds potentially applicable in food and breeding animals, the nitro group was removed, and the thiazole-moiety was modified by other functional groups. We had shown earlier that replacement of the nitro-group by a bromo-moiety did not notably affect in vitro efficacy of the drugs against N. caninum. In this study we report on the characterization of two bromo-derivatives, namely Rm4822 and its de-acetylated putative metabolite Rm4847 in relation to the nitro-compounds NTZ and TIZ. IC50 values for proliferation inhibition were 4.23 and 4.14μM for NTZ and TIZ, and 14.75 and 13.68μM for Rm4822 and Rm4847, respectively. Complete inhibition (IC99) was achieved at 19.52 and 22.38μM for NTZ and TIZ, and 18.21 and 17.66μM for Rm4822 and Rm4847, respectively. However, in order to exert a true parasiticidal effect in vitro, continuous culture of infected fibroblasts in the presence of the bromo-thiazolide Rm4847 was required for a period of 3 days, while the nitro-compound TIZ required 5 days continuous drug exposure. Both thiazolides induced rapid egress of N. caninum tachyzoites from their host cells, and egress was inhibited by the cell membrane permeable Ca2+-chelator BAPTA-AM. Host cell entry by N. caninum tachyzoites was inhibited by Rm4847 but not by TIZ. Upon release from their host cells, TIZ-treated parasites remained associated with the fibroblast monolayer, re-invaded neighboring host cells and resumed proliferation in the absence of the drug. In contrast, Rm4847 inhibited host cell invasion and respective treated tachyzoites did not proliferate further. This demonstrated that bromo- and nitro-thiazolides exhibit differential effects against the intracellular protozoan N. caninum and bromo-thiazolides could represent a valuable alternative to the nitro-thiazolyl-salicylamide drugs.

Time-dependent CYP inhibition

Time-dependent inhibition (TDI) of CYP refers to a change in potency during an in vitro incubation or dosing period in vivo. Potential mechanisms include the formation of inhibitory metabolites and mechanism-based inhibition (MBI). In vitro experiments are configured to assess TDI and MBI is inferred, at least initially. MBI is more profound after multiple-dosing and the recovery period is independent of continued drug exposure. Advances in in vitro–in vivo extrapolations for competitive inhibition and the potential relationship between MBI and reactive metabolite-mediated toxicity, have redirected emphasis to CYP TDI. In contrast, with reversible inhibition, strategies for projecting the risks from TDI are less developed and the traditional I/Ki model often yields a dramatic underprediction. This review explores the contribution of TDI to drug–drug interactions and idiosyncratic drug toxicity.

The FLT3 Inhibitor PKC412 Interacts Synergistically with Both Daunorubicin and Cytarabine in Acute Myeloid Leukemia (AML) Cells by Heterogeneous Mechanisms.

