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1.  Phase I study of GTI-2040, a ribonucleotide reductase antisense, with high dose cytarabine in patients with relapsed/refractory acute myeloid leukemia 
Leukemia & lymphoma  2013;55(6):1332-1336.
We hypothesized that GTI-2040, a 20-mer oligonucleotide complementary to the R2 subunit mRNA of ribonucleotide reductase, combined with high dose cytarabine (HiDAC) would result in enhanced cytotoxicity by favoring Ara-CTP DNA incorporation. In a phase I dose escalation trial, adults (≥60 years) with refractory or relapsed acute myeloid leukemia (AML) received daily HiDAC plus infusional GTI-2040. Using a novel assay, evidence of intracellular drug accumulation and target R2 down-regulation was observed. GTI-2040/HiDAC can be administered safely. However, with no complete remissions observed, alternative doses and schedules may need to be investigated to achieve clinical activity in older patients with AML.
doi:10.3109/10428194.2013.838764
PMCID: PMC4298748  PMID: 24015841
Acute myeloid leukemia; antisense therapy; phase I study; GTI-2040; ribonucleotide reductase
4.  Flavopiridol Can Be Safely Administered Using a Pharmacologically Derived Schedule and Demonstrates Activity in Relapsed and Refractory Non-Hodgkin’s Lymphoma 
American journal of hematology  2013;89(1):19-24.
Flavopiridol is a broad cyclin-dependent kinase inhibitor (CDKI) that induces apoptosis of malignant lymphocytes in vitro and in murine lymphoma models. We conducted a phase I dose-escalation study to determine the maximum tolerated dose (MTD) for single-agent flavopiridol administered on a pharmacokinetically derived hybrid dosing schedule to patients with relapsed and refractory non-Hodgkin’s lymphoma. Dose was escalated independently in one of four cohorts: indolent B-cell (cohort 1), mantle cell (cohort 2), intermediate grade B-cell including transformed lymphoma (cohort 3), and T-/NK-cell excluding primary cutaneous disease (cohort 4). Forty-six patients were accrued. Grade 3 or 4 leukopenia was observed in the majority of patients (60%), but infection was infrequent. Common non-hematologic toxicties included diarrhea and fatigue. Biochemical tumor lysis was observed in only 2 patients, and no patients required hemodialysis for its management. Dose escalation was completed in two cohorts (indolent and aggressive B-cell). Dose-limiting toxicities were not observed, and the MTD was not reached in either cohort at the highest dose tested (50 mg/m2 bolus + 50 mg/m2 continuous infusion weekly for 4 consecutive weeks of a 6 week cycle). Clinical benefit was observed in 26% of 43 patients evaluable for response, including 14% with partial responses (2 mantle cell, 3 indolent B-cell, and 1 diffuse large B-cell). The single-agent activity of this first-generation CDKI suggests that other agents in this class merit further study in lymphoid malignancies, both alone and in combination.
doi:10.1002/ajh.23568
PMCID: PMC4150545  PMID: 23959599
flavopiridol; non-Hodgkin’s lymphoma; cyclin-dependent kinase inhibitors; phase 1 trial; pharmacokinetics
5.  Standard Pentostatin Dose Reductions in Renal Insufficiency are not Adequate: Selected Patients with Steroid-Refractory Acute Graft-versus-Host Disease 
Clinical pharmacokinetics  2013;52(8):705-712.
Background and Objective
Pentostatin is an irreversible inhibitor of adenosine deaminase and has been used to prevent graft-versus-host disease (GVHD) and to treat both acute and chronic GVHD. Dose reduction equations for patients with renal insufficiency are based on few patients with limited pharmacokinetic and clinical results. This phase II study (NCT00201786) was conducted to assess pentostatin efficacy and infectious complications seen from our previous phase I study in steroid-refractory acute GVHD (aGVHD).
Patients and Methods
Hospitalized patients with steroid-refractory aGVHD were given pentostatin 1.5 mg/m2/day intravenously on days 1–3 of each 14 day cycle. Prior to each dose, dose modifications were based on Cockcroft-Gault estimated creatinine clearance (eCrCL) with 30–50 ml/min/1.73m2 leading to a 50% dose reduction and eCrCL< 30 ml/min/1.73m2 leading to study removal. Plasma pentostatin area under the concentration-time curve (AUC) and incidence of infectious complications were evaluated.
Results
Two of the eight patients treated demonstrated excessive pentostatin exposure as determined by measurement of AUC. One of these patients had renal impairment while the other patient demonstrated borderline renal function. Despite dose reduction to 0.75 mg/m2, AUCs were significantly increased compared to the other patients in this study. Seven of eight patients treated with pentostatin had cytomegalovirus (CMV) viremia after pentostatin treatment; however none developed proven CMV disease.
