Combining nonsense mutation suppression therapy with nonsense-mediated decay inhibition in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) results from germline mutations in the tumor-suppressor gene NF1 and predisposes patients to developing nervous system tumors. Twenty percent of NF1 patients harbor nonsense mutations resulting in premature termination codons (PTCs). Nonsense suppression therapies can facilitate ribosomal readthrough of PTCs to restore full-length protein, but their potential in NF1 is underexplored. We developed a minipig model of NF1 carrying a PTC to test whether nonsense suppression could restore expression of the NF1-encoded protein neurofibromin in vitro and in vivo. Nonsense suppression did not reliably increase neurofibromin in primary NF1-/- Schwann cells isolated from minipig neurofibromas but could reduce phosphorylated ERK. Gentamicin in vivo produced a similar plasma pharmacokinetic profile to humans and was detectable in clinically relevant tissues, including cerebral cortex, sciatic nerve, optic nerve, and skin. In gentamicin-treated animals, increased neurofibromin expression was seen in the optic nerve. Nonsense-mediated decay (NMD) causes degradation of transcripts with PTCs, which could impede nonsense suppression therapies. Nonsense suppression in combination with NMD inhibition restored neurofibromin protein expression in primary NF1-/- Schwann cells isolated from minipig neurofibromas. Thus, the effectiveness of nonsense suppression therapies can be improved in NF1 by the concurrent use of NMD inhibitors.

Influence of Renal Function on Phosphoramide Mustard Exposure: A Nonlinear Mixed-Effects Analysis.

Phosphoramide mustard (PM) is the final cytotoxic metabolite formed from the parent compound cyclophosphamide through a complex metabolic pathway, primarily through hepatic metabolism. Little is known about the effect of renal elimination on the disposition of PM. We evaluated the effect of renal function on PM exposure after single doses of cyclophosphamide in 85 patients undergoing allogeneic hematopoietic cell transplantation using nonlinear mixed-effects modeling. Mixed linear and nonlinear elimination pathways were required to adequately describe the disposition of PM. Creatinine clearance (CrCL) was incorporated as a covariate associated with first-order elimination, representing renal clearance (ClR ) of PM. For a 70-kg patient, ClR was 14.9 L/h, Volume of distribution was 525 L, maximum rate was 81.2 mg/h, and the concentration to achieve 50% of maximum rate was 0.51 mg/L. We conducted simulations to explore the impact of CrCL as a measure of renal function and observed that when CrCL decreases from 120 to 40 mL/min, PM area under the plasma concentration-time curve (AUC) from time 0 to 8 hours and AUC increases by 9.2% and 80.9% on average after a single dose, respectively. Our data suggest that renal function has limited influence on PM exposure during the first 8 hours after dosing but has a large impact on the total exposure. Dose adjustment of cyclophosphamide may not be necessary in hematopoietic cell transplant recipients with moderate to severe kidney dysfunction to attain targeted exposures based on AUC from time 0 to 8 hours. However, dose reduction may be necessary if demonstrated at some future time that total AUC is a better surrogate for safety or toxicity.

Comparison of Dose Adjustment Strategies for Obesity in High-dose Cyclophosphamide Among Adult Hematopoietic Cell Transplantation Recipients: Pharmacokinetic Analysis.

