Jeffrey S. Johnston

In Vitro and Pharmacophore-Based Discovery of Novel hPEPT1 Inhibitors

No HeadingPurpose.The human proton-coupled small peptide carrier (hPEPT1) is a low-affinity, high-capacity transporter with broad substrate specificity. We have taken an iterative in vitro and in silico approach to the discovery of molecules with hPEPT1 affinity.Methods.A pharmacophore-based approach was taken to identifying hPEPT1 inhibitors. The well-characterized and relatively high affinity ligands Gly-Sar, bestatin, and enalapril were used to generate a common features (HIPHOP) pharmacophore. This consisted of two hydrophobic features, a hydrogen bond donor, acceptor, and a negative ionizable feature.Results.The pharmacophore was used to search the Comprehensive Medicinal Chemistry (CMC) database of more than 8000 drug-like molecules and retrieved 145 virtual hits mapping to the pharmacophore features. The highest scoring compounds within this set were selected and tested in a stably transfected CHO-hPepT1 cell model. The antidiabetic repaglinide and HMG CoA reductase inhibitor fluvastatin were found to inhibit hPEPT1 with sub-millimolar potency (IC50 178 ± 1.0 and 337 ± 4 μM, respectively). The pharmacophore was also able to identify known hPEPT1 substrates and inhibitors in further database mining of more than 500 commonly prescribed drugs.Conclusions.This study demonstrates the potential of combining computational and in vitro approaches to determine the affinity of compounds for hPEPT1 and, in turn, provides insights into key molecular interactions with this transporter.

Phase 2 trial of rituximab and bortezomib in patients with relapsed or refractory mantle cell and follicular lymphoma

In vitro studies in mantle cell lymphoma (MCL) cell lines and patient‐derived cells have demonstrated synergistic apoptosis with combined rituximab and bortezomib (R‐bortezomib) compared with single‐agent bortezomib. Therefore, the authors of this report evaluated R‐bortezomib in a preclinical model and in a phase 2 clinical trial.

Combination bortezomib and rituximab treatment affects multiple survival and death pathways to promote apoptosis in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a distinct histologic subtype of B cell non-Hodgkins lymphoma (NHL) associated with an aggressive clinical course. Inhibition of the ubiquitin-proteasome pathway modulates survival and proliferation signals in MCL and has shown clinical benefit in this disease. This has provided rationale for exploring combination regimens with B-cell selective immunotherapies such as rituximab. In this study, we examined the effects of combined treatment with bortezomib and rituximab on patient-derived MCL cell lines (Jeko, Mino, SP53) and tumor samples from patients with MCL where we validate reversible proteasome inhibition concurrent with cell cycle arrest and additive induction of apoptosis. When MCL cells were exposed to single agent bortezomib or combination bortezomib/rituximab, caspase dependent and independent apoptosis was observed. Single agent bortezomib or rituximab treatment of Mino and Jeko cell lines and patient samples resulted in decreased levels of nuclear NF-kB complex(es) capable of binding p65 consensus oligonucleotides, and this decrease was enhanced by the combination. Constitutive activation of the AKT pathway was also diminished with bortezomib alone or in combination with rituximab. On the basis of in vitro data demonstrating additive apoptosis and enhanced NF-kB and phosphorylated AKT depletion in MCL with combination bortezomib plus rituximab, a phase II trial of bortezomib-rituximab in patients with relapsed/refractory MCL is underway.