Conclusion
A 50% dose reduction in patients with eCrCL 30–50 ml/min/1.73m2 seems reasonable. However, the eCrCL should be interpreted with extreme cautions in patients who are critically ill and/or with poor performance status. Renal function assessment based on the Cockcroft-Gault method could be significantly overestimated thus risking pentostatin over-dosing. These results imply a need to closely monitor pentostatin exposure in patients with renal insufficiency.
doi:10.1007/s40262-013-0064-7
PMCID: PMC3720841  PMID: 23588536
6.  A phase I study of prolonged infusion of triapine in combination with fixed dose rate gemcitabine in patients with advanced solid tumors 
Investigational new drugs  2012;31(3):685-695.
Summary
Purpose
Prolonged exposure of cancer cells to triapine, an inhibitor of ribonucleotide reductase, followed by gemcitabine enhances gemcitabine activity in vitro. Fixed-dose-rate gemcitabine (FDR-G) has improved efficacy compared to standard-dose. We conducted a phase I trial to determine the maximum tolerated dose (MTD), safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of prolonged triapine infusion followed by FDR-G.
Experimental Design
Triapine was given as a 24-hour infusion, immediately followed by FDR-G (1000 mg/m2 over 100-minute). Initially, this combination was administered days 1 and 8 of a 21-day cycle (Arm A, triapine starting dose 120 mg); but because of myelosuppression, it was changed to days 1 and 15 of a 28-day cycle (Arm B, starting dose of triapine 75 mg). Triapine steady-state concentrations (Css) and circulating ribonucleotide reductase M2-subunit (RRM2) were measured.
Results
Thirty-six patients were enrolled. The MTD was determined to be triapine 90 mg (24-hour infusion) immediately followed by gemcitabine 1000 mg/m2 (100-minute infusion), every 2 weeks of a 4-week cycle. DLTs included grade 4 thrombocytopenia, leukopenia and neutropenia. The treatment was well tolerated with fatigue, nausea/vomiting, fever, transaminitis, and cytopenias being the most common toxicities. Among 30 evaluable patients, 1 had a partial response and 15 had stable disease. Triapine PK was similar, although more variable, compared to previous studies using doses normalized to body-surface-area. Steady decline in circulating levels of RRM2 may correlate with outcome.
Conclusions
This combination was well tolerated and showed evidence of preliminary activity in this heavily pretreated patient population, including prior gemcitabine failure.
doi:10.1007/s10637-012-9863-1
PMCID: PMC3646991  PMID: 22847785
Triapine; Gemcitabine; Phase I; Clinical Trial
7.  Phase I study of azacitidine and bortezomib in adults with relapsed or refractory acute myeloid leukemia 
Leukemia & lymphoma  2013;55(6):1304-1308.
We previously reported that bortezomib indirectly modulates transcription of DNA methyltransferase 1 (DNMT). We designed a phase I study of azacitidine (a direct DNMT inhibitor) plus bortezomib in acute myeloid leukemia (AML) to determine safety and tolerability. Twenty-three adults with relapsed/refractory AML received azacitidine 75mg/m2 daily on days 1-7. Bortezomib was dose escalated from 0.7mg/m2 on days 2 and 5 to 1.3mg/m2 on days 2, 5, 9, and 12. The target dose was reached without dose limiting toxicities. Infection and/or febrile neutropenia were frequent. Patients received a median of 2 cycles of therapy (range, 1-12+). Five of 23 patients achieved remission including two with morphologic and cytogenetic complete response (CR) and three with CR and incomplete count recovery (CRi). Of CR/CRi responders with cytogenetic abnormalities at baseline, three of four achieved cytogenetic CR. The combination of azacitidine and bortezomib was tolerable and active in this cohort of poor-risk previously-treated AML patients.
doi:10.3109/10428194.2013.833333
PMCID: PMC3925754  PMID: 23952243
Relapsed AML; bortezomib; velcade
8.  A Pharmacokinetic/Pharmacodynamic Model of Tumor Lysis Syndrome in Chronic Lymphocytic Leukemia Patients Treated with Flavopiridol 
Purpose
Flavopiridol, the first clinically evaluated cyclin dependent kinase inhibitor, demonstrates activity in patients with refractory chronic lymphocytic leukemia, but prevalent and unpredictable tumor lysis syndrome (TLS) presents a major barrier to its broad clinical use. The purpose of this study was to investigate the relationships between pretreatment risk factors, drug pharmacokinetics, and TLS.
Experimental Design
A population pharmacokinetic/pharmacodynamic model linking drug exposure and TLS was developed. Plasma data of flavopiridol and its glucuronide metabolite (flavo-G) were obtained from 111 patients treated in early phase trials with frequent sampling following initial and/or escalated doses. TLS grading was modeled with logistic regression as a pharmacodynamic endpoint. Demographics, baseline disease status, and blood chemistry variables were evaluated as covariates.