Both FLT3 activating mutations and overexpression of ATP-binding cassette (ABC) transport proteins that confer multidrug resistance (MDR) are associated with inferior treatment outcomes in AML. The protein kinase C inhibitor PKC412 inhibits both FLT3 signaling and drug transport by the ABC protein P-glycoprotein (Pgp; ABCB1). The effects of PKC412 on other MDR-associated ABC proteins expressed in AML cells, including multidrug resistance protein-1 (MRP-1; ABCC1) and breast cancer resistance protein (BCRP; ABCG2), are not known. We studied the effects of PKC412 on transport of MDR protein substrate drugs and its interactions with the MDR protein substrate daunorubicin (DNR) and the non-MDR protein substrate cytarabine (AraC) in cell lines overexpressing Pgp (HL60/VCR), MRP-1 (HL60/ADR) and BCRP (8226/MR20), in wild-type HL60 cells, which express no MDR proteins, and in 8 AML patient bone marrow samples characterized for FLT3 mutations by a DNA-based assay and for expression and function of Pgp, MRP-1 and BCRP by flow cytometry using specific antibodies (MRK16, MRPm6 and BXP21) and modulators (PSC-833, probenecid and fumitemorgin C). in vitro drug sensitivity was assessed by cell survival measured by the wst-1 assay following 48- (AML samples) or 96- (cell lines) hour continuous drug exposure in 96-well microculture assays, and drug interactions were evaluated by the combination index approach of Chou and Talalay. PKC412 increased uptake of MDR protein substrate drugs in cell lines overexpressing MRP-1 and BCRP, as well as Pgp, in a concentration-dependent manner, with maximum effects at ≥5 μM. PKC412 was cytotoxic to all cell lines studied, and cell lines overexpressing Pgp, MRP-1 and BCRP were 20- to 40-fold resistant to PKC412 in relation to parental drug-sensitive cells, suggesting that PKC412 may be a substrate for Pgp, MRP-1 and BCRP and may thus modulate drug transport by competitive inhibition. The combination of PKC412 with DNR synergistically decreased cell survival (CI50 values <0.9) in cell lines overexpressing Pgp, MRP-1 and BCRP, but not in wild-type HL60 cells, whereas the combination of PKC412 and AraC synergistically decreased cell survival in wild-type HL60 cells as well as in the cell lines expressing Pgp, MRP-1 and BCRP. These data suggest that PKC412 interacts synergistically with the MDR protein substrate drug DNR at least in part by virtue of modulation of its transport, but interacts with AraC through alternative mechanisms in both wild-type and multidrug resistant cells. The eight AML patient samples studied included 4 each with FLT3 internal tandem duplication (ITD) and wild-type FLT3, and in vitro sensitivity to PKC412 did not differ between these two groups. All eight samples exhibited expression or function of at least one ABC transport protein. Synergistic cytotoxicity was observed for the combination of PKC412 with DNR in 7 of 8 AML patient samples, whereas the combination of PKC412 with AraC produced synergistic cytotoxicity in 4, including 3 with wild-type FLT3 and one with FLT3 ITD. Thus combinations of PKC412 with DNR and with AraC have the potential for synergistic activity in the treatment of AML, and synergy occurs both through modulation of Pgp, MRP-1 and BCRP and through other, likely heterogeneous, mechanisms. Interactions of PKC412 with both MDR protein substrate and non-substrate chemotherapy drugs have the potential to be exploited in novel combination regimens.

C-JUN-NH2-Terminal Kinase Promotes Apoptosis, Suppresses P-Glycoprotein Mediated Multidrug Resistance but Enhances MRP Mediated Efflux in AML Cells.

JNK has been implicated in distinct cellular events, as proliferation, cellular transformation and apoptosis. JNK has also been recently reported to reverse MDR1 mediated drug resistance and increase sensitivity to chemotherapeutic agents in non-hematopoietic cancer cells. As acquired drug resistance represents a major obstacle in successful therapy of Acute Myeloid Leukemia (AML), the significance of JNK activation in relation to apoptosis induction and drug resistance in AML was sought. JNK is active in U937 leukemia cells and undergoes further activation upon treatment with therapeutically relevant concentrations of daunomycin. Daunomycin induces massive apoptosis in these cells, which is significantly reduced if U937 cells are preincubated with JNK inhibitor 420116. MDR1 and MRP expression levels are low in U937 cells. URD cells, a U937 deritive cell line characterized by resistance to adriamycin, was tested. Interestingly, no basal JNK activity was detected in URD cells and daunomycin treatment did not activate JNK nor induced apoptosis, albeit abundant JNK protein expression. JNK has been proposed to be prerequisite for mediating drug resistance in HL60 cells, by up regulating GSTP1 and MRP efflux pump. GSTP1 was strongly expressed in URD cells in the absence of JNK activation, but MRP expression was insignificant. Moreover, GSTP1 contributed to JNK inactivation in these cells, as immunoprecipitation experiments revealed a complex of GSTP1 with JNK. MDR1 activity in URD cells was 90%, as detected by the calcein AM efflux assay. Transfection of URD cells with SEKED plasmid expressing JNK decreased MDR1 activity by 35% while MRP activity exhibited >3 fold increase, from 5% to 18,4%. 29% of URD cells underwent apoptosis after 24hr of treatment with 1μM daunomycin compared to 5% of cells transfected with empty vector, as measured by annexin V/ PI and DAPI staining. To validate our results to primary AML cells, 25 AML bone marrow samples were tested for JNK activation, apoptosis induction after daunomycin treatment and GSTP1, MDR1 and MRP expresion levels. Basal JNK activity was detected in 58% of patients and did not correlate with apoptosis susceptibility or MDR1 and MRP expression levels. JNK activation within 1 hr of treatment occurred in 9 samples (36%) and strongly correlated with statistically significant apoptosis (p=0,0044< 0,05) after 12hr of continuous drug exposure. In 16 samples (64%), JNK activation was not observed upon daunomycin treatment and apoptosis did not reach a statistically significant level (p=0,3>0,05). Chemotherapy induced JNK activation was irrelevant to basal JNK activity status. GSTP1 was strongly expressed in all samples examined. Our experimental data indicate that JNK activation is implicated in distinct cellular processes in response to chemotherapy. Activation of JNK correlated with apoptosis induction in U937 cells and in primary leukemia samples. Moreover, JNK activity was lost in resistant URD cells, perhaps due to GSTP1 inhibition, and when expressed induced MDR1 downregulation and apoptosis. The proapoptotic function of JNK in URD cells can not be attributed only to MDR1 downregulation, since it is also observed in U937 cells, characterized by low MDR1 activity. Interestingly, JNK expression in URD cells also upregulated the MRP efflux pump. Further studies are needed to elucidate whether JNK represents a possible drug target to overcome drug resistance in AML.

