Phase II and pharmacodynamic studies of pyrazine diazohydroxide (NSC 361456) in patients with advanced renal and colorectal cancer.

Pyrazine diazohydroxide (PZDH) is a novel antitumor agent that forms DNA adducts via the reactive pyrazine diazonium ion. In a recent Phase I study of PZDH, we identified a recommended Phase II dose of 100 mg/m2/day x 5, given as a 5-min i.v. bolus with the cycles repeated every 42 days (N. J. Vogelzang, et al, Cancer Res., 54: 114-119, 1994). There was a moderate negative correlation between serum chloride concentration and logarithm platelet nadir, suggesting the hypothesis that PZDH is activated in an acidic environment, leading to more toxicity in acidotic patients. Therefore, the University of Chicago Phase II cooperative network conducted two Phase II studies of PZDH in renal cancer (15 patients, 2 with liver metastases) and in 5-fluorouracil-refractory colorectal cancer (14 patients, 13 with liver metastases) to determine efficacy in each disease and to correlate safety and tolerance of the drug with PZDH pharmacokinetics/pharmacodynamics and with arterial blood gas measurements. There were no responses seen in either tumor type. The primary toxicity of PZDH was myelosuppression with neutropenia (absolute neutrophil count, < 1000/microl) and thrombocytopenia (<50,000 cells/microl), seen in 41 and 24% of all cycles, respectively. Other grade 3 and 4 toxicities were rare. Pharmacodynamic analysis revealed no significant correlation between plasma levels at 5, 60, and 120 min; WBCs; absolute neutrophil and platelet count nadirs; and initial serum chloride or blood pH levels. The colorectal patients experienced significantly more thrombocytopenia than did the renal cancer patients (median platelet nadir after cycle 1 was 151 x 10(3)/microl for renal patients versus 76 x 10(3)/microl for colon patients; P = 0.04), suggesting either that prior 5-fluorouracil and leucovorin reduced bone marrow reserve or that colorectal patients with liver metastases experienced more PZDH toxicity. Regression analyses revealed a possible relationship (P = 0.06) between serum pH and thrombocytopenia (i.e., for each increase of 0.03 in pH, there was a 34% increase in the platelet nadir), but there was no relationship between serum chloride and thrombocytopenia. Curiously, an increase in alkaline phosphatase was associated with an increase in the platelet nadir (P = 0.02). If PZDH continues to be developed as an antineoplastic agent, further studies of these relationships are suggested.

Evaluation of in vivo models for studying calcification behavior of commercially available bovine pericardium.

BACKGROUND AND AIM OF THE STUDY: A common frame of reference is essential when attempting to determine if new treatments intended to reduce calcification of bioprostheses are superior to existing processes and products. The aim of this study was to examine calcification behavior for a commercially available pericardial bioprosthesis in subcutaneous and sheep valve models, and to evaluate the importance of appropriate control treatments in comparative studies with proposed new treatments. METHODS: Samples of bovine pericardium were placed subcutaneously under the dorsal skin of weanling rats and juvenile rabbits for 30-, 60- and 90-day intervals. Samples were either commercially available pericardial tissue or tissue processed with phosphate-buffered glutaraldehyde alone. Commercially available pericardial valves were also implanted in the mitral position in juvenile sheep, with elective sacrifice at 20 weeks. Retrieved samples underwent X-ray, histologic and elemental analysis. RESULTS: Commercial samples retrieved from the subcutaneous and sheep models showed similar, minimal calcification behavior on X-ray and histologic slides, whereas pericardium exposed to glutaraldehyde alone demonstrated rapid calcification. CONCLUSIONS: The 90-day subcutaneous rabbit model produced patterns of calcification similar to those in valves explanted from juvenile sheep after 20 weeks. A statistically significant decrease (p < 10(-8)) in calcification was demonstrated for clinical pericardium when compared with pericardium exposed to glutaraldehyde alone in the subcutaneous model. This suggests that subcutaneous models may be a cost-effective, time-efficient means of evaluating and comparing various tissue treatment methods. The rabbit methodology may provide a more accurate prediction of clinical performance, offering a greater degree of sensitivity. These studies also indicate that the commercially available process shows minimal calcification in the commonly used 30-day weanling rat subcutaneous model, contradicting other reported studies that may not accurately represent commercially available processes.

The effects of chlorhexidine irrigation solution on contaminated bone-tendon allografts.