Synergy Between 3′Azido-3′deoxythymidine and Paclitaxel in Human Pharynx FaDu Cells

AbstractPurpose. We recently demonstrated simultaneous targeting of telomere and telomerase as a novel cancer therapeutic approach, and that telomerase inhibitors such as 3′azido-3′deoxythymidine (AZT) significantly enhanced the antitumor activity of paclitaxel, which causes telomere erosion, in telomerase-positive human pharynx FaDu tumors in vitro and in vivo (1). The present study evaluated the synergy between AZT and paclitaxel to identify optimal combinations for future clinical evaluation. Methods. FaDu cells were incubated with or without AZT for 24 h and then treated with AZT with or without paclitaxel for an additional 48 h. Under these conditions, single agent paclitaxel produced a 60% maximum reduction of cell number (IC50 was 7.3 nM), and single agent AZT produced a 97% reduction (IC50 was 5.6 μM). Synergy was evaluated using fixed-concentration and fixed-ratio methods, and data were analyzed by the combination index method. Results. The results indicate a concentration-dependent synergy between the two drugs; the synergy was higher for combinations containing greater paclitaxel-to-AZT concentration ratios and increased with the level of drug effect. For example, in combinations containing 1 μM AZT, synergy was 1.3-fold at the 20% effect level and 3.1-fold at the 60% effect level. Because the major antitumor activity, determined by comparing the posttreatment cell number to the pretreatment cell number, was antiproliferation at the 20% effect level and cell kill at the 60% effect level, our results suggest that AZT mainly enhances the cell kill effect of paclitaxel. Conclusion. In summary, the present study demonstrates a synergistic interaction between paclitaxel and AZT and supports a combination using a low and nontoxic AZT dose in combination with a therapeutically active dose of paclitaxel.

INTERSPECIES DIFFERENCES IN PHARMACOKINETICS AND METABOLISM OF S-3-(4-ACETYLAMINO-PHENOXY)-2-HYDROXY-2-METHYL-N-(4-NITRO-3-TRIFLUOROMETHYLPHENYL)-PROPIONAMIDE: THE ROLE OF N-ACETYLTRANSFERASE

N-Acetyltransferase (NAT) is one of the major phase II enzymes involved in drug metabolism. Both species differences and polymorphism are observed in NAT expression. During the preclinical development of a novel selective androgen receptor modulator, S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide (S4), we also observed species differences in S4 metabolism due to the interaction between the deacetylation metabolite M1 and NAT, which converted M1 back to S4 both in vitro and in vivo. During incubation with human liver cytosol or rat liver S9 fraction in the presence of acetyl-CoA, more than 50% of M1 (2 μM) was converted back to S4, but this conversion was not observed in the incubation with dog liver S9 fraction or human liver microsome. In vivo pharmacokinetic experiments showed that M1 could be rapidly converted back to S4 in rats, but a similar conversion was not observed in dogs. When S4 was administered, the formation of M1 was only observed in dogs due to the absence of NAT expression. Simultaneous fitting of the concentration-time profiles of both S4 and M1 showed that more than 50% of S4 was deacetylated to M1 in dogs after i.v. administration of S4, whereas more than 80% of M1 was converted to S4 in rats after i.v. administration of M1. Considering the polymorphism in NAT expression, the interaction between M1 and NAT may raise concerns for drug-drug interactions during clinical applications of S4. The observed species differences suggested that interspecies scaling might not be applicable for predicting the metabolism and disposition of S4 in humans.

Telomere maintenance in telomerase-positive human ovarian SKOV-3 cells cannot be retarded by complete inhibition of telomerase.

The two known mechanisms for telomere maintenance in eukaryocytes are telomerase in telomerase‐positive cells and alternative lengthening of telomeres (ALT) in telomerase‐negative cells. We report here that telomere maintenance in the telomerase‐positive human ovarian SKOV‐3 cells was not affected by inhibition of telomerase. For comparison, the effect of telomerase inhibitors on telomere maintenance in another telomerase‐positive cell line (i.e. human pharynx FaDu cells) and the telomerase‐negative human osteosarcoma Saos‐2 cells was examined. Telomerase activity was measured using a modified telomeric repeat amplification protocol and telomere length was measured using a solution hybridization‐based method and fluorescence in situ hybridization. A reverse transcriptase inhibitor (3′‐azido‐deoxythymidine or AZT) and an antisense against a component of human telomerase RNA (antisense hTR) were used to inhibit telomerase. FaDu and SKOV‐3 cells showed comparable baseline telomerase activity. Telomerase activity in both cells was inhibited about equally by AZT (maximal inhibition of ∼80%) and by expression of antisense hTR (complete inhibition in SKOV‐3 cells and maximal inhibition of ∼80% in FaDu cells). However, treatment with telomerase inhibitors resulted in ∼50% telomere shortening in FaDu cells but had no effect on SKOV‐3 nor Saos‐2 cells. SKOV‐3 cells did not show the characteristic features of ALT (i.e. heterogeneous telomere length and promyelocytic leukemia bodies), whereas these ALT features were observed in Saos‐2 cells. Collectively, these results suggest the existence of a telomerase‐independent mechanism of telomere maintenance in the telomerase‐positive SKOV‐3 cells.