Cyclophosphamide (CY) is an alkylating agent widely used in the field of oncology and hematopoietic cell transplantation (HCT). It is recommended to use an adjusted body weight with an adjustment factor of 0.25 (ABW25) for dosing of CY in obese patients undergoing HCT. However, evidence based on the pharmacokinetics (PK) of CY to support this recommendation is lacking. We aimed to identify a dosing strategy of CY that achieves equivalent exposures among obese and nonobese patients. The present study is a secondary analysis of a previously conducted observational PK study of phosphoramide mustard (PM), the final cytotoxic metabolite of CY. Data were collected from 85 adults with hematologic malignancy who received a single infusion of CY 50 mg/kg, fludarabine, ± anti-thymocyte globulin, and a single fraction of total body irradiation as HCT conditioning therapy. A previously developed population PK model in these patients was used for simulations. Using individualized PK parameters from that analysis, simulations were performed to assess cumulative exposures of PM (i.e., area-under-the-curve [AUC]) resulting from 8 different dosing strategies according to various measures of body size: (1) "mg/kg" by total body weight (TBW); (2) "mg/kg" by ideal body weight (IBW); (3) "mg/kg" by fat free mass; (4) "mg/m2" by body surface area (BSA); (5) "mg/kg" by TBW combined with ABW25 (TBW-ABW25); (6) "mg/kg" by IBW combined with ABW25 (IBW-ABW25); (7) "mg/kg" by TBW combined with ABW by adjustment factor of 0.50 (TBW-ABW50); and (8) "mg" by fixed-dose. We defined equivalent exposure as the effect of obesity on PM AUC within ±20% from the PM AUC in the nonobese group, where obesity is defined based on TBW/IBW ratio (i.e., nonobese, <1.2; mildly obese, 1.2-1.5; and moderately/severely obese, >1.5). Primary and secondary outcomes were PM AUC0-8hours and PM AUC0-infinity, respectively. In the 85 patients, with the median age of 63 years (range 21-75), 46% were classified as mildly and 25% were moderately/severely obese based on the TBW/IBW ratio. Negative correlations (i.e., higher the extent of obesity, lower the PM AUC) were shown when dosing simulations were based on IBW, TBW-ABW25, and fixed dosing (P < .05). Positive correlations were shown when dosing was simulated by TBW (P < .05). None of the 8 dosing strategies attained equivalent PM AUC0-8hours between patients with versus without obesity, whereas dosing by BSA and TBW-ABW50 attained equivalent PM AUC0-infinity (P < .05). Our study predicted that the recommended ABW25 dose adjustment may result in lower exposure of CY therapy in obese patients than in nonobese. A CY dosing strategy that would result in similar PM concentrations between obese and nonobese was not identified for early exposure (i.e., PM AUC0-8hours). The data suggest though that CY dosing based on "mg/m2" by BSA or "mg/kg" by TBW-ABW50 would result in similar total exposure (i.e., PM AUC0-infinity) and may minimize exposure differences in obese and nonobese patients.

Predictive Value of C-Reactive Protein and Albumin for Temporal Within-Individual Pharmacokinetic Variability of Voriconazole in Pediatric Patients Undergoing Hematopoietic Cell Transplantation.

Voriconazole is a widely used antifungal agent in immunocompromised patients, but its utility is limited by its variable exposure and narrow therapeutic index. Population pharmacokinetic (PK) models have been used to characterize voriconazole PK and derive individualized dosing regimens. However, determinants of temporal within-patient variability of voriconazole PK were not well established. We aimed to characterize temporal variability of voriconazole PK within individuals and identify predictive clinical factors. This study was conducted as a part of a single-institution, phase I study of intravenous voriconazole in children undergoing hematopoietic cell transplantation (NCT02227797). We analyzed voriconazole PK study data collected at week 1 and again at week 2 after the start of voriconazole therapy in 59 pediatric patients undergoing HCT (age <21 years). Population PK analysis using nonlinear mixed effect modeling was performed to analyze temporal within-individual variability of voriconazole PK by incorporating a between-occasion variability term in the model. A 2-compartment linear elimination model incorporating body weight and cytochrome P450 2C19 phenotype described the data. The ratio of individual voriconazole clearance between weeks 1 and 2 ranged from 0.11 to 3.3 (-9.1 to +3.3-fold change). Incorporation of covariate effects by serum C-reactive protein and albumin levels decreased between-occasion variability of clearance as compared to the model without them (coefficient of variation, 41.2% and 59.5%, respectively) and improved the model fit (P < .05). As significant covariates on voriconazole PK, C-reactive protein and albumin concentrations may potentially serve as useful biomarkers as part of therapeutic drug monitoring.

