Robert G. Kilbourn

Reversal of endotoxin-mediated shock by NG-methyl-L-arginine, an inhibitor of nitric oxide synthesis

Septic shock is a life-threatening condition that results from exposure to bacterial endotoxin. It is manifested by cardiovascular collapse and mediated by the release of cytokines such as tumor necrosis factor. Some of these cytokines cause the release of vasoactive substances. In the present study, administration of 40 microgram/kg of bacterial endotoxin to dogs caused a 33% decrease in peripheral vascular resistance and a 54% fall in mean arterial blood pressure within 30 to 90 minutes. Vascular resistance and systemic arterial pressure returned to normal within 1.5 minutes after intravenous administration of NG-methyl-L-arginine (20 mg/kg), a potent and selective inhibitor of nitric oxide synthesis. L-Arginine reversed the effect of L-NMA and restored the endotoxin-induced hypotension. Although NG-methyl-L-arginine injection increased blood pressure in control dogs, the hypertensive effect was much greater in endotoxemic dogs (24.8 +/- 2.7 mmHg vs 47.8 +/- 6.8 mmHg, p = 0.01, n = 4). NG-Methyl-L-arginine caused only a modest increase in blood pressure in dogs made hypotensive by continuous intravenous infusion of nitroglycerin (17.1 +/- 5.0 mm Hg, n = 3). These findings suggest that nitric oxide overproduction is an important contributor to endotoxic shock. Moreover, our findings demonstrate for the first time, the utility of nitric oxide synthesis inhibitors in endotoxic shock and suggest that such inhibitors may be of therapeutic value in the treatment of septic shock.

A prospective randomized trial comparing MVAC and CISCA chemotherapy for patients with metastatic urothelial tumors.

To evaluate the relative efficacy of cisplatin, cyclophosphamide, and Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH) (CISCA) versus methotrexate, vinblastine, Adriamycin, and cisplatin (MVAC), a prospective randomized trial was performed in patients with advanced metastatic urothelial tumors. Patients were stratified by histologic disease type and degree of tumor dissemination. Equal distribution of the clinical characteristics was achieved. One hundred ten patients with metastatic disease of the urinary tract (86 bladder, 16 renal pelvis, seven ureter, one prostatic urethra) met eligibility criteria and were enrolled on study. These represented 82% of the total patients seen during the study period in the Section of Genitourinary Oncology who met the eligibility criteria. The combined complete and partial response rate was significantly higher for patients treated with MVAC than for those treated with CISCA (65% v 46%; P less than .05). The survival duration of MVAC-treated patients was significantly longer than that of CISCA-treated patients (mean, 62.6 weeks; median, 48.3; range, 5.0+ to 162.3+ v mean, 40.4 weeks; median, 36.1; range, 7+ to 147.1+). We conclude that MVAC chemotherapy is superior to CISCA chemotherapy, achieving a higher response rate and a longer survival for equivalent patients with metastatic urothelial tumors.

Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages

We have previously reported that cultured murine brain endothelial cells (MBE) produce large quantities of nitric oxide (NO) after activation with interferon-gamma in combination with any of several immunoactivators including: bacterial endotoxin, tumor necrosis factor and interleukin-1. Since endothelial cells are the first example of a cell-type which may possess both a constitutive and an inducible type of NO synthase, it was of interest to compare the requirements of these two enzyme activities. Induction of NO synthesis in MBE by cytokines was abolished by the protein synthesis inhibitor, cycloheximide, and by 2,4-diamino-6-hydroxypyridine (DAHP), a selective inhibitor of GTP cyclohydrolase I, the rate-limiting enzyme for de novo synthesis of tetrahydrobiopterin (THB). In the presence of DAHP, NO synthesis was restored by sepiapterin (SEP), a substrate for the alternative pathway of THB synthesis occurring via pterin salvage. Moreover, SEP increased NO synthesis to greater than 150% of control values, suggesting that THB availability is rate-limiting for NO synthesis by cytokine-induced MBE. Methotrexate, an inhibitor of the pterin salvage pathway of THB synthesis, completely reversed the stimulation of NO synthesis by sepiapterin. Thus, cytokine-induced MBE NO synthase appears to have an absolute requirement for THB as cofactor. In additional studies we found that NO synthesis by cytokine-activated MBE was inhibited by NG-monosubstituted arginine analogs with a rank-order of potency NH2 greater than CH3 greater than NO2, in contrast with the rank-order of NO2 greater than NH2 greater than CH3 previously described for inhibition of the constitutive endothelial cell enzyme. Using a kinetic assay for NO synthase activity, based on oxidation of myoglobin heme-iron, we have found that these rank orders of potency also apply to cytosol preparations of cytokine-induced and untreated endothelial cells, respectively. Further differences between constitutive and cytokine-induced NO synthase were observed with regard to calmodulin requirements. Whereas constitutive NO synthase was potently inhibited by the calmodulin antagonists mellitin and trifluoperazine, cytokine-induced NO synthase was unaffected. In summary, NO synthesis by cytokine-activated MBE is THB-dependent, calmodulin-independent and inhibited by NG-substituted arginine analogs with a rank-order profile distinct from that for untreated endothelial cells but identical to that for cytokine-activated macrophages.