HPMA Copolymer-Bound Doxorubicin Induces Apoptosis in Ovarian Carcinoma Cells by the Disruption of Mitochondrial Function

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-bound doxorubicin has showed greater potency than free doxorubicin in the treatment of ovarian cancer in vivo and in vitro. The promising activity of the conjugate demonstrated in clinical trials has generated considerable interest in understanding the mechanism of action of this macromolecular therapeutic. In this study, the involvement of the mitochondrial pathway in HPMA copolymer-bound doxorubicin-induced apoptosis in the human ovarian cancer cell line A2780 was investigated. Through a series of in vitro assays, including confocal microscopy, flow cytometry, and spectrofluorimetry, a significant decrease in mitochondrial membrane potential in A2780 cells treated with HPMA copolymer-bound doxorubicin was found. The most dramatic changes in mitochondrial membrane potential were observed between 2 and 12 h of continuous drug exposure. The potential of the mitochondrial membrane remained collapsed when drug treatment continued up to 24 h. For the first time, it was shown that HPMA copolymer-bound doxorubicin induces apoptosis in ovarian cancer cells by simultaneous activation of both caspase-dependent and caspase-independent pathways of DNA damage. This was determined by monitoring the translocation of the mitochondrial proteins cytochrome c and apoptosis-inducing factor to cytosol. The altered balance between anti-apoptotic and pro-apoptotic members of the Bcl-2 family of proteins was responsible for the mitochondrial function distraction. HPMA copolymer-bound doxorubicin induced a time-dependent decrease in the expression of the anti-apoptotic Bcl-2 and Bcl-xL proteins, which control cell survival. At the same time, the expression level of pro-apoptotic members (Bax, Bad) of the Bcl-2 family was increased under the chosen experimental conditions. Altogether, these results indicate that HPMA copolymer-bound doxorubicin induced apoptosis in ovarian cancer cells through the mitochondrial pathway.

ABCG2 Harboring the Gly482 Mutation Confers High-Level Resistance to Various Hydrophilic Antifolates

ABCG2 is an ATP-binding cassette transporter that confers resistance to various chemotherapeutic agents. Recent studies have established that an Arg (wild-type) to Gly mutation at amino acid 482 in ABCG2 alters substrate specificity. Here, we explored the role of this G482 mutation in antifolate resistance using a clinically relevant 4-hour drug exposure. Stable transfectants overexpressing the mutant G482 transporter displayed 120-, 1,000-, and >6,250-fold resistance to the antifolates methotrexate, GW1843, and Tomudex, respectively, relative to parental human embryonic kidney cells. Moreover, although overexpressing equal transporter levels at the plasma membrane, G482-ABCG2 cells were 6-, 23-, and >521-fold more resistant to methotrexate, GW1843, and Tomudex, respectively, than R482-ABCG2 cells. In contrast, upon a continuous (72-hour) drug exposure, both the G482- and R482-ABCG2 cells lost almost all their antifolate resistance; this result was consistent with the inability of ABCG2 to extrude long-chain antifolate polyglutamates. Ko143, a specific and potent ABCG2 inhibitor reversed methotrexate resistance in both G482- and R482-ABCG2 cells. Consistently, whereas the pool of free methotrexate in parental human embryonic kidney cells was prominent after 4 hours of transport with 1 micromol/L [3H]methotrexate, in R482- and G482-ABCG2 cells, it was minimal. Furthermore, G482-ABCG2 cells contained marked decreases in the di- and triglutamate species of [3H]methotrexate at 4 hours of incubation with methotrexate and in the tetra- and pentaglutamates at 24 hours. These changes were not associated with any significant decrease in folylypoly-gamma-glutamate synthetase activity. These results provide the first evidence that the G482-ABCG2 mutation confers high-level resistance to various hydrophilic antifolates.