Results
Gender was the most significant pharmacokinetic covariate, with females displaying higher flavo-G exposure than males. Glucuronide metabolite exposure was predictive of TLS occurrence, and bulky lymphadenopathy was identified as a significant covariate on TLS probability. The estimated probability of TLS occurrence in patients with baseline bulky lymphadenopathy < 10 cm or > 10 cm during the first two treatments was 0.111 (SE% 13.0%) and 0.265, (SE% 17.9%) respectively, when flavo-G area under the plasma concentration vs. time curve was at its median value in whole patient group.
Conclusions
This is the first population pharmacokinetic/pharmacodynamic model of TLS. Further work is needed to explore potential mechanisms and to determine if the associations between TLS, gender and glucuronide metabolites are relevant in CLL patients treated with other cyclin dependent kinase inhibitors.
doi:10.1158/1078-0432.CCR-12-1092
PMCID: PMC3845832  PMID: 23300276
chronic lymphocytic leukemia; flavopiridol; tumor lysis syndrome; population pharmacokinetics; glucuronide metabolite; logistic regression model
9.  Cyclophosphamide, Alvocidib (Flavopiridol), and Rituximab, a Novel Feasible Chemoimmunotherapy Regimen for Patients with High-Risk Chronic Lymphocytic Leukemia 
Leukemia research  2013;37(10):1195-1199.
Alvocidib has demonstrated efficacy in high-risk chronic lymphocytic leukemia (CLL) patients. In this phase I study, we combined cyclophosphamide, alvocidib and rituximab (CAR) in a schema designed to mitigate tumor lysis syndrome (TLS) seen previously with alvocidib. Nine nucleoside analog-naïve, high-risk patients received escalating doses of CAR therapy. Dose limiting toxicity was not experienced. No instances of TLS were observed. Patient responses included three complete remissions and four partial remissions. CAR was tolerable and active in high-risk CLL patients without TLS toxicity. With continued monitoring of toxicities, a phase Ib/II study of this combination as frontline therapy is warranted.
doi:10.1016/j.leukres.2013.06.006
PMCID: PMC3934299  PMID: 23867058
chronic lymphocytic leukemia; flavopiridol; high-risk cytogenetics; cyclin-dependent kinase inhibitor; chemoimmunotherapy; alvocidib; del(17p); del(11q)
10.  Pharmacokinetics and Dose Escalation of the Heat Shock Protein Inhibitor 17-AAG in Combination with Bortezomib in Relapsed or Refractory Acute Myeloid Leukemia 
Leukemia & lymphoma  2013;54(9):10.3109/10428194.2012.760733.
This phase I study was conducted to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLT) of the heat shock protein 90 (HSP90) inhibitor 17-allyamino-17-demethoxygeldanamycin (17-AAG) in combination with bortezomib, and to provide pharmacokinetic data in relapsed or refractory acute myeloid leukemia (AML). Eleven patients were enrolled. The MTD was 17-AAG 150mg/m2 and bortezomib 0.7mg/m2. Hepatic toxicity and cardiac toxicity were dose limiting. Co-administration on day 4 led to a decrease in clearance (p=0.005) and increase in AUC (p=.032) of 17-amino-17-demethoxygeldanamycin (17-AG) not observed when 17-AAG was administered alone. Pharmacokinetic parameters of patients who developed toxicities and those who did not were not different. The combination of 17-AAG and bortezomib led to toxicity without measurable response in patients with relapsed or refractory AML. Pharmacokinetic data provide insight for studies of related agents in AML; next generation HSP90 inhibitors are appealing for further development in this area.
doi:10.3109/10428194.2012.760733
PMCID: PMC3860322  PMID: 23256542
Relapsed AML; bortezomib; 17-AAG; heat shock protein inhibition
11.  In Vivo Quantification of Active Decitabine-Triphosphate Metabolite: A Novel Pharmacoanalytical Endpoint for Optimization of Hypomethylating Therapy in Acute Myeloid Leukemia 
The AAPS Journal  2012;15(1):242-249.
Decitabine (DAC) is used for treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Following cellular uptake, DAC is activated to DAC-triphosphate (TP) and incorporated into DNA. Once incorporated into the DNA, DAC-TP binds and inactivates DNA methyltransferases (DNMTs), thereby leading to hypomethylation and re-expression of epigenetically silenced tumor suppressor genes and ultimately antileukemia activity. However, direct evidence of in vivo DAC-TP occurrence in DAC-treated patients has been difficult to demonstrate due to a lack of suitable validated analytical methodology. Thus, we developed and validated a nonradioactive sensitive and specific LC-MS/MS assay for quantification of DAC-TP. The assay is linear from 50 to 1,000 nM and from 1 to 10 μM and has a lower limit of quantitation of 50 nM and a coefficient of variation for both within- and between-day precision <20%. Following DAC treatment, we detected DAC-TP in parental and DAC-resistant AML cells (in vitro) and bone marrow (BM) and spleen of normal and leukemic mice (in vivo). Downregulation of DNMTs and correlation of DAC-TP concentration with proteins involved in mechanisms of DAC resistance were also demonstrated. The clinical applicability of this method was proven by measuring DAC-TP level in BM and blood mononuclear cells from DAC-treated AML patients. Higher levels are seemingly associated with clinical response. Monitoring the DAC-TP intracellular level may serve as a novel pharmacological endpoint for designing more effective DAC-based regimens.