Cationic liposome-mediated E1A gene transfer to human breast and ovarian cancer cells and its biologic effects : a phase I clinical trial

PURPOSE Preclinical studies have demonstrated that the adenovirus type 5 E1A gene is associated with antitumor activities by transcriptional repression of HER-2/neu and induction of apoptosis. Indeed, E1A gene therapy is known to induce regression of HER-2/neu-overexpressing breast and ovarian cancers in nude mice. Therefore, we evaluated the feasibility of intracavitary injection of E1A gene complexed with DC-Chol cationic liposome (DCC-E1A) in patients with both HER-2/neu-overexpressing and low HER-2/neu-expressing breast and ovarian cancers in a phase I clinical trial. PATIENTS AND METHODS An E1A gene complexed with DCC-E1A cationic liposome was injected once a week into the thoracic or peritoneal cavity of 18 patients with advanced cancer of the breast (n = 6) or ovary (n = 12). RESULTS E1A gene expression in tumor cells was detected by immunohistochemical staining and reverse transcriptase-polymerase chain reaction. This E1A gene expression was accompanied by HER-2/neu downregulation, increased apoptosis, and reduced proliferation. The most common treatment-related toxicities were fever, nausea, vomiting, and/or discomfort at the injection sites. CONCLUSION These results argue for the feasibility of intracavitary DCC-E1A administration, provide a clear proof of preclinical concept, and warrant phase II trials to determine the antitumor activity of the E1A gene.

Phase II study of ketoconazole combined with weekly doxorubicin in patients with androgen-independent prostate cancer.

PURPOSE A phase II clinical trial was performed to assess the antitumor activity and toxicity of ketoconazole in combination with doxorubicin (Adriamycin; Adria Laboratories, Columbus, OH) in patients with androgen-independent prostate cancer (AI PCa). PATIENTS AND METHODS Thirty-nine consecutive patients whose disease progressed following castration were treated with oral ketoconazole (1,200 mg) daily and Adriamycin (20 mg/m2 in a 24-hour infusion) once weekly. Antitumor activity was assessed by the level of prostatic-specific antigen (PSA) decline. RESULTS PSA levels decreased > or = 50% from baseline in 21 (55%; 95% confidence interval, 38% to 71%) of 38 assessable patients. We observed partial responses (PRs) in seven (58%) of 12 patients with measurable soft tissue disease (in the lung, lymph nodes, and liver). Two patients with history of atherosclerotic heart disease had a sudden cardiac death. Serious toxic reactions included grade III to V stomatitis and grade III to IV acral erythema in 11 patients (29%), and grade III to IV anal and urethral mucositis in five patients (13%). Grade III to IV neutropenia occurred in 11 patients (29%). Seventeen patients (45%) required hospitalization for complications. Fifteen patients (39%) developed hypokalemia, and 24 patients (63%) developed clinical adrenal insufficiency. CONCLUSION The combination of ketoconazole and Adriamycin has a 55% PSA response rate in patients with AI PCa and is worthy of additional study. This treatment results in frequent adrenal insufficiency. Therefore, future studies should incorporate routine corticosteroid replacement. The cardiac complications caused by this combination should be studied further before it is widely used.

