Frank C. Richardson

A Randomized, Phase II, Biomarker-Selected Study Comparing Erlotinib to Erlotinib Intercalated With Chemotherapy in First-Line Therapy for Advanced Non–Small-Cell Lung Cancer

PURPOSE Erlotinib prolongs survival in patients with advanced non-small-cell lung cancer (NSCLC). We report the results of a randomized, phase II study of erlotinib alone or intercalated with chemotherapy (CT + erlotinib) in chemotherapy-naïve patients with advanced NSCLC who were positive for epidermal growth factor receptor (EGFR) protein expression and/or with high EGFR gene copy number. PATIENTS AND METHODS A total of 143 patients were randomly assigned to either erlotinib 150 mg daily orally until disease progression (PD) occurred or to chemotherapy with paclitaxel 200 mg/m(2) intravenously (IV) and carboplatin dosed by creatinine clearance (AUC 6) IV on day 1 intercalated with erlotinib 150 mg orally on days 2 through 15 every 3 weeks for four cycles followed by erlotinib 150 mg orally until PD occurred (CT + erlotinib). The primary end point was 6-month progression-free survival (PFS); secondary end points included response rate, PFS, and survival. EGFR, KRAS mutation, EGFR fluorescent in situ hybridization and immunohistochemistry, and E-cadherin and vimentin protein levels were also assessed. RESULTS Six-month PFS rates were 26% and 31% for the two arms (CT + erlotinib and erlotinib alone, respectively). Both were less than the historical control of 45% (P = .001 and P = .011, respectively). Median PFS times were 4.57 and 2.69 months, respectively. Patients with tumors harboring EGFR activating mutations fared better on erlotinib alone (median PFS, 18.2 months v 4.9 months for CT + erlotinib). CONCLUSION The feasibility of a multicenter biomarker-driven study was demonstrated, but neither treatment arms exceeded historical controls. This study does not support combined chemotherapy and erlotinib in first-line treatment of EGFR-selected advanced NSCLC, and the patients with tumors harboring EGFR mutations had a better outcome on erlotinib alone.

Evaluation of mitochondrial DNA content and enzyme levels in tenofovir DF-treated rats, rhesus monkeys and woodchucks

The antiviral compound tenofovir DF (Gilead Sciences) was evaluated for possible mitochondrial toxicity in rats, rhesus monkeys and woodchucks. Animals were treated by oral gavage with tenofovir DF, and the levels of mitochondrial enzymes cytochrome c oxidase and citrate synthase were assayed. In rats (6/group) treated daily for 28 days with 300 mg/kg tenofovir DF the enzyme levels were unchanged versus control in liver, kidney, and skeletal muscle. In a parallel study, rats (6/group) were treated with 40 mg/kg of the antiviral adefovir dipivoxil (Gilead Sciences) and enzyme levels were also unchanged versus control. In rhesus monkeys (6/group) treated daily with 30 mg/kg or 250 mg/kg tenofovir DF for 56 days, and in woodchucks (6/group) treated daily with 15 mg/kg or 50mg/kg tenofovir DF for 90 days, the enzyme levels were unchanged in liver, kidney, skeletal muscle and cardiac muscle. Mitochondrial DNA (mtDNA) content was determined in tissue from treated versus control animals by utilizing a quantitative real-time PCR (QPCR) technique, where the relative ratios of mitochondrial cytochrome b gene to the genomic actin gene were measured. The relative mtDNA content from rats, rhesus monkeys and woodchucks were unchanged in the various treatment groups. Variations in mtDNA content between animals in the same treatment group were noted. The actual species-dependent mitochondria/genomic ratios were estimated from the QPCR assay. In summary, treatment with tenofovir DF, or with adefovir dipivoxil, did not affect mtDNA content or level of mitochondrial enzymes, and no liver, muscle or renal microscopic abnormalities were observed in tenofovir-treated animals.