doi:10.1208/s12248-012-9427-5
PMCID: PMC3535094  PMID: 23180159
acute myeloid leukemia; decitabine; metabolite; quantification method; triphosphate
12.  A dose escalation and pharmacodynamic study of triapine and radiation in patients with locally advanced pancreas cancer 
PURPOSE
Triapine (Vion Pharmaceuticals), a novel inhibitor of the M2 subunit of ribonucleotide reductase (RR), is a potent radiosensitizer. This NCI/CTEP-sponsored phase I study assessed the safety/tolerability of triapine in combination with radiation (RT) in patients with locally advanced pancreas cancer (LAPCA).
METHODS AND MATERIALS
We evaluated 3 dose levels of triapine (24 mg/m2, 48 mg/m2, 72 mg/m2) administered with 50.4 Gy of RT in 28 fractions. Patients with LAPCA received triapine thrice weekly, every other week during the course of RT. Dose-limiting toxicity (DLT) was assessed during and for 4 weeks following completion of RT. Dynamic contrast enhanced (DCE)-MRI and serum RR levels were evaluated as potential predictors for early response.
RESULTS
Twelve patients were treated. Four patients (1 non-evaluable [NE]) were enrolled at dose level 1 (DL1), three patients at DL2, and five patients (2NE) at DL3. No DLTs were observed and the MTD was not reached. Two patients (17%) achieved PR and 6 patients (50%) had SD. One patient underwent R0 resection following therapy. 92% of patients (100% on DL3) experienced freedom from local tumor progression. 75% of patients who eventually progressed developed metastases without local progression. RR levels did not appear to predict outcome. In 4 patients with available data, DCE-MRI may predict early response or resistance to therapy.
CONCLUSION
The combination of triapine at 72 mg/m2 three times weekly every other week and standard RT is tolerable with interesting activity in patients with LAPCA.
doi:10.1016/j.ijrobp.2012.06.003
PMCID: PMC3477302  PMID: 22818416
Triapine; radiation; pancreas cancer
13.  Phase I and pharmacokinetic study of erlotinib (OSI-774) in combination with docetaxel in squamous cell carcinoma of the head and neck (SSCHN) 
Cancer chemotherapy and pharmacology  2010;67(3):10.1007/s00280-010-1332-y.
Purpose
This phase I study determined the maximal-tolerated dose, dose-limiting toxicities, pharmacokinetics, and recommended dose of erlotinib with docetaxel.
Patients and methods
Twenty-eight patients with head and neck cancer were enrolled. Patients were orally given erlotinib (50 mg) daily plus 35 mg/m2 of docetaxel intravenously weekly × 3 every 4 weeks. Dose escalation of erlotinib was in 50-mg increments until toxicity. Pharmacokinetics were studied with LC–MS/MS, standard, and population pharmacokinetic methods.
Results
Ninety-five courses were successfully given (median 3, range 1–6). The most frequent side effects were diarrhea, fatigue, skin rash, anemia, and hypoalbuminemia. Dose de-escalation for both erlotinib and docetaxel was due to skin rash, neutropenia and/or severe infection with docetaxel to 25 mg/m2 and erlotinib to starting dose of 50 mg and re-escalation of docetaxel to 35 mg/m2. Responses were observed in 4/26 evaluable patients (100 mg erlotinib). In 24 patients, the mean Cmax and AUC erlotinib values increased with dose and following cumulative dosing (days 7 and 8 vs. day1, p < 0.05). The CL/F (~7 L/h), V/F (~140 L), and t1/2 (~20 h) for erlotinib were similar to the reported. The mean AUC ratio of metabolite OSI-420 to erlotinib following repetitive dosing at 100 mg (+ or − docetaxel) showed a ~50% increase (p < 0.02), possibly suggesting self-enzyme induction. Population pharmacokinetic studies showed no significant covariate affecting erlotinib pharmacokinetics.
Conclusions
The combination of erlotinib and docetaxel was associated with significant toxicity, which limited the amount of administered erlotinib. Dosing for phase II trials was docetaxel 35 mg/m2 and erlotinib 50 mg. The reason for excessive toxicity is not clear, but not due to change in pharmacokinetics.
doi:10.1007/s00280-010-1332-y
PMCID: PMC3828747  PMID: 20490801
Erlotinib; Squamous cell carcinoma of the head and neck; OSI-774; Phase I
14.  PRMT5 Is Upregulated in Malignant and Metastatic Melanoma and Regulates Expression of MITF and p27Kip1 
PLoS ONE  2013;8(9):e74710.
