Kenneth S. Bauer

Inhibition of angiogenesis by thalidomide requires metabolic activation, which is species-dependent.

Thalidomide has been shown to be an inhibitor of angiogenesis in a rabbit cornea micropocket model; however, it has failed to demonstrate this activity in other models. These results suggest that the anti-angiogenic effects of thalidomide may only be observed following metabolic activation of the compound. This activation process may be species specific, similar to the teratogenic properties associated with thalidomide. Using a rat aorta model and human aortic endothelial cells, we co-incubated thalidomide in the presence of either human, rabbit, or rat liver microsomes. These experiments demonstrated that thalidomide inhibited microvessel formation from rat aortas and slowed human aortic endothelial cell proliferation in the presence of human or rabbit microsomes, but not in the presence of rat microsomes. In the absence of microsomes, thalidomide had no effect on either microvessel formation or cell proliferation, thus demonstrating that a metabolite of thalidomide is responsible for its anti-angiogenic effects and that this metabolite can be formed in both humans and rabbits, but not in rodents.

Phase I Trial of 72-Hour Continuous Infusion UCN-01 in Patients With Refractory Neoplasms

PURPOSE To define the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of the novel protein kinase inhibitor, UCN-01 (7-hydroxystaurosporine), administered as a 72-hour continuous intravenous infusion (CIV). PATIENTS AND METHODS Forty-seven patients with refractory neoplasms received UCN-01 during this phase I trial. Total, free plasma, and salivary concentrations were determined; the latter were used to address the influence of plasma protein binding on peripheral tissue distribution. The phosphorylation state of the protein kinase C (PKC) substrate alpha-adducin and the abrogation of DNA damage checkpoint also were assessed. RESULTS The recommended phase II dose of UCN-01 as a 72-hour CIV is 42.5 mg/m(2)/d for 3 days. Avid plasma protein binding of UCN-01, as measured during the trial, dictated a change in dose escalation and administration schedules. Therefore, nine patients received drug on the initial 2-week schedule, and 38 received drug on the recommended 4-week schedule. DLTs at 53 mg/m(2)/d for 3 days included hyperglycemia with resultant metabolic acidosis, pulmonary dysfunction, nausea, vomiting, and hypotension. Pharmacokinetic determinations at the recommended dose of 42.5 mg/m(2)/d for 3 days included mean total plasma concentration of 36.4 microM (terminal elimination half-life range, 447 to 1176 hours), steady-state volume of distribution of 9.3 to 14.2 L, and clearances of 0.005 to 0.033 L/h. The mean total salivary concentration was 111 nmol/L of UCN-01. One partial response was observed in a patient with melanoma, and one protracted period ( > 2.5 years) of disease stability was observed in a patient with alk-positive anaplastic large-cell lymphoma. Preliminary evidence suggests UCN-01 modulation of both PKC substrate phosphorylation and the DNA damage-related G(2) checkpoint. CONCLUSION UCN-01 can be administered safely as an initial 72-hour CIV with subsequent monthly doses administered as 36-hour infusions.

Phase I trial of micronized formulation carboxyamidotriazole in patients with refractory solid tumors: pharmacokinetics, clinical outcome, and comparison of formulations.

PURPOSE Cytostatic agents targeted against angiogenesis and tumor cell invasive potential form a new class of investigational drugs. Orally administered carboxyamidotriazole (CAI) (NSC609974) is both antiangiogenic and antimetastatic. An encapsulated micronized powder formulation has been developed to optimize CAI administration. A phase I dose escalation trial with pharmacokinetic analysis has been performed. PATIENTS AND METHODS Twenty-one patients with refractory solid tumors and good end organ function and performance status were enrolled onto the study. Patients received a test dose followed 1 week later by daily administration of CAI in the encapsulated micronized formulation at doses of 100 to 350 mg/m2. Patients remained on CAI until disease progression or dose-limiting toxicity. Plasma samples were taken to characterize the pharmacokinetic parameters of this formulation of CAI. RESULTS All patients were assessable for toxicity and 18 were assessable for pharmacokinetics and response analysis. Grade 1 and 2 gastrointestinal side effects were observed in up to 50% of patients. Dose-limiting toxicity was observed in both patients treated at 350 mg/m2/d, consisting of reversible grade 2 to 3 cerebellar ataxia (n = 1) and confusion (n = 1). One minor response (MR) was observed in a patient with renal cell carcinoma and another nine patients had disease stabilization (MR + SD = 47%). Pharmacokinetic analysis demonstrated reduced bioavailability (58% reduction) compared with the PEG-400 liquid formulation previously reported. CONCLUSION The better toxicity profile of encapsulated micronized CAI with similar frequency of disease stabilization and ease of administration compared with the liquid or gelatin capsule, suggests that the micronized formulation is a preferable formulation for subsequent studies. A dose of 300 mg/m2/d is proposed for phase II investigations.