Mice deficient for cytosolic thymidine kinase gene develop fatal kidney disease

The thymidine kinase (Tk) gene codes for a cytosolic protein involved in the pyrimidine nucleotide salvage pathway. A functional Tk gene is not necessary for cells in culture, and a naturally occurring Tk deficient phenotype has not been described in humans or animal models. In order to determine the biological significance of the Tk gene, we created Tk(-/-) knockout (KO) mice through homologous recombination in mouse embryonic stem cells. Tk KO mice have shortened life spans compared with their wild-type or Tk heterozygous (HET) siblings. All Tk KO mice develop sclerosis of kidney glomeruli and die before one year of age of kidney failure. Among other changes in KO animals, the most consistent is a switch from exclusively mucous secretion to predominantly serous secretion in the sublingual salivary gland. HET parents can produce KO mice at a frequency approaching Mendelian inheritance. Other observations in KO animals include an elevated level of serum thymidine, a significant decrease in the cloning efficiency of splenic lymphocytes, an increase in the frequency of hypoxanthine guanine phosphoribosyl transferase gene mutant lymphocytes, and histological alteration in the lymphoid structure of the spleen. In addition, KO animals sporadically exhibit inflammation of the arteries, which taken together with the lymphocyte and spleen abnormalities, suggest an abnormal immune system. Alterations in Tk KO mice indicate that the pyrimidine nucleotide salvage pathway is indispensable in vivo.

OSI-930: A Novel Selective Inhibitor of Kit and Kinase Insert Domain Receptor Tyrosine Kinases with Antitumor Activity in Mouse Xenograft Models

OSI-930 is a novel inhibitor of the receptor tyrosine kinases Kit and kinase insert domain receptor (KDR), which is currently being evaluated in clinical studies. OSI-930 selectively inhibits Kit and KDR with similar potency in intact cells and also inhibits these targets in vivo following oral dosing. We have investigated the relationships between the potency observed in cell-based assays in vitro, the plasma exposure levels achieved following oral dosing, the time course of target inhibition in vivo, and antitumor activity of OSI-930 in tumor xenograft models. In the mutant Kit-expressing HMC-1 xenograft model, prolonged inhibition of Kit was achieved at oral doses between 10 and 50 mg/kg and this dose range was associated with antitumor activity. Similarly, prolonged inhibition of wild-type Kit in the NCI-H526 xenograft model was observed at oral doses of 100 to 200 mg/kg, which was the dose level associated with significant antitumor activity in this model as well as in the majority of other xenograft models tested. The data suggest that antitumor activity of OSI-930 in mouse xenograft models is observed at dose levels that maintain a significant level of inhibition of the molecular targets of OSI-930 for a prolonged period. Furthermore, pharmacokinetic evaluation of the plasma exposure levels of OSI-930 at these effective dose levels provides an estimate of the target plasma concentrations that may be required to achieve prolonged inhibition of Kit and KDR in humans and which would therefore be expected to yield a therapeutic benefit in future clinical evaluations of OSI-930.

Polymerization of 2′-fluoro- and 2′-O-methyl-dNTPs by human DNA polymerase α, polymerase γ, and primase

Studies were undertaken to assess the ability of human polymerase α (pol α) and polymerase γ (pol γ) to incorporate 2′-fluoro- and 2′-O-methyldeoxynucleotides into DNA. In vitro DNA synthesis systems were used to detect incorporation and determine Km and Vmax for 2′-FdATP, 2′-FdUTP, 2′-FdCTP, 2′-FdGTP, 2′-O-MedATP, 2′-O-MedCTP, 2′-O-MedGTP, 2′-O-MedUTP, dUTP, UTP, and FIAUTP, in addition to normal deoxynucleotides. Pol α incorporated all 2′-FdNTPs except 2′-FdATP, but not 2′-O-MedNTPs. Pol γ incorporated all 2′-FdNTPs, but not 2′-O-MedNTPs. In general, 2′-fluorine substitution decreased Vmax/Km; however, the magnitude of the changes was nucleotide dependent, with dATP and dUTP being the most affected. Misinsertion frequencies for pol α and pol γ of 2′-FdNTPs compared with their normal nucleotides were: FIAUTP > 2′-FdCTP > 2′-FdGTP > 2′-FdATP (pol γ only) > 2′-FdUTP. Because kinetics of insertion of pol α can be affected by the nature of the primer, we examined the ability of pol α to polymerize 2′-fluoro- and 2′-O-MedATP and dGTP when elongating a primer synthesized by DNA primase. Under these conditions, both 2′-FdATP and 2′-FdGTP were polymerized, but 2′-O-MedATP and 2′-O-MedGTP were not. Primase alone could not readily polymerize these analogs into RNA primers. Previous studies showed that 2′-deoxy-2′-fluorocytosine (2′-FdC) is incorporated by several non-human DNA polymerases. The current studies showed that human polymerases can polymerize numerous 2′-FdNTPs but cannot polymerize 2′-O-MedNTPs.