Protein arginine methyltransferase-5 (PRMT5) is a Type II arginine methyltransferase that regulates various cellular functions. We hypothesized that PRMT5 plays a role in regulating the growth of human melanoma cells. Immunohistochemical analysis indicated significant upregulation of PRMT5 in human melanocytic nevi, malignant melanomas and metastatic melanomas as compared to normal epidermis. Furthermore, nuclear PRMT5 was significantly decreased in metastatic melanomas as compared to primary cutaneous melanomas. In human metastatic melanoma cell lines, PRMT5 was predominantly cytoplasmic, and associated with its enzymatic cofactor Mep50, but not STAT3 or cyclin D1. However, histologic examination of tumor xenografts from athymic mice revealed heterogeneous nuclear and cytoplasmic PRMT5 expression. Depletion of PRMT5 via siRNA inhibited proliferation in a subset of melanoma cell lines, while it accelerated growth of others. Loss of PRMT5 also led to reduced expression of MITF (microphthalmia-associated transcription factor), a melanocyte-lineage specific oncogene, and increased expression of the cell cycle regulator p27Kip1. These results are the first to report elevated PRMT5 expression in human melanoma specimens and indicate this protein may regulate MITF and p27Kip1 expression in human melanoma cells.
doi:10.1371/journal.pone.0074710
PMCID: PMC3786975  PMID: 24098663
15.  Therapeutic Potential of the Translation Inhibitor Silvestrol in Hepatocellular Cancer 
PLoS ONE  2013;8(9):e76136.
Background & Aims
Although hepatocellular cancers (HCC) frequently arise in the setting of fibrosis and a hepatic regenerative response requiring new cell growth, therapeutic strategies for these cancers have not targeted protein synthesis. Silvestrol, a rocaglate isolated from Aglaiafoveolata, can inhibit protein synthesis by modulating the initiation of translation through the eukaryotic initiation factor 4A. In this study, we evaluated the therapeutic efficacy of silvestrol for HCC.
Methods
The efficacy of silvestrol was examined using human HCC cells in vitro using an orthotopic tumor cell xenograft model in a fibrotic liver. The impact of silvestrol on the liver was assessed in vivo in wild-type mice.
Results
Silvestrol inhibited cell growth with an IC50 of 12.5-86 nM in four different HCC cell lines. In vitro, silvestrol increased apoptosis and caspase 3/7 activity accompanied by loss of mitochondrial membrane potential and decreased expression of Mcl-1 and Bcl-xL. A synergistic effect was observed when silvestrol was combined with other therapeutic agents, with a dose-reduction index of 3.42-fold with sorafenib and 1.75-fold with rapamycin at a fractional effect of 0.5. In vivo, an antitumor effect was observed with 0.4 mg/kg silvestrol compared to controls after one week, and survival of tumor-bearing mice was improved with a median survival time of 42 and 28 days in the silvestrol and control groups, respectively. The effect on survival was not observed in orthotopic xenografts in non-fibrotic livers. Silvestrol treatment in vivo did not alter liver structure.
Conclusions
These data identify silvestrol as a novel, structurally unique drug with potent anticancer activity for HCC and support the potential value of targeting initiation of translation in the treatment of HCC.
doi:10.1371/journal.pone.0076136
PMCID: PMC3784426  PMID: 24086701
16.  Development and validation of a rapid and sensitive high-performance liquid chromatography-mass spectroscopy assay for determination of 17-(allylamino)-17-demethoxygeldanamycin and 17-(amino)-17-demethoxygeldanamycin in human plasma 
A sensitive method was developed and validated for the measurement of 17-(allylamino)-17-demethoxygeldanamycin (17AAG) and its active metabolite 17-amino-17-demethoxygeldanamycin (17AG) in human plasma using 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG) as an internal standard. After the addition of internal standard, 200 µL of plasma was extracted using ice cold acetonitrile followed by analysis on a Thermo Finnigan triple-quadruple mass spectrometer coupled to an Agilent 1100 HPLC system. Chromatography was carried out on a 50 × 2.1 mm Agilent Zorbax SB-phenyl 5 µm column coupled to a 3mm Varian metaguard diphenyl pre-column using glacial acetic acid 0.1% and a gradient of acetonitrile and water at a flow rate of 500 µL/min. Atmospheric pressure chemical ionization and detection of 17AAG, 17AG and 17DMAG were accomplished using selected reaction monitoring of m/z 584.3 > 541.3, 544.2 > 501.2, and 615.3 >572.3 respectively in negative ion mode. Retention times for 17AAG, 17AG, and 17DMAG were 4.1, 3.5, and 2.9 minutes, respectively, with a total run time of 7 minutes. The assay was linear over the range 0.5–3000 ng/mL for 17AAG and 17AG. Replicate sample analysis indicated within- and between-run accuracy and precision within 15%. The recovery of 17AAG and 17AG from 200 µL of plasma containing 1, 25, 300, and 2500 ng/mL was 93% or greater. This high performance liquid chromatographic tandem mass spectroscopy (HPLC/MS/MS) method is superior to previous methods. It is the first analytical method reported to date for the quantitation of both 17AAG and its metabolite 17AG and can reliably quantitate concentrations of both compounds as low as 0.5 ng/mL.
