Phase II trial of vatalanib in patients with advanced or metastatic pancreatic adenocarcinoma after first-line gemcitabine therapy (PCRT O4-001).

PURPOSE: Vatalanib (PTK 787/ZK22584) is an oral poly-tyrosine kinase inhibitor with strong affinity for platelet-derived growth factor and vascular endothelial growth factor (VEGF) receptors. We conducted an open-label, phase II multicenter therapeutic trial investigating the efficacy and tolerability of vatalanib in patients with metastatic or advanced pancreatic cancer who failed first-line gemcitabine-based therapy. METHODS: Vatalanib treatment consisted of a twice daily oral dosing using a "ramp-up schedule," beginning with 250 mg bid during week 1,500 mg bid during week 2, and 750 mg bid on week three and thereafter. The primary objective of this study was to evaluate the 6-month survival rate. RESULTS: Sixty-seven patients were enrolled. The median age was 64, and 66% (N = 43) had only one prior regimen. Common grade 3/4 adverse events included hypertension (20%; N = 13), fatigue (17%; N = 11), abdominal pain (17%; N = 11), and elevated alkaline phosphatase (15%; N = 10). Among the 65 evaluable patients, the 6-month survival rate was 29% (95% CI 18-41%) and the median progression-free survival was 2 months. Fifteen patients survived 6 months or more. Two patients had objective partial responses, and 28% of patients had stable disease. Changes in biomarkers including soluble VEGF and vascular endothelial growth factor receptor did not correlate with response to drug. CONCLUSION: Vatalanib was well tolerated as a second-line therapy and resulted in favorable 6-month survival rate in patients with metastatic pancreatic cancer, compared with historic controls.

A phase IB trial of 24-hour intravenous PX-12, a thioredoxin-1 inhibitor, in patients with advanced gastrointestinal cancers.

We investigated the safety, pharmacokinetics, and pharmacodynamics of PX-12, a thioredoxin-1 (Trx-1) inhibitor, administered as a 24-hour infusion every 7 or 14 days in patients with gastrointestinal malignancies. PX-12 is the first Trx-1 inhibitor to undergo clinical development. The first Phase 1 study of PX-12 demonstrated promising clinical activity, but the 1 and 3 hour-infusion schedules investigated were associated with a strong and irritating odor due to exhalation of one of its metabolites, 2-butanethiol. In an effort to achieve tolerability and achieve a drug exposure level necessary for biological activity, the current study was undertaken. While the maximally tolerated dose was estimated to be 300 mg/m(2) /24 h once a week as the 2-butanethiol expirate was tolerable at that dose level, no evidence of clinical activity was observed. Pharmacokinetic studies of the parent compound PX-12 demonstrated rapid, irreversible binding to plasma components, resulting in low (ng/ml) peak plasma concentrations of non-bound PX-12 during infusion. DCE-MRI was performed pre-and post-infusion in three patients. There were no significant trends observed in changes in plasma Trx-1, vascular endothelial growth factor (VEGF), or beta fibroblast growth factor (FGF-2) pre- or post-treatment. However, there was a trend for a decrease in circulating Trx-1 during the first four PX-12 treatment cycles in patients that had a Trx-1 baseline level >18 ng/mL. Aggregate clinical trial results suggest that further clinical development of PX-12, as an intravenous infusion, is not feasible. However, the Trx-1 pathway remains a target of interest in patients with gastrointestinal malignancies.

Drug interactions with the taxanes: clinical implications.