Paclitaxel, Cisplatin and Methotrexate Combination Chemotherapy is Active in the Treatment of Refractory Urothelial Malignancies

PURPOSE We investigated the activity of combination chemotherapy consisting of paclitaxel, cisplatin and methotrexate in patients with advanced urothelial cancers. MATERIALS AND METHODS A total of 25 consecutive patients with metastatic refractory urothelial malignancies was treated with a combination of 200 mg./m.2 paclitaxel, 30 mg./m.2 methotrexate and 70 mg./m.2 cisplatin in a pilot study. RESULTS There were no complete responses. Of 25 patients 10 (40%), including 3 of 7 with liver metastases, had a partial response. Hematological and nonhematological toxicity was tolerable. CONCLUSIONS The combination chemotherapeutic regimen of paclitaxel, cisplatin and methotrexate is active in patients with advanced urothelial cancer and warrants further study.

Escalated MVAC with or without recombinant human granulocyte-macrophage colony-stimulating factor for the initial treatment of advanced malignant urothelial tumors: results of a randomized trial.

PURPOSE Hematopoietic growth factors have been shown to ameliorate the side effects of chemotherapy. Here we assess the ability of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to increase the dose-intensity and reduce the side effects of escalated methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) chemotherapy. PATIENTS AND METHODS A prospective randomized trial to compare escalated MVAC versus escalated MVAC with rhGM-CSF was conducted. All patients were treated at The University of Texas M.D. Anderson Cancer Center (UTMDACC) and had a metastatic or unresectable urothelial tumor. Forty-eight patients were randomized (25 to MVAC with rhGM-CSF and 23 to escalated MVAC alone). The clinical characteristics of the study populations were similar (ie, degree of tumor dissemination and performance status). RESULTS The dose-intensity in the two arms of the study did not differ significantly. No difference in the frequency of bacteriologically documented infections occurred between the two study arms. CONCLUSION The use of the hematopoietic growth factor rhGM-CSF did not result in an increased dose-intensity of escalated MVAC. The inability to increase the dose-intensity of MVAC further was a result of nonhematologic side effects of the chemotherapy. Escalation of treatment delivered at its median-tolerated dose is unlikely to result in additional therapeutic benefit for patients with common solid tumors. Future development of therapy may require the development of new agents or concepts, rather than modification of existing therapies.

NG-methyl-L-arginine, an inhibitor of nitric oxide synthase, reverses interleukin-2-induced hypotension

OBJECTIVE To evaluate the role of NG-methyl-L-arginine as a modulator of the hyperdynamic shock induced by the administration of interleukin-2 (IL-2). DESIGN A prospective, pilot clinical study. SETTING Intensive care unit of a tertiary care center. PATIENTS Three sequential patients with metastatic renal cell carcinoma who developed hypotension during their first course of treatment with high-dose IL-2 (18 x 10(6) IU/m2/day by continuous infusion for 5 days). INTERVENTIONS Upon developing hypotension during their subsequent therapy with IL-2, patients were administered 12 mg/kg of NG-methyl-L-arginine. Thereafter, a dose of 4 mg/kg was given every 4 hrs, as needed, to maintain the systolic blood pressure above 100 mm Hg. MEASUREMENTS AND MAIN RESULTS Invasive hemodynamic monitoring was instituted before the initiation of treatment with IL-2. Differences noted before, and 15 mins after, the administration of NG-methyl-L-arginine were analyzed using the paired t-test. NG-methyl-L-arginine (12 mg/kg) induced a significant antihypotensive effect (mean blood pressure increased from 87 +/- 4 to 121 +/- 7 mm Hg), accompanied by an increase of the systemic vascular resistance (549 +/- 51 to 860 +/- 167 dyne.sec/cm5) and pulmonary vascular resistance (81 +/- 16 to 117 +/- 29 dyne.sec/cm5). A decrease in the cardiac index was also documented (4.5 +/- 0.5 to 3.6 +/- 0.3 L/min/m2). No significant changes in pulmonary artery occlusion and central venous pressures were observed. Maintenance doses of 4 mg/kg of NG-methyl-L-arginine induced similar hemodynamic results, although the duration of the antihypotensive effect of NG-methyl-L-arginine decreased with sequential doses. CONCLUSIONS The hemodynamic effects induced by IL-2 administration are reversed by NG-methyl-L-arginine, a nitric oxide synthesis inhibitor. These results provide evidence for the biological activity of NG-methyl-L-arginine when administered alone to hypotensive patients. While no adverse effects were observed in this preliminary study, issues of toxicity and effectiveness need to be defined further in formal clinical trials. NG-methyl-L-arginine may play a therapeutic role in the modulation of the extreme vasodilation induced by cytokine administration or in septic shock.