doi:10.1016/j.jchromb.2008.06.029
PMCID: PMC3782378  PMID: 18635408
17.  Phase II Trial of Temsirolimus in Patients with Relapsed or Refractory Multiple Myeloma 
Leukemia research  2009;33(11):1475-1480.
In a phase II trial, 16 patients with relapsed refractory multiple myeloma received temsirolimus 25 mg IV weekly until progression. One partial response and 5 minor responses were observed for a total response rate of 38%. The median time to progression was 138 days. Grade 3–4 toxicity included fatigue (n=3), neutropenia (n=2), thrombocytopenia (n=2), interstitial pneumonitis (n=1), stomatitis (n=1) and diarrhea (n=1). Clinical activity was associated with a higher area under the curve (AUC) and maximal reduction in phosphorylated p70S6K and 4EBP1 in peripheral blood mononuclear cells. At the dose and schedule used, temsirolimus had low single agent activity. Investigation of alternate dosing schedules and use in combinations is indicated.
doi:10.1016/j.leukres.2009.01.039
PMCID: PMC3771631  PMID: 19261329
Multiple myeloma; mTOR; temsirolimus; pharmacokinetics
18.  Dual Targeting of the Cyclin/Rb/E2F and Mitochondrial Pathways in Mantle Cell Lymphoma with the Translation Inhibitor Silvestrol 
Purpose
During cell cycle progression, D-cyclins activate cyclin-dependent kinases (CDKs) 4/6 to inactivate Rb, permitting E2F1-mediated S-phase gene transcription. This critical pathway is typically deregulated in cancer, and novel inhibitory strategies would be effective in a variety of tumors. The protein synthesis inhibitor silvestrol has potent activity in B-cell leukemias via the mitochondrial pathway of apoptosis, and also reduces cyclin D1 expression in breast cancer and lymphoma cell lines. We hypothesized that this dual activity of silvestrol would make it especially effective in malignancies driven by aberrant cyclin D1 expression.
Experimental Design
Mantle Cell Lymphoma (MCL), characterized by elevated cyclin D1, was used as a model to test this approach. The cyclin D/Rb/E2F1 pathway was investigated in vitro using MCL cell lines and primary tumor cells. Silvestrol was also evaluated in vivo using an aggressive model of MCL.
Results
Silvestrol showed low nanomolar potency both in MCL cell lines and primary MCL tumor cells. D-cyclins were depleted with just 10 nM silvestrol at 16 hr, with subsequent reductions of phosphorylated Rb, E2F1 protein, and E2F1 target transcription. As demonstrated in other leukemias, silvestrol caused Mcl-1 depletion followed by mitochondrial depolarization and caspase-dependent apoptosis, effects not related to inhibition of CDK4/6. Silvestrol significantly (P<0.0001) prolonged survival in a MCL xenograft model without detectable toxicity.
Conclusions
These data indicate that silvestrol effectively targets the cyclin/CDK/Rb pathway, and additionally induces cytotoxicity via intrinsic apoptosis. This dual activity may be an effective therapeutic strategy in MCL and other malignancies.
doi:10.1158/1078-0432.CCR-12-0839
PMCID: PMC3677230  PMID: 22791882
translation; cyclin; Rb; lymphoma; cell cycle
19.  Development and Validation of a Highly Sensitive Liquid Chromatography/Mass Spectrometry Method for Simultaneous Quantification of Lenalidomide and Flavopiridol in Human Plasma 
Therapeutic drug monitoring  2008;30(5):620-627.
Lenalidomide, an immunomodulatory agent, and flavopiridol, a broad cyclin-dependent kinase inhibitor, are both active therapies for clinical use in genomic high risk chronic lymphocytic leukemia (CLL). A high-performance liquid chromatographic assay with tandem mass spectrometric detection has been developed to simultaneously quantify lenalidomide and flavopiridol in human and mouse plasma to facilitate their combined clinical development. Samples were prepared by liquid-liquid extraction with acetonitrile- (ACN) containing internal standard (IS), genistein, followed by evaporation of solvent and reconstitution in 95/5 H2O/ACN. Lenalidomide and IS were separated by reverse phase liquid chromatography on a C-18 column using a gradient of H2O and ACN, each with 0.1% formic acid. Atmospheric pressure chemical ionization (APCI) in positive-ion mode with single reaction monitoring on a triple quadrupole mass spectrometer were applied to detect transitions of lenalidomide (260.06 > 149.10) and flavopiridol (402.09 > 341.02). Lower limits of quantification (LLOQ) of lenalidomide and flavopiridol were 1nM and 0.3nM respectively. Recoveries of lenalidomide and flavopiridol from human plasma ranged from 99% to 116% throughout their linear ranges. Within and between-run precision and accuracy of replicate samples were all less than 15%. This is the most sensitive analytical method reported to date for both lenalidomide and flavopiridol. This sensitivity will enable late terminal phase concentration measurements and accurate pharmacokinetic parameter estimation in a planned clinical trial with lenalidomide and flavopiridol in CLL patients.