CYP2C19 Phenotype and Body Weight-Guided Voriconazole Initial Dose in Infants and Children after Hematopoietic Cell Transplantation.

Prophylactic voriconazole use is recommended for children undergoing hematopoietic cell transplantation (HCT). Dosing considerations are essential, due to the narrow therapeutic window of voriconazole. Known covariates do not sufficiently explain the large interindividual pharmacokinetic (PK) variability of voriconazole. Moreover, knowledge of voriconazole PK for age <2 years is limited. We investigated genetic and clinical covariate associations with voriconazole interindividual PK variability and subsequently simulated dosing regimens in children. This study was conducted as part of a single-institution, phase I study of intravenous voriconazole therapy for children undergoing HCT. We conducted a population PK analysis and tested covariate effects on voriconazole PK, including 67 genetic variants and clinical variables. We analyzed plasma voriconazole and N-oxide metabolite concentrations from 58 children <21 years of age (including 12 children <2 years of age). A two-compartment parent mixed linear/nonlinear model best described our data. The CYP2C19 phenotype and body weight were significant covariates (P < 0.05 for both). Our model performance for age <2 years was comparable to that for other age groups. Simulation of the final model suggested the following doses to attain target steady-state trough concentrations of 1.5 to 5.0 mg/liter for the CYP2C19 normal phenotype: 16 mg/kg (weight of <15 kg), 12 mg/kg (weight of 15 to 30 kg), or 10 mg/kg (weight of >30 kg); doses were 33 to 50% lower for CYP2C19 poor/intermediate phenotypes and 25 to 50% higher for CYP2C19 rapid/ultrarapid phenotypes. We propose a new starting-dose regimen, combined with therapeutic drug monitoring, for intravenous voriconazole therapy in children of all ages. Future studies should validate this dosing regimen.

Selumetinib normalizes Ras/MAPK signaling in clinically relevant neurofibromatosis type 1 minipig tissues in vivo.

BACKGROUND: The MEK1/2 inhibitor selumetinib was recently approved for neurofibromatosis type 1 (NF1)-associated plexiform neurofibromas, but outcomes could be improved and its pharmacodynamic evaluation in other relevant tissues is limited. The aim of this study was to assess selumetinib tissue pharmacokinetics (PK) and pharmacodynamics (PD) using a minipig model of NF1. METHODS: WT (n = 8) and NF1 (n = 8) minipigs received a single oral dose of 7.3 mg/kg selumetinib. Peripheral blood mononuclear cells (PBMCs), cerebral cortex, optic nerve, sciatic nerve, and skin were collected for PK analysis and PD analysis of extracellular regulated kinase phosphorylation (p-ERK) inhibition and transcript biomarkers (DUSP6 & FOS). RESULTS: Key selumetinib PK parameters aligned with those observed in human patients. Selumetinib concentrations were higher in CNS tissues from NF1 compared to WT animals. Inhibition of ERK phosphorylation was achieved in PBMCs (mean 60% reduction), skin (95%), and sciatic nerve (64%) from all minipigs, whereas inhibition of ERK phosphorylation in cerebral cortex was detected only in NF1 animals (71%). Basal p-ERK levels were significantly higher in NF1 minipig optic nerve compared to WT and were reduced to WT levels (60%) with selumetinib. Modulation of transcript biomarkers was observed in all tissues. CONCLUSIONS: Selumetinib reduces MAPK signaling in tissues clinically relevant to NF1, effectively normalizing p-ERK to WT levels in optic nerve but resulting in abnormally low levels of p-ERK in the skin. These results suggest that selumetinib exerts activity in NF1-associated CNS tumors by normalizing Ras/MAPK signaling and may explain common MEK inhibitor-associated dermatologic toxicities.