A novel class of achiral seco-analogs of CC-1065 and the duocarmycins: design, synthesis, DNA binding, and anticancer properties

The synthesis, DNA binding properties, and in vitro and in vivo anticancer activity of fifteen achiral seco-cyclopropylindoline (or achiral seco-CI) analogs (5a–o) of CC-1065 and the duocarmycins are described. The achiral seco-CI analogs contain a 4-hydroxyphenethyl halide moiety that is attached to a wide range of indole, benzimidazole, pyrrole, and pyridyl-containing noncovalent binding components. The 4-hydroxyphenethyl halide moiety represents the simplest mimic of the seco-cyclopropylpyrroloindoline (seco-CPI) pharmacophore found in the natural products, and it lacks a chiral center. The sequence and minor groove specificity of the achiral compounds was ascertained using a Taq DNA polymerase stop assay and a thermal induced DNA cleavage experiment using either a fragment of pBR322 or pUC18 plasmid DNA. For example, seco-CI-InBf (5a) and seco-CI-TMI (5c) demonstrated specificity for AT-rich sequences, particularly by reacting with the underlined adenine-N3 position of 5′-AAAAA(865)-3′. This is also the sequence that CC-1065 and adozelesin prefer to alkylate. The achiral seco-CI compounds were subjected to cytotoxicity studies against several human (K562, LS174T, PC3, and MCF-7) and murine cancer cell lines (L1210 and P815). Following continuous drug exposure, the achiral compounds were found to be cytotoxic, with IC50 values in the μM range. Interestingly, the carbamate protected compound 5p was significantly less cytotoxic than agent 5c, supporting the hypothesis that loss of HCl and formation of a spiro[2,5]cyclopropylcyclohexadienone intermediate is necessary for biological activity. The achiral seco-CI compounds 5a and 5c were submitted to the National Cancer Institute for further cytotoxicity screening against a panel of 60 different human cancer cell lines. Both compounds showed significant activity, particularly against several solid tumor cell lines. Flow cytometry studies of P815 cells that were incubated with compound 5c at its IC50 concentration for 24h showed induction of apoptosis in a large percentage of cells. Compounds 5a and 5c were selected by the NCI for an in vivo anticancer hollow-fiber test, and received composite scores of 18 and 22, respectively. These two compounds were subsequently evaluated for in vivo anticancer activity against the growth of a human advanced stage SC UACC-257 melanoma in skid mice. At a dose of 134mg/kg administered IP, compound 5c gave a T/C value of 40% (for day 51), and the median number of days of doubling tumor growth was 27.7, versus 15.8 for untreated animals. For compound 5a, at 200mg/kg, the T/C was 58% and the median number of days of doubling tumor growth was 20.0 versus 8.7 for untreated animals. At these doses no toxicity or weight loss was observed for either compound. Furthermore, compound 5c was not toxic to murine bone marrow cell growth in culture, at a dose that was toxic for the previously reported seco-CBI (cyclopropylbenzoindoline)-TMI (4).

The antitumor triazoloacridone C-1305 is a topoisomerase II poison with unusual properties.

C-1305 [S-[[3-(dimethylamino)propyl]amino]-8-hydroxy-6H-v-triazolo[4,5,1-de]acridin-6-one] is a triazoloacridone with excellent activity in colon cancer models. The mechanism of C-1305 is unknown, although similarities in the chemical structure between C-1305 and amsacrine suggest common cellular targets. Here, we report that C-1305 is a topoisomerase II poison that is able to induce cleavable complexes with topoisomerase II in vitro as well as in living cells. Even at optimal concentrations, C-1305 is a much weaker inducer of cleavable complexes than amsacrine. Because the cytotoxic activities of the two compounds after continuous drug exposure are comparable, these findings suggest that the low levels of cleavable complexes induced by C-1305 may be unusually toxic. In contrast to amsacrine, the cytotoxicity of C-1305 is strongly time-dependent, with at least 24 h of drug exposure required for optimal cytotoxicity. The p53 tumor suppressor is inactivated in the majority of human tumors, including colorectal cancers. We therefore compared the long-term cytotoxic effects of C-1305, amsacrine, and doxorubicin on human cell lines in which the p53 or p21 pathways have been specifically disrupted by targeted homologous recombination. Disruption of p53 and p21 had minor influence on the cytotoxicity of doxorubicin, whereas p53 but not p21 disruption was associated with increased resistance to amsacrine. In marked contrast, disruption of p53 and p21 was associated with increased sensitivity to C-1305. Taken together, our results show that exposure to C-1305 is accompanied by the formation of low levels of potent cleavable complexes that are selectively toxic toward tumor cells with defective p53 function.

