Jianxia Guo

Phase I and Pharmacodynamic Study of 17-(Allylamino)-17-Demethoxygeldanamycin in Adult Patients with Refractory Advanced Cancers

Purpose: The primary objective was to establish the dose-limiting toxicity (DLT) and recommended phase II dose of 17-(allylamino)-17-demethoxygeldanamycin (17AAG) given twice a week. Experimental Design: Escalating doses of 17AAG were given i.v. to cohorts of three to six patients. Dose levels for schedule A (twice weekly × 3 weeks, every 4 weeks) were 100, 125, 150, 175, and 200 mg/m2 and for schedule B (twice weekly × 2 weeks, every 3 weeks) were 150, 200, and 250 mg/m2. Peripheral blood mononuclear cells (PBMC) were collected for assessment of heat shock protein (HSP) 90 and HSP90 client proteins. Results: Forty-four patients were enrolled, 32 on schedule A and 12 on schedule B. On schedule A at 200 mg/m2, DLTs were seen in two of six patients (one grade 3 thrombocytopenia and one grade 3 abdominal pain). On schedule B, both patients treated at 250 mg/m2 developed DLT (grade 3 headache with nausea/vomiting). Grade 3/4 toxicities seen in >5% of patients were reversible elevations of liver enzymes (47%), nausea (9%), vomiting (9%), and headache (5%). No objective tumor responses were observed. The only consistent change in PBMC proteins monitored was a 0.8- to 30-fold increase in HSP70 concentrations, but these were not dose dependent. The increase in PBMC HSP70 persisted throughout the entire cycle of treatment but returned to baseline between last 17AAG dose of cycle 1 and first 17AAG dose of cycle 2. Conclusions: The recommended phase II doses of 17AAG are 175 to 200 mg/m2 when given twice a week and consistently cause elevations in PBMC HSP70.

Efficacy, pharmacokinetics, tisssue distribution, and metabolism of the Myc–Max disruptor, 10058-F4 [Z,E]-5-[4-ethylbenzylidine]-2-thioxothiazolidin-4-one, in mice

Objectivesc-Myc is commonly activated in many human tumors and is functionally important in cellular proliferation, differentiation, apoptosis and cell cycle progression. The activity of c-Myc requires noncovalent interaction with its client protein Max. In vitro studies indicate the thioxothiazolidinone, 10058-F4, inhibits c-Myc/Max dimerization. In this study, we report the efficacy, pharmacokinetics and metabolism of this novel protein–protein disruptor in mice.MethodsSCID mice bearing DU145 or PC-3 human prostate cancer xenografts were treated with either 20 or 30 mg/kg 10058-F4 on a qdx5 schedule for 2 weeks for efficacy studies. For pharmacokinetics and metabolism studies, mice bearing PC-3 or DU145 xenografts were treated with 20 mg/kg of 10058-F4 i.v. Plasma and tissues were collected 5–1440 min after dosing. The concentration of 10058-F4 in plasma and tissues was determined by HPLC, and metabolites were characterized by LC-MS/MS.ResultsFollowing a single iv dose, peak plasma 10058-F4 concentrations of approximately 300 μM were seen at 5 min and declined to below the detection limit at 360 min. Plasma concentration versus time data were best approximated by a two-compartment, open, linear model. The highest tissue concentrations of 10058-F4 were found in fat, lung, liver, and kidney. Peak tumor concentrations of 10058-F4 were at least tenfold lower than peak plasma concentrations. Eight metabolites of 10058-F4 were identified in plasma, liver, and kidney. The terminal half-life of 10058-F4 was approximately 1 h, and the volume of distribution was >200 ml/kg. No significant inhibition of tumor growth was seen after i.v. treatment of mice with either 20 or 30 mg/kg 10058-F4.ConclusionThe lack of significant antitumor activity of 10058-F4 in tumor-bearing mice may have resulted from its rapid metabolism and low concentration in tumors.