CYP51A1 polymorphism and voriconazole-associated hepatotoxicity in children undergoing hematopoietic cell transplant.

Fungal CYP51A (14α-sterol demethylase) is the target of an azole antifungal, voriconazole (VCZ), which also partially inhibits human CYP51A1. Hepatotoxicity is a common adverse effect of azoles, which is reported to be caused by altered gene expressions secondary to cholesterol synthesis inhibition by azoles. This is a post-hoc analysis of a previously conducted phase 1 dose-finding study of prophylactic VCZ in 56 pediatric hematopoietic cell transplant recipients. We explored an association between variants in human CYP51A1 (rs2282976 and rs6465348) and VCZ-induced hepatotoxicity. Genotype A/G or G/G in rs6465348 showed lower odds of hepatotoxicity after adjusting for VCZ area-under-the-curve (OR: 0.10, 95% CI: 0.01 - 0.79, vs. A/A).

Impact of Obesity on Voriconazole Pharmacokinetics among Pediatric Hematopoietic Cell Transplant Recipients.

Voriconazole (VCZ) is an antifungal agent with wide inter- and intrapatient pharmacokinetic (PK) variability and narrow therapeutic index. Although obesity was associated with higher VCZ trough concentrations in adults, the impact of obesity had yet to be studied in children. We characterized the PK of VCZ in obese patients by accounting for age and CYP2C19 phenotype. We conducted intensive PK studies of VCZ and VCZ N-oxide metabolite in 44 hematopoietic stem cell transplantation (HSCT) recipients aged 2 to 21 years who received prophylactic intravenous VCZ every 12 hours (q12h). Blood samples were collected at 5 and 30 minutes; at 1, 3, 6, and 9 hours after infusion completion; and immediately before the next infusion start. We estimated PK parameters with noncompartmental analysis and evaluated for an association with obesity by multiple linear regression analysis. The 44 participants included 9 (20%) with obesity. CYP2C19 metabolism phenotypes were identified as normal in 22 (50%), poor/intermediate in 13 (30%), and rapid/ultrarapid in 9 patients (21%). Obesity status significantly affects the VCZ minimum concentration of drug in serum (Cmin) (higher by 1.4 mg/liter; 95% confidence interval [CI], 0.0 to 2.8; P = 0.047) and VCZ metabolism ratio (VCZRATIO) (higher by 0.4; 95% CI, 0.0 to 0.7; P = 0.03), while no association was observed with VCZ area under the curve (AUC) (P = 0.09) after adjusting for clinical factors. A younger age and a CYP2C19 phenotype were associated with lower VCZ AUC. Obesity was associated with decreased metabolism of VCZ to its inactive N-oxide metabolite and, concurrently, increased VCZ Cmin, which is deemed clinically meaningful. Future research should aim to further characterize its effects and determine a proper dosing regimen for the obese.

A phase I dose finding study of intravenous voriconazole in pediatric patients undergoing hematopoietic cell transplantation.

To optimize voriconazole dosing in pediatric hematopoietic cell transplantation (HCT), we conducted a phase I study with a modified 3 + 3 dose-escalation followed by an expansion cohort at the maximum tolerated, minimum efficacious dose (MTD/MED). Patients ≤21 years who required voriconazole for prevention or treatment of an invasive fungal infection were assigned to three age groups. Of the 59 evaluable patients, 13 were <2 years, 23 were 2-11, and 23 were 12-21. Therapeutic serum voriconazole troughs (1.5-5 µg/mL) drawn at 7 days after initiation determined efficacy. The MTD/MED was 12 mg/kg/dose q12 h × 2 loading doses, then 10 mg/kg/dose q12 h in patients <2, and was 10 mg/kg/dose q12 h in patients 2-11. The 12-21 age group had no dose-limiting toxicity at 8 mg/kg/dose q12 h; however, the MED was not reached. Drug-related AEs ≥grade 3 included increased bilirubin, transaminases, and creatinine, all occurring in <10%. There was no significant association between supra-therapeutic troughs and AEs. Five of 17 patients who had supra-therapeutic troughs (29%) had an AE, compared to 8 of 42 who did not (19%, p = 0.38). Observational population pharmacokinetic analysis demonstrated that inter-individual variability on voriconazole clearance was >100% CV, and clearance increased with age.