BACKGROUND: The taxanes paclitaxel and docetaxel are among the most active antitumor agents. Clinically important pharmacodynamic interactions have been reported to occur with these agents that are sequence or schedule dependent. Because the taxanes undergo hepatic oxidation via the cytochrome P450 system, pharmacokinetic interactions due to enzyme induction or inhibition can also occur. METHODS: A comprehensive literature search was conducted using Medline to identify clinically important drug-interactions with the taxanes. RESULTS: Clinically significant taxane interactions were identified for carboplatin, cisplatin, doxorubicin, docetaxel, epirubicin and anticonvulsants. Doxorubicin and epirubicin should be administered 24 h before paclitaxel, and the cumulative anthracycline dose limited to 360 mg/m(2). This will prevent the enhanced toxicities due to sequence and schedule dependent interactions between anthracyclines and paclitaxel. Conversely, paclitaxel should be administered at least 24 h before cisplatin to avoid a decrease in clearance and increase in myelosuppression. With concurrent anticonvulsant therapy, cytochrome p450 enzyme induction results in decreased paclitaxel plasma steady state concentrations, possibly requiring an increased dose of paclitaxel. A number of other drug interactions have been reported in preliminary studies for which clinical significance has yet to be established. CONCLUSION: Clinically significant drug interactions have been reported to occur when paclitaxel is administered with doxorubicin, cisplatin, or anticonvulsants (phenytoin, carbamazepine, and phenobarbital).

Thioredoxin peroxidase-1 (peroxiredoxin-1) is increased in thioredoxin-1 transfected cells and results in enhanced protection against apoptosis caused by hydrogen peroxide but not by other agents including dexamethasone, etoposide, and doxorubicin.

Thioredoxin-1 (Trx-1) is a small redox oncoprotein whose expression is increased in a number of human primary cancers where it is associated with aggressive tumor growth, inhibition of apoptosis and decreased patient survival. We report that Trx-1-transfected MCF-7 human breast cancer cells have increased expression of thioredoxin peroxidase-1 (TrxP-1) a peroxiredoxin family member that scavenges H(2)O(2) using Trx-1 as a source of reducing equivalents. Our work shows that TrxP-1 is more effective than selenium-dependent glutathione peroxidase in protecting cells against H(2)O(2) damage. Transfection of mouse WEHI7.2 lymphoma cells with human TrxP-1 or TrxP-2, but not TrxP-4, protects the cells against H(2)O(2) induced apoptosis but does not protect against apoptosis induced by dexamethasone, etoposide, or doxorubicin. The results show that an increase in TrxP-1 expression contributes to the protection against H(2)O(2) induced apoptosis caused by Trx-1, but does not protect against apoptosis induced by other agents.

Catalase-overexpressing thymocytes are resistant to glucocorticoid-induced apoptosis and exhibit increased net tumor growth.

Glucocorticoids are used for the treatment of lymphoid neoplasms, taking advantage of the well-known ability of these compounds to cause apoptosis in lymphoid tissues. Previously, we have shown that dexamethasone, a synthetic glucocorticoid, causes a down-regulation of several antioxidant defense enzymes and proteins, including catalase and thioredoxin, concomitant with the induction of apoptosis in WEHI7.2 mouse thymoma cells. To test whether this down-regulation plays a critical role in the mechanism of steroid-induced apoptosis, WEHI7.2 cells were transfected with rat catalase. Two clones, expressing 1.4-fold and 2.0-fold higher catalase specific activity, respectively, when compared with vectoronly transfectants were selected for further study. An increase to 1.4-fold parental cell catalase activity delayed cell loss after dexamethasone treatment, whereas a 2.0-fold parental catalase activity prevented dexamethasone-induced cell loss for 48 h after treatment. Dexamethasone treatment of the WEHI7.2 cells stimulated a release of cytochrome c into the cytosol. Catalase-overexpressing cells showed a delay or lack of cytochrome c release from the mitochondria, which correlated temporally with the delay or prevention of cell loss in the culture after dexamethasone treatment. A decreased amount of cell death from WEHI7.2 cells overexpressing catalase was also seen in tumor xenografts in severe combined immunodeficient mice when compared with tumors from vector-only transfected cells. Similarly, thioredoxin-overexpressing WEHI7.2 cells, shown previously to be apoptosis resistant, showed decreased cell death in tumor xenografts. This resulted in larger tumors from cells overexpressing these proteins. Cell death in control transfectant tumor xenografts was primarily attributable to apoptosis. In contrast, the cell death we observed in tumors from thioredoxin- or catalase-overexpressing cells had a higher frequency of a nonapoptotic, nonnecrotic type of cell death termed para-apoptosis. These data suggest that: (a) oxidative stress plays a critical role in steroid-induced apoptosis prior to the commitment of the cells to undergo apoptosis; and (b) resistance to oxidative stress can contribute to tumor growth.