DNA base changes induced following in vivo exposure of unadapted, adapted or ada- Escherichia coli to N-methyl-N'-nitro-N-nitrosoguanidine.

SummaryThe adaptive response is one of the major repair pathways in Escherichia coli that removes DNA alkylation damage. To investigate the role of the adaptive response in mutagenesis, the E. coli gpt forward mutation assay system was used to determine the mutation spectrum of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in MNNG-adapted and unadapted GP120 (wild-type) and unadapted PJ5 (ada-5) cells. We observed that 34/37 mutations in the unadapted GP120 cells, 38/40 mutations in the adapted GP120 cells, and 10/10 mutations in the PJ5 cells were GC → AT transitions. The remaining 3/37 mutations in the unadapted GP120 cells were large insertions. The remaining 2/40 mutations in the adapted GP120 cells were transversions with one a GC → CG and the other an AT → CG. A surrounding sequence specificity of mutagenesis was observed for the GC → AT transitions in both the unadapted (GP120 and PJ5) and adapted (GP120) cells, with 70% of the unadapted PJ5, 68% of the unadapted GP120, and 61% of the adapted GP120 mutations occurring at the middle G of the sequence 5′—GG(A or T)—3′. Both strains also displayed a statistically significant preference for mutagenesis at guanine bases in the non-transcribed strand. The overall distribution of mutated sites in the gpt gene in adapted and unadapted cells was similar, although the rate of mutations at certain sites appeared different. These minor differences could result from either non-uniform repair of alkylation damage at different sites on the DNA, or altered processing of the alkylated bases to mutations in the adapted state. However, these results indicate that there are no major differences in the types, surrounding sequence specificity, or distribution of mutations in the adapted GP120, unadapted GP120, or the ada− PJ5 cells.

Biodistribution of NX211, liposomal lurtotecan, in tumor-bearing mice.

Prolonging tumor exposure to topoisomerase I inhibitors has been correlated to enhance the efficacy of those agents. Lurtotecan, a water-soluble camptothecin analog, was formulated as a liposomal drug, NX211, to enhance the delivery of drug to tumors. Tumor-bearing mice were treated with either [14C]NX211 containing [14C]lurtotecan, [3H]NX211 containing [3H]phosphatidylcholine or [14C]lurtotecan, euthanized at selected times post-injection, and tissues, plasma, urine and feces were collected. These studies demonstrated that KB tumors of [14C]NX211-treated mice had approximately 70-fold greater concentrations of [14C]lurtotecan at 24 h, respectively, compared to concentrations of [14C]lurtotecan of the KB tumors of [14C]lurtotecan-treated mice. The area under curve (AUC) from 0 to 48 h of [14C]lurtotecan for the KB tumors of [14C]NX211-treated animals was over 17-fold greater than the AUC of [14C]lurtotecan for the tumors of [14C]lurtotecan-treated animals. Treatment with [3H]NX211 demonstrated that the lipid component continually accumulated over 24 h in the tissues. HPLC analysis of extracted material from tumors of [14C]NX211-treated mice showed that more than 95% of the radioactive material was intact [14C]lurtotecan. These findings are one of the keys justifying the development of a liposomal formulation of lurtotecan, which has the intent to increase tumor exposure and increase antitumor efficacy.

Novel Application of Layered Expression Scanning for Proteomic Profiling of Plucked Hair Follicles

Background: There is a need for a technology that can quantitatively assay multiple proteins from a single hair follicle while preserving the morphology of the follicle. For proteomic profiling, the technology should be less labor intensive, with a higher throughput, more quantitative and more reproducible than immunohistochemistry. Objective: To test the ability of a novel method, layered expression scanning of hair (LES-hair) to detect the levels and localization of proteins in plucked hair follicles. Methods: LES-hair was used to assay proteins in the plucked hair follicle. Results: LES-hair detected differential expression of proteins within discrete regions of the plucked hair follicle. These proteins included cleaved caspase 3, Ki-67 and the phosphorylated forms of c-Kit, epidermal growth factor receptor and vascular endothelial growth factor receptor. Conclusion: LES-hair provides a research tool for studying the basic biology of plucked hair follicles and has potential clinical applications such as monitoring treatment of alopecia or using plucked hair follicles as a surrogate tissue to monitor pharmacodynamic effects of targeted cancer therapies.