The purpose of this study was to determine an expedient and effective method for disinfecting contaminated human bone-tendon allografts. The first part of this study used beef muscle and cadaveric human tissues to determine the most effective solution and volume to decontaminate tissues inoculated with four different organisms: Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Of the solutions tested (benzalkonium chloride, castile soap, castile soap followed by benzalkonium chloride, triple antibiotic, chlorhexidine gluconate, and chlorhexidine gluconate/triple antibiotic), only the 4% chlorhexidine power irrigation solution and 4% chlorhexidine/triple antibiotic bath completely disinfected all tissues. Work in part 2 revealed that a 2% chlorhexidine irrigation solution was equally effective as the 4% solutions. Part 3 of the study involved human Achilles tendon-calcaneus allografts. We found similar results: 3 liters of 2% chlorhexidine power irrigation solution thoroughly removed all microorganisms from the contaminated tissues. All control allografts irrigated with normal saline solution alone revealed positive bacterial growth for all four organisms after 72 hours' growth on sheep blood agar. Total decontamination time was 10 to 12 minutes. Two percent chlorhexidine irrigation solution may be an effective method for decontaminating human bone-tendon allografts challenged with a polymicrobial inoculum. This method of disinfecting bone-tendon allografts is at least five times more expeditious than methods in previously reported studies.

Interaction of platelet glycoprotein V with glycoprotein Ib-IX regulates expression of the glycoproteins and binding of von Willebrand factor to glycoprotein Ib-IX in transfected cells.

The goal of the present study was to determine whether platelet glycoprotein (GP) V interacts directly with the von Willebrand factor receptor GP Ib-IX and, if so, whether it affects the expression and function of this receptor. A melanoma cell line that does not contain actin-binding protein was transfected with the cDNAs coding for GP V and for each of the three subunits of GP Ib-IX. GP V co-immunoprecipitated and co-localized with GP Ib-IX. Although GP V could be expressed in the absence of GP Ib-IX, the amount incorporated in the membrane was markedly increased when GP Ib-IX was present. Similarly, there was an enhanced expression of GP Ib-IX on the cell surface in the presence of GP V. The binding affinity of botrocetin-induced von Willebrand factor to GP Ib-IX was unaffected by the presence or absence of GP V. However, the binding capacity was increased by the presence of GP V. We conclude that GP V interacts directly with GP Ib-IX, that GP V must associate with GP Ib-IX to be efficiently expressed in the membrane, and that GP V increases the binding capacity of the cells for von Willebrand factor by enhancing the surface expression of the GP Ib-IX complex.

The cytoplasmic domain of the alpha-subunit of glycoprotein (GP) Ib mediates attachment of the entire GP Ib-IX complex to the cytoskeleton and regulates von Willebrand factor-induced changes in cell morphology.

The glycoprotein (GP) Ib-IX complex is one of the major platelet membrane glycoproteins. Its extracellular domain binds von Willebrand factor at a site of injury, an interaction that leads to activation of intracellular pathways. Its intracellular domain associates tightly with the platelet cytoskeleton through actin-binding protein. The goal of the present study was to investigate the role of the cytoplasmic domain of the GP Ib-IX complex and its interaction with the cytoskeleton. Cultured cells were transfected with the cDNAs coding for GP Ib(beta), GP IX, and full-length or truncated forms of GP Ib(alpha). Western blots of detergent-insoluble fractions of Triton X-100-lysed cells showed that deletion of amino acids Trp-570 to Ser-590 from the cytoplasmic domain of GP IB(alpha) abolished the interaction of the entire GP Ib-IX complex with the cytoskeleton. Truncated GP Ib(alpha) that was unable to associate with the cytoskeleton retained its ability to associate with GP Ib(beta), to be inserted into the membrane, and to bind von Willebrand factor. Cells expressing GP Ib(alpha) changed their shape following adhesion to immobilized von Willebrand factor. Cells expressing truncated GP Ib(alpha) also changed their shape following adhesion but showed a very different morphology as compared to cells expressing full-length GP Ib(alpha). These results show that GP Ib-IX-von Willebrand factor interactions lead to cytoskeletal reorganizations, that the cytoplasmic domain of GP Ib(alpha) regulates these reorganizations, and that the cytoplasmic domain of GP Ib(alpha) is absolutely required for attachment of the GP Ib-IX complex to the cytoskeleton.

Enhanced in vitro activity of pyrimethamine in combination with dapsone against Mycobacterium avium complex.

The in vitro activities of pyrimethamine and dapsone alone and in combination were evaluated against 23 clinical isolates of Mycobacterium avium complex. The broth dilution MICs of dapsone and pyrimethamine alone ranged from 16 to > 64 micrograms/ml. Pyrimethamine in combination with a fixed concentration of dapsone at 0.5 microgram/ml showed enhanced activity, with an MIC range of 0.5 to 16 micrograms/ml.

In vitro activities of PS-15, a new dihydrofolate reductase inhibitor, and its cyclic metabolite against Mycobacterium avium complex.

PS-15, a new dihydrofolate reductase inhibitor, and its cyclic metabolite were evaluated for in vitro activity against 31 clinical Mycobacterium avium complex isolates. Broth dilution MICs of PS-15 ranged from 16 to 64 micrograms/ml. The cyclic metabolite was three to five times more active than the parent compound. Further evaluation of these compounds in an M. avium-infected murine test system will be of interest.

Role of actin-binding protein in insertion of adhesion receptors into the membrane.