Gemcitabine and metabolite pharmacokinetics in advanced NSCLC patients after bronchial artery infusion and intravenous infusion.

PURPOSE: We investigated the safety, pharmacokinetics, and efficacy of gemcitabine administered via bronchial artery infusion (BAI) and IV infusion in advanced NSCLC patients. METHODS: Patients were eligible if they had received at least two prior cytotoxic chemotherapy regimens. Gemcitabine was administered via BAI as 600 mg/m2 on day one of cycle one, followed by IV as 1000 mg/m2 on day eight of cycle one, and IV on days one and eight of all subsequent cycles. Pharmacokinetics for gemcitabine and dFdU metabolite in plasma, and dFdCTP active metabolite in peripheral blood mononuclear cells (PBMC) were evaluated. Intensive pharmacokinetic sampling was performed after BAI and IV infusions during cycle one. RESULTS: Three male patients (age range 59-68 years) were evaluated. All patients responded with stable disease or better. One PR was observed after cycle three, and the remaining had SD. Cmax (mean ± SD) following BAI for gemcitabine, dFdCTP, and dFdU were 7.71 ± 0.13, 66.5 ± 40.6, and 38 ± 6.27 µM and following IV infusion, 17 ± 2.36, 50.8 ± 3.61, and 83.2 ± 12.3 µM, respectively. The AUCinf (mean ± SD) following BAI for gemcitabine, dFdCTP, and dFdU were 6.89 ± 1.2, 791.1 ± 551.2, and 829.9 ± 217.8 µM h and following IV infusion, 12.5 ± 3.13, 584 ± 86.6, and 1394.64 ± 682.2 µM h, respectively. The AUC and Cmax of dFdCTP after BAI were higher than IV. The median OS was 6.27 months. No grade 3 or 4 toxicity was observed. The most common side effects were all grade ≤ 2 involving nausea, vomiting, rigor, thrombocytopenia, and anemia. CONCLUSIONS: Systemic exposure to dFdCTP was higher after BAI than IV in two out of three patients.

Genetically engineered minipigs model the major clinical features of human neurofibromatosis type 1.

Neurofibromatosis Type 1 (NF1) is a genetic disease caused by mutations in Neurofibromin 1 (NF1). NF1 patients present with a variety of clinical manifestations and are predisposed to cancer development. Many NF1 animal models have been developed, yet none display the spectrum of disease seen in patients and the translational impact of these models has been limited. We describe a minipig model that exhibits clinical hallmarks of NF1, including café au lait macules, neurofibromas, and optic pathway glioma. Spontaneous loss of heterozygosity is observed in this model, a phenomenon also described in NF1 patients. Oral administration of a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor suppresses Ras signaling. To our knowledge, this model provides an unprecedented opportunity to study the complex biology and natural history of NF1 and could prove indispensable for development of imaging methods, biomarkers, and evaluation of safety and efficacy of NF1-targeted therapies.

Personalized fludarabine dosing to reduce nonrelapse mortality in hematopoietic stem-cell transplant recipients receiving reduced intensity conditioning.