Pharmacokinetics and dose escalation of the heat shock protein inhibitor 17-allyamino-17-demethoxygeldanamycin in combination with bortezomib in relapsed or refractory acute myeloid leukemia

Abstract This phase I study was conducted to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLTs) 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 150 mg/m2 and bortezomib 0.7 mg/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 = 0.032) 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.

Anesthesia for brain tumor resection using intraoperative magnetic resonance imaging (iMRI) with the Polestar N-20 system: experience and challenges

The Polestar N-20 Scanner (Medtronic Navigation, Louisville, CO) is an intraoperative magnetic resonance image (iMRI) guidance system designed for neurosurgery. Sixty-five patients underwent craniotomy for tumor resection using the iMRI during the period from April 2005 to December 2006. Anesthesiologists used an iMRI-compatible patient monitoring system (Veris MR, MEDRAD, Indianola, PA), anesthesia machine (Aestiva/5 MRI, Datex-Ohmeda, Madison, WI), and infusion pumps (Continuum; MEDRAD). Average setup time for each case (from intubation to incision) was one hour, 33 minutes and showed learning improvement over the 21-month period. The challenges facing the anesthesiologists in these cases included the need to use longer intravenous (IV) catheters and gas delivery and sampling lines, which increased dead space. Electrocardiographic signals became contaminated with electrical noise during iMRI scan procedures, which made it difficult to distinguish rhythm changes. None of our iMRI patients underwent a repeat resection, whereas the repeat resection rate for conventional tumor resection is reported to be as high as 30%. The use of a small, low-field iMRI system provided adequate imaging for resection of lesions without the need of repeat resections in the weeks following the initial procedure, and did not significantly alter the anesthetic procedure. A team effort between the anesthesiologists, surgeons, nurses, and MRI technicians is paramount for the practical and efficient use of these iMRI systems.

Development and validation of a sensitive liquid chromatography/mass spectrometry method for quantitation of flavopiridol in plasma enables accurate estimation of pharmacokinetic parameters with a clinically active dosing schedule

A high-performance liquid chromatographic assay with tandem mass spectrometric detection was developed and validated for quantitation of the broad spectrum kinase inhibitor, flavopiridol, in human plasma. Sample preparation conditions included liquid-liquid extraction in acetonitrile (ACN), drying, and reconstitution in 20/80 water/ACN. Flavopiridol and the internal standard (IS), genistein, were separated by reversed phase chromatography using a C-18 column and a gradient of water with 25 mM ammonium formate and ACN. Electrospray ionization and detection of flavopiridol and genistein were accomplished with single reaction monitoring of m/z 402.09>341.02 and 271.09>152.90, respectively in positive-ion mode [M+H](+) on a triple quadrupole mass spectrometer. Recovery was greater than 90% throughout the linear range of 3-1000 nM. Replicate sample analysis indicated within- and between-run accuracy and precision to be less than 13% throughout the linear range. This method has the lowest lower limit of quantitation (LLOQ) reported to date for flavopiridol, and it allows for more accurate determination of terminal phase concentrations and improved pharmacokinetic parameter estimation in patients receiving an active dosing schedule of flavopiridol.

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 microL 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 mm x 2.1 mm Agilent Zorbax SB-phenyl 5 microm 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 microL/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 min, respectively, with a total run time of 7 min. 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 microL 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.