doi:10.1097/FTD.0b013e318185813d
PMCID: PMC3740534  PMID: 18708993
Lenalidomide; Pharmacokinetics; Flavopiridol; LCMS
20.  Complex karyotype predicts for inferior outcomes following reduced-intensity conditioning allogeneic transplant for chronic lymphocytic leukaemia 
British journal of haematology  2012;159(1):82-87.
Summary
Complex karyotype (CK) on metaphase cytogenetics discriminates poor outcome in chronic lymphocytic leukaemia (CLL) patients undergoing salvage treatment; we hypothesized that it might provide prognostic information for patients undergoing allogeneic stem cell transplant. Fifty-one CLL patients were analysed following transplant; 18-month overall survival (OS), event-free survival (EFS) and cumulative incidence of progression estimates were 35%, 14% and 63%, respectively, in patients with CK (n = 19) versus 83%, 68% and 29% in patients without (n = 32) (P ≤ 0.0001, P ≤ 0.0001, and P = 0.02). In patients with high-risk interphase cytogenetics, CK remained predictive of worse OS (P = 0.02) and EFS (P = 0.009). These findings support further evaluation of metaphase karyotype in transplant risk assessment.
doi:10.1111/j.1365-2141.2012.09239.x
PMCID: PMC3719859  PMID: 22831395
chronic lymphocytic leukaemia; transplant; fluorescent insitu hybridization; metaphase cytogenetics; conditioning
21.  Phase I/II trial of non-cytotoxic suramin in combination with weekly paclitaxel in metastatic breast cancer treated with prior taxanes 
Purpose
Suramin, a polysulfonated naphthylurea, inhibits the actions of polypeptide growth factors including acidic and basic fibroblast growth factors (aFGF and bFGF), which confer broad spectrum chemotherapy resistance. We hypothesized that suramin at non-cytotoxic doses in combination with weekly paclitaxel would be well tolerated and demonstrate anti-tumor activity.
Methods
Women with metastatic breast cancer who had been previously treated with a taxane in the adjuvant or metastatic setting were eligible. The primary objective of the phase I was to determine the dose of intravenous (IV) weekly suramin that resulted in plasma concentrations between 10 and 50 umol/l over 8–48 h (or the target range) in combination with IV 80 mg/m2 of weekly paclitaxel. The primary objective of the phase II trial was to determine the anti-tumor activity of the dosing regimen defined in phase I. Therapy was continued until disease progression or development of unacceptable toxicity.
Results
Thirty-one patients were enrolled (9: phase I; 22: phase II). In phase I, no dose-limiting toxicities were observed. Pharmacokinetics during the first cycle showed suramin concentrations within the target range for 21 of 24 weekly treatments (88 %). In phase II, the objective response rate (ORR) was 23 % (95 % CI 8–45 %), the median progression-free survival was 3.4 months (95 % CI 2.1–4.9 months), and the median overall survival was 11.2 months (95 % CI 6.6–16.0 months).
Conclusions
Non-cytotoxic doses of suramin in combination with weekly paclitaxel were well tolerated. The efficacy was below the pre-specified criteria required to justify further investigation.
doi:10.1007/s00280-012-1887-x
PMCID: PMC3466596  PMID: 22729159
Suramin; Paclitaxel; Metastatic; breast cancer; Phase I; Phase II
23.  The Novel Cyclin-Dependent Kinase Inhibitor Dinaciclib (SCH727965) Promotes Apoptosis and Abrogates Microenvironmental Cytokine Protection in Chronic Lymphocytic Leukemia Cells 
Leukemia  2012;26(12):2554-2557.