Phase I pharmacokinetic trial of docetaxel and thalidomide: A regimen based on anti-angiogenic therapeutic principles

3135 Background: Preclinical models suggest that chronic, low-dose administration of anticancer therapy (cytotoxic agent and an angiogenesis inhibitor) may result in prolonged periods of freedom from progression (FFP) and tumor regression. This outcome may derive from continuous drug exposure and more pronounced anti-angiogenic rather than cytotoxic effects. Methods: Patients (pts) with advanced cancer for whom there is no curative or meaningful palliative therapy are eligible and enrolled in cohorts of 3. Pts are treated with a weekly dose of docetaxel (IV) with thalidomide (PO) given every 12 hours for 84 days (12 weeks) of continuous therapy. Since we sought to pilot a metronomic schedule over 12 weeks dose-limiting toxicity (DLT) was defined as ≥ grade 2 non-hematologic or ≥ grade 3 hematologic. Plasma concentrations of both drugs were determined by HPLC / MS-MS and pts were observed for response. Results: Sixteen pts have been enrolled; 5 are not evaluable [docetaxel hypersensitivity not considered DLT (3 pts) and 2 pts withdrew before 12 weeks]. All pts received thalidomide 100 mg bid. Docetaxel was escalated from 10, 20, 25, and 30 mg/m2 in 3, 3, 3, and 2 pts respectively. A total of 17 cycles have been completed. A single DLT (grade 3 fatigue) occurred at 30 mg/m2. There has been no myelosuppression. There has been 1 PR (SCLC), and 4 pts with FFP of >115 (prostate), 182 (papillary thyroid), >230 (prostate), and 321 days (melanoma). Measured trough plasma thalidomide concentrations of 466 ng/ml (range 344–682) have been achieved. Docetaxel plasma concentrations are consistent with previous reports of weekly schedules. Conclusions: The 12-week metronomic schedule of this regimen is feasible with a very acceptable safety profile. Clinical activity and prolonged FFP have been observed in a highly refractory population early in this study. (This trial is supported in part by the NIH grant MO1 RR00080, Aventis Oncology and Celgene.) No significant financial relationships to disclose.

Effects of sodium stibogluconate on differentiation and proliferation of human myeloid leukemia cell lines in vitro

PTPases are key signaling molecules and targets for developing novel therapeutics. We have studied the in vitro biological activity of PTPase inhibitor sodium stibogluconate (SS) on differentiation and proliferation of myeloid leukemia cell lines (NB4, HL-60 and U937). SS (250 microg/ml, 6 days) induced 87% of NB4 cells to reduce nitroblue tetrazolium (NBT), in comparison to the 90% induced by ATRA (1 microM, 6 days). SS treatment of NB4 cells resulted in an increase of CD11b expression and of a morphologically more mature population, coincident with growth arrest at S phase and increased cell death. The effect of SS on NB4 differentiation was irreversible and required continuous drug exposure. SS (400 microg/ml, 6 days) induced 60% and 55% of NBT-positive cells in HL-60 and U937 cell lines, which were augmented in the presence of GM-CSF (25 ng/ml) to levels (85% and 81%, respectively) comparable to those induced by ATRA. SS induced increased tyrosine phosphorylation of cellular proteins in the AML cell lines and inactivated SHP-1 PTPase in NB4 cells, consistent with SS functioning as a PTPase inhibitor in the leukemia cells. These results provide the first evidence of an anti-leukemia activity of SS as a PTPase inhibitor.

In vitro pharmacodynamics of CKD-602 in HT-29 cells.