Phase II Study of Interferon-α and Chemotherapy (5-Fluorouracil and Mitomycin C) in Metastatic Renal Cell Cancer

AbstractA total of 49 patients with metastatic renal cell cancer underwent recombinant interferon-α2a therapy combined with chemotherapy. Before therapy the patients without nephrectomy underwent angioinfarction of the primary renal tumor. Combined treatment included interferon at 5 × 106 units per m.2 intramuscularly daily, 5-fluorouracil at 750mg./m.2 daily by continuous infusion intravenously (days 1 to 5) and mitomycin C at 5mg./m.2 per day intravenously (days 1 and 2) repeated every 28 days. Of the patients 17 (35%, 95% confidence interval 22 to 49%) responded, and all 17 had partial remission that lasted a median of 7.1 months (range 4.2 to 20.9+ months). Response rate differed by metastatic sites: lung 46% (18 of 39 patients), lymph nodes 46% (6 of 13), mediastinum 20% (2 of 10) and liver 18% (2 of 11). Grade 3 to 4 toxicity (World Health Organization) included neutropenia (79% of the patients), thrombocytopenia (45%), stomatitis (34%), diarrhea (8%), nausea (18%) and central nervous system disorde...

Drug-induced vasodilation in an in vitro and in vivo study: the effects of nicardipine, papaverine, and lidocaine on the rabbit carotid artery.

&NA; Extreme arterial vasoconstriction (vasospasm) is a common problem encountered in microvascular surgery. An ideal pharmacologic tool able to counteract ischemia during microsurgery should be easy to apply and exert its action both locally and distally in the microcirculation of the flap. We have compared in vitro and in vivo vascular properties of nicardipine, papaverine, and lidocaine in the rabbit carotid artery. In vitro, rings from the rabbit carotid artery (n = 7) were bathed in Krebs‐Ringers solution and stretched progressively to an optimal tension of 3.7 to 4.2 g. The specimens were contracted with norepinephrine (1 &mgr;M), and a cumulative dose response curve was established. In vivo, microvascular anastomoses were performed bilaterally in the rabbit carotid artery in 35 animals using 9‐0 nylon suture and standard microsurgical techniques. During and after the anastomoses, nicardipine (0.1, 0.01 mg topical, or 0.1 mg/hour IV), papaverine (30 mg/cc topical), and lidocaine (2% with and without epinephrine) were applied (blinded) at the anastomotic site in five rabbits each. Heparinized sodium chloride was used as topical irrigation for control and to clean the anastomosis. Blood flow changes were monitored continuously with the transonic Doppler for 30 minutes after the procedure. The systemic blood pressure was also monitored in a group of pilot experiments. A documented decrease in blood flow was noted in all animals after the microvascular anastomosis. Nicardipine and papaverine evoked a concentrationdependent relaxation to precontracted rings to norepinephrine. Nicardipine was greater than papaverine in inducing relaxation. Lidocaine demonstrated a biphasic response with low concentrations potentiating contraction. Systemic nicardipine and papaverine significantly increased the blood flow in the rabbit carotid artery. Topical application of nicardipine and lidocaine did not significantly alter the blood flow; however, the application of nicardipine demonstrates a trend toward increased flow. Lidocaine with epinephrine significantly decreased the blood flow. No drug was found to alter the blood pressure of the animals. Our results demonstrate that nicardipine and papaverine seem to be pharmacologic tools able to increase the blood flow in anastomotic arteries. In contrast, the use of 2% lidocaine as a spasmolytic agent should be re‐evaluated, since this substance may act as a partial agonist. (Plast. Reconstr. Surg. 100: 1475, 1997.)