Modulation of antioxidant defenses during apoptosis.

Understanding the fundamental mechanism of apoptosis is crucial to developing therapeutic strategies for controlling apoptosis in diseased tissues. We are using model systems with relevance to cancer treatment to investigate the mechanism of apoptosis. Subtraction hybridization cloning was used to identify transcripts present at higher levels in regressing vs. normal prostate; these may be important for apoptosis. One of the genes cloned from regressing prostate is also upregulated in the murine W7.2 lymphocyte cell line induced to undergo apoptosis by treatment with the synthetic glucocorticoid, dexamethasone. This gene encodes a mu class glutathione S-transferase (EC 2.5.1.18), a protein that can protect the cell against oxidative stress by repairing oxidized lipids, proteins, and DNA. Glutathione S-transferase expression does not increase with dexamethasone treatment of lymphocyte cell lines expressing nonfunctional glucocorticoid receptors or a mutation in the apoptotic pathway. Other antioxidant defenses, including catalase (EC 1.11.1.6) and superoxide dismutase (EC 1.15.1.1), decline following dexamethasone treatment of W7.2 cells. Overexpression of the bcl-2 oncogene protects these cells against dexamethasone-mediated apoptosis and prevents the decrease in antioxidant enzyme activity. These findings support the hypothesis that control of the cellular redox state is important to the mechanism of glucocorticoid-mediated lymphocyte apoptosis. Another model system we are using is tumor necrosis factor-alpha treatment of MCF-7 human breast cancer cells. Our preliminary results suggest that, in this system, activation of nuclear factor-kappa B and increased expression of manganese superoxide dismutase may afford protection from apoptosis.

Decreased antioxidant defence and increased oxidant stress during dexamethasone-induced apoptosis: bcl-2 prevents the loss of antioxidant enzyme activity.

When the WEHI7.2 mouse lymphoid cell line was treated with dexamethasone to induce apoptosis the activities and transcript levels of the antioxidant defence enzymes catalase, superoxide dismutase (SOD) and DT-diaphorase exhibited a progressive decrease over 48 hours. Catalase activity was maintained and total SOD and DT-diaphorase activity showed smaller decreases following dexamethasone treatment of WEHI7.2 cells transfected with the bcl-2 oncogene, which protects the cells against apoptosis. Treatment of wild-type WEHI7.2 and bcl-2 transfected cells with a catalase inhibitor, aminotriazole, was not sufficient to induce apoptosis. Antioxidants, including bovine liver catalase, bovine erythrocyte CuZn-SOD, sodium selenite and Trolox, a water soluble vitamin E analogue, as well as hypoxia, inhibited dexamethasone-induced apoptosis. These results suggest that oxidant stress due to the decreased activity of antioxidant defence enzymes may play a role in dexamethasone-mediated lymphoid cell apoptosis and that bcl-2 may prevent apoptosis by maintaining the level of critical antioxidant defence mechanisms, which include catalase.

Modulation of the antioxidant defence as a factor in apoptosis.

This review focuses on evidence that oxidative stress during apoptosis is controlled, at least in part, by modulating cellular antioxidant defences. Evidence is presented from studies of apoptosis induced by glucocorticoids, HIV-1 infection and tumour necrosis factor-alpha. Glucocorticoid treatment of murine lymphocyte cell lines leads to the down-regulation of primary antioxidant defence enzymes, including catalase, superoxide dismutases, thioredoxin and DT-diaphorase. Following HIV-1 infection, disturbances in glutathione metabolism are seen, and decreased antioxidant enzyme activities have been reported for HIV-1-infected cell lines. The viral protein Tat may mediate these effects. Cellular resistance to apoptosis induced by tumour necrosis factor-alpha is modulated by the expression of manganese superoxide dismutase or Bcl-2. The loss of antioxidant defences is predicted to lead to oxidative stress, which could contribute to the mechanism of apoptosis through an effect on redox-sensitive transcription factors, calcium homeostasis or cysteine proteases.