The cyclin dependent kinase (CDK) inhibitor flavopiridol has demonstrated promising clinical results in relapsed CLL patients leading to efforts to develop improved CDK inhibitors. Dinaciclib (SCH727965) is a pan-CDK inhibitor, derived from a detailed screen in ovarian xenograft mouse models for therapeutic index, whose toxicity in solid tumor phase I studies appears favorable. Dinaciclib in CLL cells demonstrates concentration dependent apoptosis that is superior to flavopiridol following a clinically relevant 2-hour exposure. Dinaciclib potently down-regulates expression of Mcl-1 in CLL cells and antagonizes protection mediated by multiple soluble proteins important in the microenvironment of CLL including TNF-α IL-4, BAFF, and CD40-ligand. In contrast, contact with stromal cells or fibronectin abrogates the cytotoxicity of dinaciclib that is antagonized by a pan inhibitor and p110 alpha isoform specific inhibitor of the phosphatidylinositol 3-kinase pathway suggesting potential for combination strategies. These data justify clinical development of dinaciclib in CLL.
doi:10.1038/leu.2012.144
PMCID: PMC3645353  PMID: 22791353
Dinaciclib; CDK; chronic lymphocytic leukemia
24.  A dose-finding, pharmacokinetic and pharmacodynamic study of a novel schedule of flavopiridol in patients with advanced solid tumors 
Investigational new drugs  2010;30(2):629-638.
Purpose
Based on the promising activity and tolerability of flavopiridol administered with a pharmacokinetically-derived dosing schedule in chronic lymphocytic leukemia (CLL), we conducted a phase I study using this schedule in patients with advanced solid tumors.
Experimental Design
Flavopiridol was given IV as a 30-min loading dose followed by a 4-hr infusion weekly for 4 weeks repeated every 6 weeks. Dose-escalation was in cohorts of three patients using the standard 3+3 phase I study design. Blood samples were obtained for pharmacokinetic and pharmacodynamic studies.
Results
Thirty-four eligible patients with advanced solid tumors received a total of 208 doses (median 7, range 1–24). Total doses ranged from 40 – 105 mg/m2. The primary dose limiting toxicity was cytokine release syndrome (CKRS). No antitumor responses were observed. The mean peak plasma concentration across all doses was 1.65 ± 0.86 µM. Area under the concentration-versus-time curve (AUC0–∞) ranged from 4.31 to 32.2 µM·hr with an overall mean of 13.6 ± 7.0 µM·hr. Plasma flavopiridol concentrations and AUC increased proportionally with dose. There was no correlation between cytokine levels and clinical outcomes.
Conclusions
The maximum-tolerated dose of flavopiridol is 20 mg/m2 bolus followed by 20 mg/m2 infusion over 4 hours given weekly for 4 weeks on a 6-week cycle in patients with advanced solid tumors. Flavopiridol PK was notably different, and there was a higher frequency of CKRS, despite prophylactic steroids, seen in this patient group compared to previous studies with CLL using a similar dosing schedule.
doi:10.1007/s10637-010-9563-7
PMCID: PMC3486515  PMID: 20938713
Flavopiridol; CDK inhibitor; Phase I trial; Solid tumors
25.  Silvestrol exhibits significant in vivo and in vitro antileukemic activities and inhibits FLT3 and miR-155 expressions in acute myeloid leukemia 
Background
Activating mutations [internal tandem duplication (ITD)] or overexpression of the FMS-like tyrosine kinase receptor-3 (FLT3) gene are associated with poor outcome in acute myeloid leukemia (AML) patients, underscoring the need for novel therapeutic approaches. The natural product silvestrol has potent antitumor activity in several malignancies, but its therapeutic impact on distinct molecular high-risk AML subsets remains to be fully investigated. We examined here the preclinical activity of silvestrol in FLT3-ITD and FLT3 wild-type (wt) AML.
Methods
Silvestrol in vitro anti-leukemic activity was examined by colorimetric cell viability assay, colony-forming and flow cytometry assays assessing growth inhibition and apoptosis, respectively. Pharmacological activity of silvestrol on FLT3 mRNA translation, mRNA and protein expression was determined by RNA-immunoprecipitation, qRT-PCR and immunoblot analyses, respectively. Silvestrol in vivo efficacy was investigated using MV4-11 leukemia-engrafted mice.
Results
Silvestrol shows antileukemia activity at nanomolar concentrations both in FLT3-wt overexpressing (THP-1) and FLT3-ITD (MV4-11) expressing AML cell lines (IC50 = 3.8 and 2.7 nM, respectively) and patients’ primary blasts [IC50 = ~12 nM (FLT3-wt) and ~5 nM (FLT3-ITD)]. Silvestrol increased apoptosis (~4fold, P = 0.0001), and inhibited colony-formation (100%, P < 0.0001) in primary blasts. Silvestrol efficiently inhibited FLT3 translation reducing FLT3 protein expression by 80–90% and decreased miR-155 levels (~60%), a frequently co-regulated onco-miR in FLT3-ITD-positive AML. The median survival of silvestrol-treated vs vehicle-treated mice was 63 vs 29 days post-engraftment, respectively (P < 0.0001).
Conclusions
Silvestrol exhibits significant in vivo and in vitro antileukemic activities in AML through a novel mechanism resulting in inhibition of FLT3 and miR-155 expression. These encouraging results warrant a rapid translation of silvestrol for clinical testing in AML.
doi:10.1186/1756-8722-6-21
PMCID: PMC3623627  PMID: 23497456

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