Patients undergoing hematopoietic cell transplantation (HCT) with reduced intensity conditioning (RIC) commonly receive fludarabine. Higher exposure of F-ara-A, the active component of fludarabine, has been associated with a greater risk of nonrelapse mortality (NRM). We sought to develop a model for fludarabine dosing in adult HCT recipients that would allow for precise dose targeting and predict adverse clinical outcomes. We developed a pharmacokinetic model from 87 adults undergoing allogeneic RIC HCT that predicts F-ara-A population clearance (Clpop) accounting for ideal body weight and renal function. We then applied the developed model to an independent cohort of 240 patients to identify whether model predictions were associated with NRM and acute graft versus host disease (GVHD). Renal mechanisms accounted for 35.6% of total F-ara-A Clpop. In the independent cohort, the hazard ratio of NRM at day 100 was significantly higher in patients with predicted F-ara-A clearance (Clpred) <8.50 L/h (P < 0.01) and area under the curve (AUCpred) >6.00 µg × h/mL (P = 0.01). A lower Clpred was also associated with more NRM at month 6 (P = 0.01) and trended toward significance at 12 months (P = 0.05). In multivariate analysis, low fludarabine clearance trended toward higher risk of acute GVHD (P = 0.05). We developed a model that predicts an individual's systemic F-ara-A exposure accounting for kidney function and weight. This model may provide guidance in dosing especially in overweight individuals and those with altered kidney function.

Pharmacokinetic-pharmacodynamic modelling of acute N-terminal pro B-type natriuretic peptide after doxorubicin infusion in breast cancer.

AIMS: The aim of the present study was to develop a pharmacokinetic-pharmacodynamic (PK-PD) model to characterize the relationship between plasma doxorubicin and N-terminal pro B-type natriuretic peptide (NT-proBNP) concentrations within 48 h of doxorubicin treatment. METHODS: The study enrolled 17 female patients with stages 1-3 breast cancer and receiving adjuvant doxorubicin (60 mg m(-2) ) and cyclophosphamide (600 mg m(-2) ) every 14 days for four cycles. In two consecutive cycles, plasma concentrations of doxorubicin, doxorubicinol, troponin and NT-proBNP were collected before infusion, and up to 48 h after the end of doxorubicin infusion. Nonlinear mixed-effects modelling was used to describe the PK-PD relationship of doxorubicin and NT-proBNP. RESULTS: A three-compartment parent drug with a one-compartment metabolite model best described the PK of doxorubicin and doxorubicinol. Troponin concentrations remained similar to baseline. An indirect PD model with transit compartments best described the relationship of doxorubicin exposure and acute NT-proBNP response. Estimated PD parameters were associated with large between-subject variability (total assay variability 38.8-73.9%). Patient clinical factors, including the use of enalapril, were not observed to be significantly associated with doxorubicin PK or NT-proBNP PD variability. CONCLUSION: The relationship between doxorubicin concentration and the acute NT-proBNP response was successfully described with a population PK-PD model. This model will serve as a valuable framework for future studies to identify clinical factors associated with the acute response to doxorubicin. Future studies are warranted to examine the relationship between this acute response and subsequent heart failure. Should such a relationship be established, this model could provide useful information on patients' susceptibility to doxorubicin-induced long-term cardiotoxicity.

Randomized, blinded trial of vitamin D3 for treating aromatase inhibitor-associated musculoskeletal symptoms (AIMSS).

The purpose of the study was to evaluate the efficacy and safety of vitamin D3 at 4000 IU/day as a treatment option for aromatase inhibitor-associated musculoskeletal symptoms (AIMSS) when compared with the usual care dose of 600 IU D3. We conducted a single site randomized, double-blind, phase 3 clinical trial in women with AIMSS comparing change in symptoms, reproductive hormones and AI pharmacokinetics. Postmenopausal women ≥18 years with stages I-IIIA breast cancer, taking AI and experiencing AIMSS [breast cancer prevention trial symptom scale-musculoskeletal (BCPT-MS) subscale ≥1.5] were admitted. Following randomization, 116 patients had a run-in period of 1 month on 600 IU D3, then began the randomized assignment to either 600 IU D3 (n = 56) or 4000 IU D3 (n = 57) daily for 6 months. The primary endpoint was a change in AIMSS from baseline (after 1 month run-in) on the BCPT-MS (general MS pain, joint pain, muscle stiffness, range for each question: 0 = not at all to 4 = extremely). Groups had no statistically significant differences demographically or clinically. There were no discernable differences between the randomly allocated treatment groups at 6 months in measures of AIMSS, pharmacokinetics of anastrozole and letrozole, serum levels of reproductive hormones, or adverse events. We found no significant changes in AIMSS measures between women who took 4000 IU D3 daily compared with 600 IU D3. The 4000 IU D3 did not adversely affect reproductive hormone levels or the steady state pharmacokinetics of anastrozole or letrozole. In both groups, serum 25(OH)D remained in the recommended range for bone health (≥30 ng/mL) and safety (<50 ng/mL).