Cumulus removal and addition of follicular fluid possibly improves pregnancy rates with in vitro fertilization for male factor.

This study evaluated the use of in vitro fertilization (IVF) for patients with subnormal semen parameters without the use of micromanipulation. All patients were characterized as having male factor as follows: normal morphology (NM) < or = 10% according to strict criteria [15] and motile density (MD) < or = 10 x 10(6)/mL. Strict morphology was divided into three groups: group I (n = 72), < or = 2% group II (n = 24), 3-5%; and group III (n = 29), 6-10%. Modification of standard IVF techniques included manual cumulus removal (CR) from oocytes, pooling up to ten oocytes together in 1 mL of media, and supplementing media with 20% human follicular fluid (FF). Rates of fertilization and pregnancy were compared. The overall fertilization rate (FR) was 57.7% and the pregnancy rate (PR) per retrieval cycle was 14.8%. There was no significant improvement in the fertilization or PRs when IVF was modified using CR and FF, although the FR was higher in group I for patients who received the modified procedures. In patients with < or = 5 x 10(6) sperm/mL, there were no pregnancies in five cycles and four transfers following the conventional method, but two sets of twins with the modified protocols in seven cycles. Clinical pregnancies were achieved with male factor without the need for micromanipulation. The most severe cases were automatically assigned to modified IVF techniques, e.g., CR with or without FF. Prospective randomized studies are needed to determine if modified procedures are superior to conventional therapy.

Serum progestagen-associated endometrial protein (PEP) levels in conception versus nonconception cycles following in vitro fertilization-embryo transfer.

The human endometrium synthesizes a specific protein known as the progestagen-dependent endometrium protein (PEP) which rises from early to late luteal phase. The PEP levels follow the pattern of the endometrial biopsy more than the serum progesterone (P) levels (4). Late luteal-phase serum PEP levels were evaluated as well as serum P in mid-luteal phase in patients undergoing IVF-ET. Comparisons were made between conceivers and nonconceivers and between aborters and nonaborters. Both serum PEP and late luteal P levels were significantly higher in pregnant patients but no differences in mid luteal P levels were seen. No difference was seen in aborters vs nonaborters. It is still inconclusive whether the higher late luteal PEP levels contribute to the greater likelihood of pregnancy or are a result of the pregnancy.

Transmission of sexually transmitted diseases by donor semen.

Therapeutic insemination by donor (TID) is being used with increasing frequency. Because many diseases, some of which are lethal, can be transmitted through semen, the American Fertility Society established guidelines for use of donor sperm. They limit TID to cases of male infertility or hereditary/genetic disorders. Donor selection requires good health and absence of genetic abnormalities; criteria for semen including normal sperm motility, concentration, and normal morphology, and blood screening for infectious agents. Human immunodeficiency virus (HIV) testing should be performed initially in donors for fresh semen inseminations. If positive, the assay is verified with a Western blot test; if negative, the donor should be screened at 6-month intervals. Frozen samples should not be used until the 180 day reevaluation of the donor. Many studies show higher pregnancy rates using fresh rather than frozen semen samples for insemination. New methods of cryopreservation minimize the deleterious effects of freezing. If these effects, namely decreased sperm motility and impaired penetration ability, are eliminated, pregnancy rates can be expected to rise. Frozen semen is preferable because it allows time for sexually transmitted diseases to manifest themselves and for specimens from those donors to be rejected prior to use.

beta-Glucosidase Activity in Corn Roots: Problems in Subcellular Fractionation.

Preliminary results from differential centrifugation experiments, washing treatments, and enrichment in linear sucrose gradients at a density of 1.09 grams per cubic centimeter all indicated that beta-glucosidase activity in corn root homogenates was associated with a membrane such as tonoplast. A subsequent sucrose density gradient centrifugation time course showed that the beta-glucosidase was actually a soluble enzyme which moved into the gradients. The problem of soluble enzymes contaminating light density membranes in sucrose gradients and the question of centrifugation time necessary for membrane vesicles to reach isopycnic conditions are addressed.