CKD-602 (7-[2-(N-isopropylamino)ethyl]-(20S)-camptothecin) is a recently-developed synthetic camptothecin analogue and currently under clinical development by Chong Kun Dang Pharm (Seoul, Korea). CKD-602 showed potent topoisomerase inhibitory activity in vitro and broad antitumor activity against various human tumor cells in vitro and in vivo in animal models. This study describes the pharmacodynamics of the immediate and delayed cytotoxicity induced by CKD-602 in a human colorectal adenocarcinoma cell line, HT-29, and its intracellular drug accumulation by HPLC. The present study was designed to address whether the higher activity of CKD-602 with prolonged exposure is due to delayed exhibition of cytotoxicity and/or an accumulation of antiproliferative effect on continuous drug exposure. The drug uptake study was performed to determine whether the delayed cytotoxicity is due to a slow drug accumulation in cells. CKD-602 produced a cytotoxicity that was exhibited immediately after treatment (immediate effect) and after treatment had been terminated (delayed effect). Both the immediate and delayed effects of CKD-602 showed a time dependent decrease in IC50 values. Drug uptake was biphasic and the second equilibrium level was obtained as early as at 24 hr, indicating that the cumulative and delayed antitumor effects of CKD-602 were not due to slow drug uptake. On the other hand, CKD-602 treatment was sufficient to induce delayed cytotoxicity after 4 hr, however, longer treatment (>24 hr) enhanced its cytotoxicity due to the intracellular accumulation of the drug, which requires 24 hr to reach maximum equilibrium concentration. In addition, Cn x T=h analysis (n=0.481) indicated that increased exposure times may contribute more to the overall antitumor activity of CKD-602 than drug concentration. Additional studies to determine the details of the intracellular uptake kinetics (e.g., concentration dependency and retention studies) are needed in order to identify the optimal treatment schedules for the successful clinical development of CKD-602.

Transient DNA double-strand breakage in 4-hydroperoxyifosfamide-treated mammalian cells in vitro does not interact with the rejoining of radiation-induced double-strand breaks.

4-Hydroxyifosfamide is the primary metabolite in vivo of the bifunctional alkylating cytostatic ifosfamide. DNA interstrand cross-linking induced by bifunctional alkylators may be repaired through an intermediate with unligated repair patches on both strands which should uncover analytically as DNA double-strand breaks and allow to measure the rejoining kinetic of this repair intermediate. Additionally, the combined effects of drug and radiation treatment on rejoining of double-strand breaks was investigated with two different mammalian cell lines. V79 (rodent fibroblasts) and Widr (human colon carcinoma) cells were treated for 2 hours with 4-hydroperoxyifosfamide which rapidly decays to 4-hydoxyifosfamide in aqueous solution or were exposed in combination with ionizing radiation followed by incubation for repair with or without the drug. DNA double-strand breakage was measured by pulsed-field electrophoresis. The 2 hours 4-hydroperoxyifosfamide treatment (30 micrograms/ml) resulted in a pronounced DNA fragmentation that, 2-4 hours after drug removal, declined with an estimated half-live of about 4 hours for both cell lines. When the cells were additionally irradiated with 10 Gy given in the middle of drug exposure, the residual fragmentation after 12 or 24 hours incubation for repair was only marginally increased, roughly corresponding to the respective value after radiation, alone. A continuous drug exposure of 6 hours (at 10 micrograms/ml) resulted in a fragmentation that was independent of a preirradiation with a high dose of 30 Gy, immediately before drug addition. The present data support the idea that unligated/unrejoined double-stranded DNA ends are generated during the repair of lesions from bifunctional alkylators. The rate of subsequent rejoining is in the order of magnitude of the slow rejoining of radiation-induced double-strand breaks. Processing of double-stranded DNA damage from either 4-hydroperoxyifosfamid or radiation exposure is apparently unaffected in combined treatments.

Role of intracellular calcium stores in cell death from oxygen-glucose deprivation in a neuronal cell line.