In Vitro Cytotoxicity and In Vivo Efficacy, Pharmacokinetics, and Metabolism of 10074-G5, a Novel Small-Molecule Inhibitor of c-Myc/Max Dimerization

The c-Myc oncoprotein is overexpressed in many tumors and is essential for maintaining the proliferation of transformed cells. To function as a transcription factor, c-Myc must dimerize with Max via the basic helix-loop-helix leucine zipper protein (bHLH-ZIP) domains in each protein. The small molecule 7-nitro-N-(2-phenylphenyl)-2,1,3-benzoxadiazol-4-amine (10074-G5) binds to and distorts the bHLH-ZIP domain of c-Myc, thereby inhibiting c-Myc/Max heterodimer formation and inhibiting its transcriptional activity. We report in vitro cytotoxicity and in vivo efficacy, pharmacodynamics, pharmacokinetics, and metabolism of 10074-G5 in human xenograft-bearing mice. In vitro, 10074-G5 inhibited the growth of Daudi Burkitts lymphoma cells and disrupted c-Myc/Max dimerization. 10074-G5 had no effect on the growth of Daudi xenografts in C.B-17 SCID mice that were treated with 20 mg/kg 10074-G5 intravenously for 5 consecutive days. Inhibition of c-Myc/Max dimerization in Daudi xenografts was not seen 2 or 24 h after treatment. Concentrations of 10074-G5 in various matrices were determined by high-performance liquid chromatography-UV, and metabolites of 10074-G5 were identified by liquid chromatography/tandem mass spectrometry. The plasma half-life of 10074-G5 in mice treated with 20 mg/kg i.v. was 37 min, and peak plasma concentration was 58 μM, which was 10-fold higher than peak tumor concentration. The lack of antitumor activity probably was caused by the rapid metabolism of 10074-G5 to inactive metabolites, resulting in tumor concentrations of 10074-G5 insufficient to inhibit c-Myc/Max dimerization. Our identification of 10074-G5 metabolites in mice will help design new, more metabolically stable small-molecule inhibitors of c-Myc.

Blocking the ZZ domain of sequestosome1/p62 suppresses myeloma growth and osteoclast formation in vitro and induces dramatic bone formation in myeloma-bearing bones in vivo

We reported that p62 (sequestosome 1) serves as a signaling hub in bone marrow stromal cells (BMSCs) for the formation of signaling complexes, including NFκB, p38MAPK and JNK, that are involved in the increased osteoclastogenesis and multiple myeloma (MM) cell growth induced by BMSCs that are key contributors to multiple myeloma bone disease (MMBD), and demonstrated that the ZZ domain of p62 (p62-ZZ) is required for BMSC enhancement of MMBD. We recently identified a novel p62-ZZ inhibitor, XRK3F2, which inhibits MM cell growth and BMSC growth enhancement of human MM cells. In the current study, we evaluate the relative specificity of XRK3F2 for p62-ZZ, characterize XRK3F2’s capacity to inhibit growth of primary MM cells and human MM cell lines, and test the in vivo effects of XRK3F2 in the immunocompetent 5TGM1 MM model. We found that XRK3F2 induces dramatic cortical bone formation that is restricted to MM containing bones and blocked the effects and upregulation of tumor necrosis factor alpha (TNFα), an osteoblast (OB) differentiation inhibitor that is increased in the MM bone marrow microenvironment and utilizes signaling complexes formed on p62-ZZ, in BMSC. Interestingly, XRK3F2 had no effect on non-MM bearing bone. These results demonstrate that targeting p62 in MM models has profound effects on MMBD.

Pharmacokinetics and tissue distribution of inositol hexaphosphate in C.B17 SCID mice bearing human breast cancer xenografts.

Inositol hexaphosphate (IP(6)) is effective in preclinical cancer prevention and chemotherapy. In addition to cancer, IP(6) has many other beneficial effects for human health, such as reduction in risk of developing cardiovascular disease and diabetes and inhibition of kidney stone formation. Studies presented here describe the pharmacokinetics, tissue distribution, and metabolism of IP(6) following intravenous (IV) or per os (PO) administration to mice. SCID mice bearing MDA-MB-231 xenografts were treated with 20 mg/kg IP(6) (3 μCi per mouse [(14)C]-uniformly ring-labeled IP(6)) and euthanized at various times after IP(6) treatment. Plasma and tissues were analyzed for [(14)C]-IP(6) and metabolites by high-performance liquid chromatography with radioactivity detection. Following IV administration of IP(6), plasma IP(6) concentrations peaked at 5 minutes and were detectable until 45 minutes. Liver IP(6) concentrations were more than 10-fold higher than plasma concentrations, whereas other normal tissue concentrations were similar to plasma. Only inositol was detected in xenografts. After PO administration, IP(6) was detected in liver; but only inositol was detectable in other tissues. After both IV and PO administration, exogenous IP(6) was rapidly dephosphorylated to inositol; however, alterations in endogenous IPs were not examined.

