Improved sensitivity for methotrexate analysis using enzyme multiplied immunoassay technique on the Siemens Viva-E instrument.

BACKGROUND: The available assay kit for methotrexate (MTX) using the Syva enzyme multiplied immunoassay technique (EMIT) reagents on the Siemens Viva-E instrument allows for the detection of MTX in serum or plasma to concentrations as low as 0.3 µmole/L. Current clinical decision points for MTX therapeutic drug monitoring and leucorvorin rescue exist at concentrations below that limit. OBJECTIVE: The goal of this study was to lower the limit of MTX quantitation to 0.05 µmole/L using the EMIT assay technology. METHODS: EMIT MTX assay parameters were modified on the Viva-E instrument to increase the sample volume, alter the calibration method, and employ an alternate calibrator set created to achieve lower detection. Intraassay and interassay precision was assessed for MTX controls. RESULTS: We observed a CV of 9.4% for intraassay precision with a bias of <0.01% and a CV of 15.7% for interassay precision with a bias of 22.5% for the 0.05 µmole/L control. Precision data for all other controls were <4%. The modified EMIT MTX assay and the unmodified approved assay were compared with a high sensitivity fluorescence polarization immunoassay method. Linear regression of correlation data revealed that both the modified and the commercial EMIT assays produced positive bias compared with the high sensitivity fluorescence polarization immunoassay method (y-int = 0.03 and 0.08, respectively). However, the modified EMIT assay had the best correlation in the low range (0.03-2 µmole/L). Additionally, endogenous and chemical interference testing demonstrated that the modified assay was not affected to a clinically significant extent. CONCLUSIONS: The described modifications have enhanced the sensitivity of the Syva EMIT assay for MTX measurements down to 0.05 µmole/L with acceptable precision that can be used in clinical practice for monitoring MTX therapy.

Biodistribution of HPMA copolymer-aminohexylgeldanamycin-RGDfK conjugates for prostate cancer drug delivery.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-RGD (Arg-Gly-Asp) conjugates targeting the alpha(v)beta(3) integrin present on angiogenic blood vessels and some tumor types have shown increased accumulation in solid tumors and possess properties that suggest their use for site-specific drug delivery. Geldanamycin (GDM) is a benzoquinoid ansamycin that binds to heat-shock protein 90 (HSP90), effective for the treatment of multiple cancer types including prostate, but has dose-limiting cytotoxicity. We recently reported the synthesis of HPMA copolymer-aminohexyl-geldanamycin (AH-GDM) conjugates containing RGDfK that demonstrated favorable properties of drug release, in vitro binding to the alpha(v)beta(3) integrin, cytotoxicity in human prostate cancer cells, and tolerability in nude mice greater than 2-fold equivalent free drug doses. In this study the biodistribution of 125I-radiolabeled HPMA copolymer-AH-GDM conjugates with and without RGDfK in both non-tumor-bearing and DU145 prostate tumor xenograft-bearing nude mice was evaluated. At 60 mg/kg drug equivalent polymer doses in non-tumor-bearing mice both conjugates showed fast elimination from blood and decreasing accumulation in all other organs. Kidney accumulation predominated and was higher for the conjugate containing RGDfK. In tumor-bearing mice, trace quantities of the conjugate containing RGDfK showed increased tumor accumulation as compared to the conjugate without RGDfK. Also evaluated were free drug concentrations in prostate tumor xenografts following treatments of 30 and 60 mg/kg drug-equivalent copolymer conjugates (with and without RGDfK) compared with 30 mg/kg free AH-GDM. Overall, 60 mg/kg treatment of RGDfK-containing conjugate showed significantly higher (p < 0.001) tumor drug concentrations compared with all other treatments. The targetable conjugates can effectively deliver higher amounts of geldanamycin to the tumor compared to nontargetable systems.

Noninvasive monitoring of HPMA copolymer-RGDfK conjugates by magnetic resonance imaging.

PURPOSE: To evaluate the tumor targeting potential of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-gadolinium(Gd)-RGDfK conjugates by magnetic resonance (MR) T1-mapping. METHODS: HPMA copolymers with and without RGDfK were synthesized to incorporate side chains for Gd chelation. The conjugates were characterized by their side-chain contents and r(1) relaxivity. In vitro integrin-binding affinities of polymeric conjugates were assessed via competitive cell binding assays on HUVEC endothelial cells and MDA-MB-231 breast cancer cells. In vivo MR imaging was performed on MDA-MB-231 tumor-bearing SCID mice at different time points using non-targetable and targetable polymers. The specificity of alphavbeta3 targeting was assessed by using non-paramagnetic targetable polymer to block alphavbeta3 integrins followed by injection of paramagnetic targetable polymers after 2 h. RESULTS: The polymer conjugates showed relaxivities higher than Gd-DOTA. Endothelial cell binding studies showed that IC(50) values for the copolymer with RGDfK binding to alphavbeta3 integrin-positive HUVEC and MDA-MB-231 cells were similar to that of free peptide. Significantly lower T1 values were observed at the tumor site after 2 h using targetable conjugate (p < 0.012). In vivo blocking study showed significantly higher T1 values (p < 0.045) compared to targetable conjugate. CONCLUSION: These results demonstrate the potential of this conjugate as an effective targetable MR contrast agent for tumor imaging and therapy monitoring.

Targetable HPMA copolymer-aminohexylgeldanamycin conjugates for prostate cancer therapy.