Pathway-based pharmacogenomics of gemcitabine pharmacokinetics in patients with solid tumors.

AIM: The aim of this study was to evaluate the association of gemcitabine pathway SNPs with detailed pharmacokinetic measures obtained from solid tumor patients receiving gemcitabine-based therapy. MATERIALS & METHODS: SNPs within nine gemcitabine pathway genes, namely CDA, CMPK, DCK, DCTD, NT5C2, NT5C3, SLC28A1, SLC28A3 and SLC29A1 were analyzed for association with gemcitabine pharmacokinetics. RESULTS: Significant association of gemcitabine clearance with SNPs in NT5C2 was identified. Clearance of 2´,2´-difluorodeoxyuridine, a gemcitabine metabolite was significantly predicted by CDA, SLC29A1 and NT5C2 SNPs. This study reports an association of formation clearance of 2´,2´-difluoro-2´-deoxycytidine triphosphate, an active form of gemcitabine with SNPs within uptake transporters SLC28A1, SLC28A3 and SLC29A1. CONCLUSION: Genetic variation in gemcitabine pathway genes is associated with its pharmacokinetics and hence could influence gemcitabine response. Our study identified pharmacogenetic markers that could be further tested in larger patient cohorts and could open up opportunities to individualize therapy in solid tumor patients.

Cytotoxic purine nucleoside analogues bind to A1, A2A, and A3 adenosine receptors.

Fludarabine, clofarabine, and cladribine are anticancer agents which are analogues of the purine nucleoside adenosine. These agents have been associated with cardiac and neurological toxicities. Because these agents are analogues of adenosine, they may act through adenosine receptors to elicit their toxic effects. The objective of this study was to evaluate the ability of cytotoxic nucleoside analogues to bind and activate adenosine receptor subtypes (A(1), A(2A), A(2B), and A(3)). Radioligand binding studies utilizing Chinese hamster ovary cells, stably transfected with adenosine A(1), A(2A), or A(3) receptor subtype, were used to assess the binding affinities of these compounds, whereas adenylyl cyclase activity was used to assess the binding to A(2B) receptors. Clofarabine and cladribine both bound to the A(2A) receptor with a K (i) of 17 and 15 µM, respectively. Clofarabine was the only adenosine analogue to bind to the A(3) receptor with a K (i) of 10 µM, and none of these compounds bound to the A(2B) receptor. Results show that clofarabine, cladribine, and fludarabine bind to the A(1) receptor. In addition, clofarabine, cladribine, and fludarabine were A(1) agonists (IC(50) 3.1, 30, and 30 µM, respectively). Neither pyrimidine nucleoside analogues gemcitabine nor cytarabine associated with any of the adenosine receptor subtypes (K (i) > 100µM). This is the first report of an interaction between all adenosine receptor subtypes and chemotherapeutic nucleoside analogues commonly used in the treatment of cancer. Therefore, activation of these receptors may be at least one mechanism through which fludarabine-associated toxicity occurs.

Pharmacodynamic modeling of sequence-dependent antitumor activity of insulin-like growth factor blockade and gemcitabine.