The goal of this study was to determine whether actin-binding protein (ABP) regulates membrane composition. ABP-deficient and ABP-containing cells were transfected with the cDNAs coding for glycoprotein (GP) Ib-IX, a platelet receptor that interacts with ABP. Most of the GP Ib-IX remained inside the ABP-deficient cells. When ABP was present, functional GP Ib-IX was inserted into the membrane. GP Ib-IX lacking the domain that interacts with ABP also showed increased membrane insertion in ABP-expressing cells. Furthermore, a fragment of ABP that lacks the dimerization and GP Ib-IX-binding sites restored the spreading of the cells and increased the amount of GP Ib-IX in the membrane. Finally, expression of ABP also increased endogenous beta1 integrin in the membrane. These results indicate that 1) ABP maintains the properties of the cell such that adhesion receptors can be efficiently expressed in the membrane; 2) increased receptor expression is accompanied by increased ability of the cell to spread; and 3) ABP exerts its effect by a mechanism that does not appear to involve direct cross-linking of actin filaments or direct interaction with receptors.

Signaling across the platelet adhesion receptor glycoprotein Ib-IX induces alpha IIbbeta 3 activation both in platelets and a transfected Chinese hamster ovary cell system.

In platelets, alpha(IIb)beta(3) exists in a form that cannot bind adhesive proteins in the plasma; although it can interact with immobilized fibrinogen it cannot interact with immobilized von Willebrand factor in the vessel wall. Soluble agonists such as thrombin convert alpha(IIb)beta(3) to a form that recognizes soluble and immobilized ligands. Attempts to reconstitute alpha(IIb)beta(3) activation in a non-hematopoietic, nucleated cell system have been unsuccessful. In the present study, we have developed a transfected Chinese hamster ovary cell model in which alpha(IIb)beta(3) activation is induced by signaling across glycoprotein (GP) Ib-IX by its ligand, von Willebrand factor. GPIb-IX activates not only the transfected alpha(IIb)beta(3) but also endogenous alpha(v)beta(3). Activation of the pathways leading to integrin activation occurred even in cells transfected with GPIb-IX lacking the domain on GPIbalpha that binds 14-3-3 or that which binds actin-binding protein. These studies demonstrate that signals induced by interaction of GPIb-IX with von Willebrand factor lead to alpha(IIb)beta(3) activation and suggest that the signaling pathways by which GPIb-IX induces alpha(IIb)beta(3) activation are different to those used by thrombin. Elucidation of these differences may provide insights into therapeutic ways in which to inhibit integrin activation in selective clinical settings.

Identification of the region in actin-binding protein that binds to the cytoplasmic domain of glycoprotein IBalpha.

Actin-binding protein (ABP-280) is a component of the submembranous cytoskeleton and interacts with the glycoprotein (GP) Ibalpha subunit of the GP Ib-IX complex in platelets. In the present studies, we have identified the binding site for GP Ibalpha in ABP-280. A melanoma cell line lacking ABP-280 was stably transfected with the cDNAs coding for GP Ib-IX, then transiently transfected with cDNA coding for various carboxyl-truncates of ABP-280. Immunocapture assays and co-immunoprecipitation experiments from detergent-lysed cells showed that deletion of the carboxyl-terminal repeats 20-24 of ABP-280 had no effect on GP Ib-IX binding, but deletion of residues 2099 through 2136 within repeat 19 abolished binding. In the yeast two-hybrid system, an ABP-280 fragment comprising repeats 17-19 bound GP Ibalpha. Deletion from either end abolished binding. Individual or multiple repeats of ABP-280 were expressed as fusion protein in bacteria and purified; structural folding was evaluated, and binding to GP Ib-IX was assessed. Binding depended on the presence of repeats 17-19. None of the individual repeats were able to bind to GP Ib-IX. These findings demonstrate that residues 1850-2136 comprising repeats 17-19 contain the binding site for GP Ib-IX.

Methotrexate, vinblastine, doxorubicin, and cisplatin in metastatic breast cancer. A phase II trial of the Hoosier Oncology Group.

Forty-six eligible patients with metastatic breast cancer (MBC) were treated with a combination of methotrexate, vinblastine, doxorubicin, and cisplatin (M-VAC) as first-line chemotherapy. Of 44 patients evaluable for response, 28 (64%) had an objective response, including seven (16%) who had a complete response. The median duration of response was 4 months (range, 0 to 38 months), and the median survival from the time of entry was 14 months (range, less than 1 to greater than 45 months). Myelosuppression was the most common dose-limiting toxicity, with 54% of patients experiencing Grade 3 or 4 leukopenia (including 28% with granulocytopenic fever and one septic death), and cumulative Grade 3 anemia occurred in 28% of patients. Grades 3 to 4 stomatitis was observed in 18% of patients. An active, although highly toxic regimen when used as first-line therapy in MBC, M-VAC has a response rate and survival duration similar to existing, less toxic combination regimens. As such, M-VAC cannot be recommended in preference to other combination chemotherapy regimens in this clinical setting.