The Relationship of Phthalocyanine 4 (Pc 4) Concentrations Measured Noninvasively to Outcome of Pc 4 Photodynamic Therapy in Mice

The ability to noninvasively measure photosensitizer concentration at target tissues will allow optimization of photodynamic therapy (PDT) and could improve outcome. In this study, we evaluated whether preirradiation tumor phthalocyanine 4 (Pc 4) concentrations, measured noninvasively by the optical pharmacokinetic system (OPS), correlated with tumor response to PDT. Mice bearing human breast cancer xenografts were treated with 2 mg kg−1 Pc 4 iv only, laser irradiation (150 J cm−2) only, Pc 4 followed by fractionated irradiation or Pc 4 followed by continuous irradiation. Laser irradiation treatment was initiated when the tumor to skin ratio of Pc 4 concentration reached a maximum of 2.1 at 48 h after administration. Pc 4 concentrations in tumor, as well as in Intralipid in vitro, decreased monoexponentially with laser fluence. Pc 4‐PDT resulted in significant tumor regression, and tumor response was similar in the groups receiving either fractionated or continuous irradiation treatment after Pc 4. Tumor growth delay following Pc 4‐PDT correlated with OPS‐measured tumor Pc 4 concentrations at 24 h prior to PDT (R2 = 0.86). In excised tumors, OPS‐measured Pc 4 concentrations were similar to the HPLC‐measured concentrations. Thus, OPS measurements of photosensitizer concentrations can be used to assist in the scheduling of Pc 4‐PDT.

Evaluation of Plasma Insulin-like Growth Factor Binding Protein 2 and Her-2 Extracellular Domain as Biomarkers for 17-Allylamino-17-Demethoxygeldanamycin Treatment of Adult Patients with Advanced Solid Tumors

Purpose: Interaction of 17-allylamino-17-demethoxygeldanamycin (17-AAG) with heat shock protein 90 results in proteasomal degradation of many proteins, including Her-2-neu, with subsequent decreased expression of insulin-like growth factor binding protein-2 (IGFBP-2). Concentrations of both IGFBP-2 and Her-2 extracellular domain (Her-2 ECD) in sera of mice bearing BT474 human breast cancer xenografts decrease after 17-AAG treatment. We investigated whether this phenomenon occurred in patients. Materials and Methods: Eight to 15 plasma samples were obtained between 0 and 72 h from 27 patients treated with single-agent 17-AAG at doses between 10 and 307 mg/m2 and 18 patients treated with 17-AAG at doses between 220 and 450 mg/m2 combined with 70 to 75 mg/m2 of docetaxel. Pretreatment plasma samples were also obtained from 12 healthy volunteers. Plasma IGFBP-2 and Her-2 ECD concentrations were quantitated by ELISA. Results: Pretreatment plasma IGFBP-2 concentrations in patients (171 ± 116 ng/mL) were 2-fold higher than those in healthy volunteers (85 ± 44 ng/mL; P < 0.05). Following 17-AAG treatment, there were no consistent dose-dependent or time-dependent changes in plasma IGFBP-2 and Her-2 ECD concentrations. IGFBP-2 concentrations decreased by ≥40% in 8 patients, increased 2- to 5-fold in 8 patients, and remained essentially unchanged in 29 patients. Her-2 ECD concentrations decreased by ≥40% in 10 patients, increased 1.5- to 5-fold in 2 patients, and remained essentially unchanged in 25 patients. Conclusions: As previously reported, IGFBP-2 concentrations in plasma of cancer patients are significantly higher than those in healthy volunteers. In contrast to a mouse model, 17-AAG treatment was not consistently associated with decreases in IGFBP-2 or Her-2 ECD concentrations in patient plasma.