Agents that block insulin-like growth factor (IGF) signaling are under investigation in clinical trials. Antitumor effects are likely to be enhanced when combined with other agents, but administration sequence effects on activity are not well-described. Three breast cancer cell lines (MCF-7, MDA-MB-231, and Hs-578T) were treated with Gemcitabine and small molecule receptor tyrosine kinase inhibitor cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]1-(2-phenyl-quinolin-7-yl)-imidazo [1,5-a]pyrazin-8-ylamine (PQIP) as single agents and then in combination in the forward (Gemcitabine followed by PQIP) and reverse (PQIP followed by Gemcitabine) sequences. Antitumor effects were assessed longitudinally by Bayesian analysis using WinBUGS. The pharmacodynamic model adequately predicted the observed data. The differences in the cell-kill rate constants for the forward vs. reverse sequence ranged from 0.11 to 0.64 (day(-1)), and statistical significance was generally dependent on cell line and PQIP concentration. These data indicate that treatment with Gemcitabine first, followed by PQIP is superior to the reverse sequence in vitro.

Combinatorial pharmacologic effects of gemcitabine and its metabolite dFdU.

Recent evidence has shown that the gemcitabine metabolite, dFdU, is pharmacologically active. Though less potent, dFdU has a longer half-life and could potentiate or antagonize the activity of gemcitabine. Hence, studies were undertaken to evaluate the combined effects. Following chemical synthesis, an improved purification procedure for dFdU was developed (80 % yield; >99 % purity). Zebrafish phenotype-based embryo screens revealed no acute toxicity after gemcitabine or dFdU treatment. Only gemcitabine affected zebrafish development in a dose-dependent manner. Synergy or antagonism for the combination was not observed. Antitumor effects for dFdU were dose dependent. Antagonism was tumor cell-line dependent and did not depend on formation of the intracellular active metabolite of gemcitabine, suggesting that the drug-metabolite interaction occurs later. These studies highlight a platform for testing the pharmacologic activity for anticancer agent and metabolite combinations. Such analyses are expected to provide insight into the beneficial or harmful effect(s) of metabolites towards parent drug activity.

Phase 1 trial of gemcitabine with bortezomib in elderly patients with advanced solid tumors.

INTRODUCTION: Bortezomib, a proteasome inhibitor, has synergistic antitumor activity with gemcitabine, an antimetabolite, in preclinical and clinical studies. The safety of this combination has not yet been established in elderly patients; therefore, this dose-escalation study was designed to assess the maximum-tolerated dose of bortezomib and gemcitabine in patients aged 70 years or older with advanced-stage solid tumors. PATIENTS AND METHODS: Gemcitabine was administered intravenously (800 to 1000 mg/m) over 30 minutes on days 1 and 8, followed 60 minutes later by bortezomib administered as an intravenous push over 3 to 5 seconds (1.0 to 1.8 mg/m) on a 21-day cycle. This study used a standard phase 1 dose-escalation design with 3 or 6 patients per dose level. RESULTS: Seventeen patients with stage IV solid tumors were treated. Median age was 73 years (range: 70 to 87 y). All patients had an Eastern Cooperative Oncology Group (ECOG) performance status less than 2. Median number of earlier chemotherapy regimens was 2 (range: 0 to 6). Dose-limiting toxicities were seen in 2 of 8 patients enrolled at the second dose level of gemcitabine (1000 mg/m) and bortezomib (1.0 mg/m), which consisted of grade ≥3 lower extremity edema, thrombocytopenia, fatigue, and dehydration. The most common grade ≥3 toxicities included thrombocytopenia (n=9), neutropenia (n=6), and anemia (n=5). Partial response (n=3) or disease stabilization (n=3) was seen in 6 of 14 evaluable patients. CONCLUSIONS: Concurrent weekly gemcitabine (800 mg/m) and bortezomib (1 mg/m) is the recommended schedule for future phase 2 trials in elderly patients with stage IV solid tumors.