To determine the role of calcium homeostasis in ischemic neuronal death, the authors used an in vitro model of oxygen-glucose deprivation in neuronal cell lines. Exposure of human neuroblastoma SH-SY5Y cells to 10-to 16-hour oxygen-glucose deprivation decreased viability to 50% or less, and longer exposure times killed almost all cells. The death following 10-to 16-hour oxygen-glucose deprivation was not manifested until 24 to 72 hours after exposure. Deprivation of both glucose and oxygen together was required for expression of toxicity at these exposure times. Dantrolene, which blocks the release of endoplasmic reticulum Ca2+ stores, partially protected SH-SY5Y cells from oxygen-glucose deprivation toxicity. The addition of dantrolene during the deprivation phase alone produced the maximal drug effect; no further protection was obtained by continued drug exposure during the recovery phase. Prevention of Ca2+ influx by chelation or channel blockade or the chelation of cytosolic Ca2+ did not inhibit oxygen-glucose deprivation toxicity. In contrast, increasing extracellular Ca2+ or stimulating Ca2+ influx did inhibit toxicity. Calcium measurements with fura-2 acetoxymethylester revealed that oxygen-glucose deprivation caused a significant reduction in thapsigargin-releasable endoplasmic reticular stores of Ca2+. These studies suggest that an important component of the neuronal toxicity in cerebral ischemia is due to disruption of calcium homeostasis, particularly to the depletion of intracellular Ca2+ stores.

In vitro activity of the novel cytotoxic agent CHS 828 in childhood acute leukemia.

CHS 828, a pyridyl cyanoguanidine, is a new drug candidate now in phase I/II trials, that has shown promising anticancer activity in experimental tumor models and primary cultures of cancer cells from patients. In this study the fluorometric microculture cytotoxicity assay was used for evaluation of CHS 828 in primary cell cultures from children with acute leukemia. The activity of and interaction with the standard drugs, doxorubicin, melphalan, etoposide and cytosine arabinoside (Ara-C), were also assessed. Samples from 65 patients, 42 with acute lymphocytic leukemia (ALL) and 23 with acute myelocytic leukemia (AML) were tested with 72-h continuous drug exposure. There was 50% cell kill at very low CHS 828 concentrations; median IC50 was 0.01 microM in ALL and 0.03 in AML samples (NS) with large interindividual variability in both groups. ALL samples were significantly more sensitive than AML samples to melphalan, doxorubicin and etoposide, but not to Ara-C. In AML samples, combinations between CHS 828 and each of the four standard drugs resulted in significantly lower cell survival than either drug alone. This was also observed in ALL samples, except for Ara-C. Using the additive interaction model, CHS 828 showed a synergistic effect with melphalan in 67%, doxorubicin in 47%, etoposide in 38% and Ara-C in 14% of AML samples. In most ALL samples subadditive effects were found. Further exploration of CHS 828 in childhood leukemia is warranted, especially in AML.

Huanglian, A chinese herbal extract, inhibits cell growth by suppressing the expression of cyclin B1 and inhibiting CDC2 kinase activity in human cancer cells.

Huanglian is an herb that is widely used in China for the treatment of gastroenteritis. We elected to determine whether huanglian could inhibit tumor cell growth by modulating molecular events directly associated with the cell cycle. Huanglian inhibited tumor growth and colony formation of gastric, colon, and breast cancer cell lines in a time- and dose-dependent manner. Cell growth was completely inhibited after 3 days of continuous drug exposure to 10 microg/ml of herb. This degree of growth inhibition was significantly greater than that observed with berberine, the major constituent of the herb. The inhibition of cell growth by huanglian was associated with up to 8-fold suppression of cyclin B1 protein. This resulted in complete inhibition of cdc2 kinase activity and accumulation of cells in G(2). The mRNA expression of cyclin B1 was not changed after huanglian treatment. There was no change in the protein expression of cyclins A or E. Therefore, the effect of huanglian on inhibiting tumor growth seems to be mediated by the selective suppression of cyclin B1, which results in the inhibition of cdc2 kinase activity. Inhibition of cyclin dependent kinase (cdk) activity is emerging as an attractive target for cancer chemotherapy. Huanglian represents a class of agents that can inhibit tumor cell growth by directly suppressing the expression of a cyclin subunit that is critical for cell cycle progression. These results indicate that traditional Chinese herbs may represent a new source of agents designed for selective inhibition of cyclin dependent kinases in cancer therapy.