Evaluation of Silicon Phthalocyanine 4 Photodynamic Therapy against Human Cervical Cancer Cells in Vitro and in Mice

BACKGROUND Cervical cancer is the second most common cancer in women worldwide [1]. Photodynamic therapy has been used for cervical intraepithelial neoplasia with good responses, but few studies have used newer phototherapeutics. We evaluated the effectiveness of photodynamic therapy using Pc 4 in vitro and in vivo against human cervical cancer cells. METHODS CaSki and ME-180 cancer cells were grown as monolayers and spheroids. Cell growth and cytotoxicity were measured using a methylthiazol tetrazolium assay. Pc 4 cellular uptake and intracellular distrubtion were determined. For in vitro Pc 4 photodynamic therapy cells were irradiated at 667nm at a fluence of 2.5 J/cm2 at 48 h. SCID mice were implanted with CaSki and ME-180 cells both subcutaneously and intracervically. Forty-eight h after Pc 4 photodynamic therapy was administered at 75 and 150 J/cm2. RESULTS The IC50s for Pc 4 and Pc 4 photodynamic therapy for CaSki and ME-180 cells as monolayers were, 7.6μM and 0.016μM and >10μM and 0.026μM; as spheroids, IC50s of Pc 4 photodynamic therapy were, 0.26μM and 0.01μM. Pc 4 was taken up within cells and widely distributed in tumors and tissues. Intracervical photodynamic therapy resulted in tumor death, however mice died due to gastrointestinal toxicity. Photodynamic therapy resulted in subcutaneous tumor death and growth delay. CONCLUSIONS Pc 4 photodynamic therapy caused death within cervical cancer cells and xenografts, supporting development of Pc 4 photodynamic therapy for treatment of cervical cancer. Support: P30-CA47904, CTSI BaCCoR Pilot Program.

Liquid chromatography–tandem mass spectrometric assay for the quantitation of the novel radiation protective agent and radiation mitigator JP4-039 in murine plasma

JP4-039 radio-protects prior to, and radio-mitigates after ionizing radiation by neutralizing reactive oxygen species. We developed and validated an LC-MS/MS assay for the quantitation of JP4-039 in murine plasma. Methanol protein precipitation of 50μL plasma was followed by isocratic reverse phase chromatography for a 6min run time, and electrospray positive mode ionization mass spectrometric detection. The plasma assay was linear from 1 to 1000ng/mL with appropriate accuracy (97.1-107.6%) and precision (3.7-12.5%CV), and fulfilled FDA guidance criteria. Recovery was 77.2-136.1% with moderate ionization enhancement (10.9-39.5%). Plasma freeze-thaw stability (98.8-104.2%), stability for 13.5 months at -80°C (93.1-105.6%), and stability for 4h at room temperature (94.2-97.6%) were all acceptable. Limited cross-validation to tissue homogenates suggested that these could also be analyzed for JP4-039 accurately. This assay has been directly applied to determine the pharmacokinetics of JP4-039 in C57BL/6 male mice after IV administration of 20mg/kg JP4-039 and will be extended to other studies of this agent.

LC–MS/MS assay for the quantitation of the ribonucleotide reductase inhibitor triapine in human plasma

HIGHLIGHTSTriapineis a ribonucleotide reductase inhibitor and radiosensitizer in oncology Phase I and Phase II trials.An LC–MS/MS assay from 3–3000 ng/mL triapinein 0.05 mL plasma was validated.Triapine is chelated by metal ions. MS responses were increased by EDTA and decreased by Fe3+, while stable isotope IS corrected this.This assay was utilized to quantitate triapine in preclinical samples and will be applied to clinical samples. ABSTRACT The ribonucleotide reductase inhibitor and radiosensitizer triapine (3‐aminopyridine‐2‐carboxaldehyde thiosemicarbazone (3‐AP), NSC 663249) is clinically being evaluated via the intravenous (IV) route for the treatment of cervical and vulvar cancer in combination with primary cisplatin chemoradiation. The need for a 2‐h infusion and frequent administration of triapine is logistically challenging, prompting us to pursue oral (PO) administration. In support of the clinical trial investigating oral triapine in combination with chemoradiation, we developed and validated a novel LC–MS/MS assay for the quantification of triapine in 50 &mgr;L human plasma. After protein precipitation, chromatographic separation of the supernatant was achieved with a Shodex ODP2 column and an isocratic acetonitrile‐water mobile phase with 10% ammonium acetate. Detection with an ABI 4000 mass spectrometer utilized electrospray positive mode ionization. The assay was linear from 3 to 3,000 ng/mL and proved to be accurate (97.1–103.1%) and precise (<7.4% CV), and met the U.S. FDA guidance for bioanalytical method validation. This LC–MS/MS assay will be an essential tool to further define the pharmacokinetics and oral bioavailability of triapine.