PURPOSE: This study focuses on the synthesis and characterization of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-cyclo-RGD (Arg-Gly-Asp) conjugates for delivery of geldanamycin to prostate tumors. MATERIALS AND METHODS: HPMA copolymers containing aminohexylgeldanamycin (AH-GDM) with and without the targeting peptide RGDfK were synthesized and characterized. Drug release from copolymers was evaluated using cathepsin B. Competitive binding of copolymer conjugates to alpha(v)beta(3) integrin was evaluated in prostate cancer (PC-3) and endothelial (HUVEC) cell lines and in vitro growth inhibition was assessed. The maximum tolerated dose for single i.v. injections of free drug and the conjugates was established in nude mice. RESULTS: HPMA copolymers containing AH-GDM and RGDfK showed active binding to the alpha(v)beta(3) integrin similar to that of free peptide. Similarly, growth inhibition of cells by conjugates was comparable to that of the free drug. Single intravenous doses of HPMA copolymer-AH-GDM-RGDfK conjugates in mice were tolerated at 80 mg/kg drug equivalent, while free drug caused morbidity at 40 mg/kg. No signs of toxicity were present in mice receiving HPMA copolymer-AH-GDM-RGDfK over the 14-day evaluation period. CONCLUSION: Results of in vitro activity and in vivo tolerability experiments hold promise for the utility of HPMA copolymer-AH-GDM-RGDfK conjugates for treatment of prostate cancer with greater efficacy and reduced toxicity.

An assessment of radionuclidic impurities of (210)Po produced via neutron irradiation of (209)Bi for use in targeted alpha-particle radiotherapy.

Radionuclidic impurities of (210)Po prepared by neutron irradiation of (209)Bi via the (209)Bi(n,gamma)(210)Bi reaction were investigated. Following irradiation and ingrowth, a pure (210)Po solution was obtained by sublimation and dissolution. Results were obtained by liquid scintillation (LS) counting, isotope dilution alpha (alpha)-spectrometry, and high-purity germanium gamma-ray spectrometry. No alpha-emitting (3-10MeV) or gamma-emitting (30-3600keV) impurities were detected, with calculated lower limits of detection for impurities of approximately 0.01% (210)Po activity. LS spectra revealed no identifiable beta-emitting impurity. LS sources prepared using Opti-Phase 'Hi Safe' III and Opti-Fluor LS cocktails were stable over a 4-day multi-cycle counting period for (210)Po dissolved in 0.1% trifluoracetic acid (pH approximately 2, water fraction approximately 2%). The radioactivity concentration determined by LS counting was verified by isotope dilution alpha spectrometry. These results suggest that neutron irradiation of (209)Bi (followed by sublimation) can produce (210)Po in a highly pure form that is suitable for radiopharmaceutical preparations.

Prospective pilot trial of PerMIT versus standard anticoagulation service management of patients initiating oral anticoagulation.

We performed a randomised pilot trial of PerMIT, a novel decision support tool for genotype-based warfarin initiation and maintenance dosing, to assess its efficacy for improving warfarin management. We prospectively studied 26 subjects to compare PerMIT-guided management with routine anticoagulation service management. CYP2C9 and VKORC1 genotype results for 13 subjects randomly assigned to the PerMIT arm were recorded within 24 hours of enrolment. To aid in INR interpretation, PerMIT calculates estimated loading and maintenance doses based on a patient's genetic and clinical characteristics and displays calculated S-warfarin plasma concentrations based on planned or administered dosages. In comparison to control subjects, patients in the PerMIT study arm demonstrated a 3.6-day decrease in the time to reach a stabilised INR within the target therapeutic range (4.7 vs. 8.3 days, p = 0.015); a 12.8% increase in time spent within the therapeutic interval over the first 25 days of therapy (64.3% vs. 55.3%, p = 0.180); and a 32.9% decrease in the frequency of warfarin dose adjustments per INR measurement (38.3% vs. 57.1%, p = 0.007). Serial measurements of plasma S-warfarin concentrations were also obtained to prospectively evaluate the accuracy of the pharmacokinetic model during induction therapy. The PerMIT S-warfarin plasma concentration model estimated 62.8% of concentrations within 0.15 mg/l. These pilot data suggest that the PerMIT method and its incorporation of genotype/phenotype information may help practitioners increase the safety, efficacy, and efficiency of warfarin therapeutic management.

Tumor-targeted HPMA copolymer-(RGDfK)-(CHX-A''-DTPA) conjugates show increased kidney accumulation.

N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-RGDfK conjugates targeting the alpha(v)beta(3) integrin have shown increased accumulation in solid tumors and promise for selective delivery of radiotherapeutics to sites of angiogenesis- or tumor-expressed alpha(v)beta(3) integrin. An unresolved issue in targeting radiotherapeutics to solid tumors is toxicity to non-target organs. To reduce toxicity of radiolabeled conjugates, we have synthesized HPMA copolymer-RGDfK conjugates with varying molecular weight and charge content to help identify a polymeric structure that maximizes tumor accumulation while rapidly clearing from non-targeted organs. Endothelial cell binding studies showed that copolymer conjugates of approximately 43, 20 and 10 kD actively bind to the alpha(v)beta(3) integrin. Scintigraphic images showed rapid clearance of indium-111 ((111)In) radiolabeled conjugates from the blood pool and high kidney accumulation within 1 h in tumor bearing mice. Biodistribution data confirms images with high accumulation in kidney (max 210% ID/g for 43 kD conjugate) and lower tumor accumulation (max 1.8% ID/g for 43 kD conjugate). While actively binding to the alpha(v)beta(3) integrin in vitro, HPMA copolymer-RGDfK conjugates with increased negative charge through increased CHX-A''-DTPA chelator content in the side chains causes increased kidney accumulation with a loss of tumor accumulation in vivo.