A Rodent Model of Cocaine Abstinence Syndrome

This study introduces a rat model of cocaine abstinence syndrome based on quantitation of spontaneously emitted behaviors following termination of continuous drug exposure (analogous to established methods of assessing morphine and nicotine abstinence). Groups of eight male S-D rats were infused SC for 7 days via an osmotic minipump with saline alone or with 40 or 60 mg/kg/day cocaine HCl. Pumps were removed and rats were observed at 12, 24, 36, and 48 h postremoval. Each 15-min observation employed a checklist of abstinence signs including ptosis, chews, teeth chatters, gasps, writhes, seminal ejaculations, head shakes, and tremors. The high infusion rate group displayed significantly more signs than the low infusion rate group, which in turn, displayed significantly more signs than the saline group. Cocaine injection significantly reduced signs by 83.3%, while saline injection reduced them by only 4.9%. In another experiment, rats infused with 60 mg/kg/day showed significantly more signs 36 h postinfusion than before infusion, during infusion and 84 h postinfusion. Finally, 6.5 days of infusion resulted in significantly more abstinence signs than did 1.5 days of infusion. This rapid and simple model quantitated cocaine abstinence syndrome in a manner that was cocaine-reversible and related to the rate and duration of drug infusion.

Antiproliferative effects of compounds derived from plants of Northeast Brazil.

Ten compounds derived from plants indigenous to Northeast Brazil were examined for antiproliferative effects on human cells in vitro. The effects of these phytochemicals on cell growth were determined by the MTT microtitre assay with 3-day continuous drug exposure. Three human cell lines were used: CEM leukaemia, SW1573 lung tumour and CCD922 normal skin fibroblasts. Four active compounds were found with IC(50) values less than 10 microg/mL in the two cancer cell lines. Oncocalyxones A and C, both 1,4-anthracenediones from Auxemma oncocalyx (Boraginaceae), showed cytotoxicity with mean IC(50) values of 0.8-2, 7-8 and 12-13 microg/mL against CEM, SW1573 and CCD922, respectively. One diterpene and one flavonoid, both from Egletes viscosa (Compositae), were also active. 12-Acetoxy-hawtriwaic acid lactone was cytotoxic with mean IC(50) values of 6, 10 and 10 microg/mL, respectively. 4,5-Dihydroxy-3,3,7, 8-tetramethoxy flavone (ternatin) was only growth-inhibitory with mean IC(50) values of 2, 1 and 10 microg/mL, respectively. These four most active compounds were examined further for their effects on DNA integrity and on DNA synthesis. All but ternatin caused substantial DNA damage and marked inhibition of 5-bromo-2'-deoxyuridine incorporation within 24 h. This study demonstrated the antiproliferative activity of four novel phytochemicals, three of which are DNA-reactive and inhibit DNA synthesis. Further studies are warranted to evaluate these compounds for antitumour potential.

Combretastatin A-4 prodrug: a potent inhibitor of malignant hemangioendothelioma cell proliferation.

Anti-vascular treatment by targeting proliferating endothelial cells has become a promising option in anti-neoplastic therapy. Combretastatin A-4 prodrug (CA-4PD) has been identified as a selective inhibitor of endothelial cell proliferation, acting by the interruption of microtubule assembly. In this study, the effect of CA-4PD on proliferating endothelial cells derived from a primary tumor of the vascular endothelium was investigated in vitro and in vivo. In vitro, CA-4PD was an effective inhibitor of endothelial cell proliferation in a time- and dose-dependent manner, displaying a certain selectivity toward endothelial cells in comparison to proliferating fibroblasts. Analysis of DNA profiles by FACS revealed an increasing proportion of cells arrested in the G(2) cell-cycle phase with correlation to the duration of drug exposure. A decrease in cell viability correlated with duration of drug exposure, whereas FACS analysis, DNA fragmentation assay, and DNA gel electrophoresis failed to demonstrate that DNA fragmentation was indicative of apoptosis up to 48 hr of continued drug exposure. In vivo, CA-4PD induced excessive regressive alterations in experimentally allotransplanted hemangioendotheliomas within 24 hr after singular i. p. injection of 100 mg CA-4PD/kg body weight. This represented less than one-tenth of the maximum tolerated dose. In conclusion, our findings characterize CA-4PD as a potent inhibitor of malignant endothelial cell proliferation in vitro, effecting arrest of proliferating cells in the G(2) cell-cycle phase with subsequent cell death on a pathway different from apoptosis. in vivo, CA-4PD induces extensive intratumoral cell loss within 24 hr following systemic administration, suggesting a synergistic effect of direct cell killing and the induction of vascular shutdown.