IMP dehydrogenase inhibitor mycophenolate mofetil induces caspase-dependent apoptosis and cell cycle inhibition in multiple myeloma cells

Multiple myeloma is an incurable disease for the majority of patients, therefore requiring new biological targeted therapies. In primary myeloma cells, IMP dehydrogenase (IMPDH) was shown to be consistently overexpressed. We therefore tested the IMPDH inhibitor mycophenolate mofetil (MMF) currently available as a clinical therapeutic agent for its antimyeloma activity in vitro. MMF depleted intracellular guanosine 5′-triphosphate (GTP) levels in myeloma cells. We showed apoptosis induction in myeloma cell lines and primary myeloma cells between 1 and 5 μmol/L MMF. MMF was also cytotoxic at this concentration in dexamethasone-resistant and Mcl-1-overexpressed myeloma cell lines shown by the tetrazolium salt XTT assay along with cell survival measured by a modified flow cytometric assay. Apoptosis was not inhibited by the presence of an antioxidant, suggesting that MMF-induced apoptosis is less likely to be associated with reactive oxygen species. However, apoptosis was abrogated by exogenously added guanosine, which activates an alternative pathway for GTP formation, implicating that this effect is directly mediated by IMPDH inhibition. MMF-induced G1-S phase cell cycle arrest and its apoptosis induction mechanism were associated with a caspase-dependent pathway as shown by alteration of mitochondrial membrane potential and cytochrome c release followed by activation of the caspases. MMF-induced apoptosis was also inhibited by a pan-caspase inhibitor Z-VAD-fmk. MMF-treated myeloma cells showed an up-regulation of Bak, which most likely together with Bax resulted in the release of cytochrome c. In summary, MMF attenuates G1-S phase cell cycle progression and activates the pathway of mitochondrial dysfunction, leading to cytochrome c release followed by activation of caspases. [Mol Cancer Ther 2006;5(2):457–66]

Clinical Pharmacology of Flavopiridol following a 72-Hour Continuous Infusion

Background Flavopiridol, a novel flavone derivative, inhibits cyclin-dependent kinase-1. We initiated a Phase I trial in patients with refractory solid tumors to determine the maximum tolerated dose and characterize the adverse effect profile. Objective To characterize the clinical pharmacology of flavopiridol. Methods Serial plasma samples were collected and analyzed by HPLC using electrochemical detection. The pharmacokinetics were analyzed by noncompartmental analysis. Enterohepatic recirculation was studied by analyzing fecal samples, with an attempt to correlate cholecystokinin and post-infusional peak concentrations. The plasma protein binding was studied using equilibrium dialysis. Results Seventy-six patients were treated with flavopiridol at 13 dose levels for a total of 504 cycles of treatment. The average steady-state concentration was 26.5 and 253 nM at 4 and 122.5 mg/m2, respectively. The clearance ranged from 49.9 to 2943 mL/min, with nonlinearity at doses >50 mg/m2/d. A post-infusional increase in plasma flavopiridol concentrations was noted in a subset of patients and generally occurred between 3 and 24 hours after the end of infusion. Flavopiridol was found in fecal matter, suggesting enterohepatic recirculation. There was nonsaturable plasma protein binding of flavopiridol (fu = 6%). Conclusions The dose-limiting toxicity for the Phase I trial of flavopiridol was secretory diarrhea. We failed to identify a clear relationship between dose or concentration and diarrhea. At 50 and 78 mg/m2/d, the mean steady-state plasma concentrations were 278 and 390 nM. These concentrations were well above those noted for in vitro antiproliferative activity. Nonlinear elimination was observed at doses above 50 mg/m2/d, and postinfusional peaks appear to be related to enterohepatic recirculation.

Phase I and Pharmacokinetic Study of 7-Hydroxystaurosporine and Carboplatin in Advanced Solid Tumors

Purpose: Based on preclinical data showing synergy between 7-hydroxystaurosporine (UCN-01) and platinum agents, a phase I trial of carboplatin with UCN-01 administered as a 3 h infusion in patients with advanced solid tumors was done. The primary goals of this trial were to evaluate the tolerability of this combination and the pharmacokinetics of UCN-01 when administered over 3 h and to compare the tolerability and pharmacokinetics with previously described schedules. Patients and Methods: Patients with advanced solid tumors, good performance status, normal organ function, and no potentially curative therapy were eligible for the trial. Carboplatin was escalated from an area under the curve (AUC) of 3 to an AUC of 5. UCN-01 was escalated from 50 to 90 mg/m2. Results: Twenty-three patients with advanced solid tumors (20 with prior platinum treatment) received a total of 60 cycles of therapy. Full doses of both agents (carboplatin AUC 5, UCN-01 90 mg/m2 in cycle 1, 45 mg/m2 in subsequent cycles) could be administered. The major toxicity noted was hypotension, which could be abrogated with the use of saline prehydration and posthydration. No responses were seen; however, seven patients were able to receive more than two courses of therapy. Of note, two of three patients with refractory, progressive small cell lung cancer were able to receive six cycles of therapy without evidence of progression. One patient experienced resolution of paraneoplastic syndrome of inappropriate antidiuretic hormone. The pharmacokinetic variables Cmax and t1/2 of the 3 h infusion were essentially identical to those previously observed when UCN-01 was administered over 72 h. The average t1/2 for cycle 1 was 506 ± 301 h, and the mean Cmax for all dose levels was >30 μmol/L. The mean AUC over the dosing interval for each dose level ranged from ∼6,000 to 9,000 μmol/L h. Thus, the AUC of UCN-01 after the 3 h infusion was lower than was observed after a 72 h infusion. Conclusion: The regimen of carboplatin and UCN-01 (administered as a 3 h infusion) was well tolerated. Further development of this combination, particularly in small cell lung cancer, is warranted.

Pharmacokinetics of Cisplatin Administered by Continuous Hyperthermic Peritoneal Perfusion (CHPP) to Patients with Peritoneal Carcinomatosis

The pharmacokinetics of cisplatin administered by continuous hyperthermic peritoneal perfusion (CHPP) was characterized in patients with peritoneal carcinomatosis. Cisplatin was added into the perfusate with escalating doses from 100 mg/m2 to 400 mg/m2. The hyperthermic perfusion was maintained for 90 minutes with a flow rate of 1.5 L/min and a target peritoneal temperature of 42.5°C after a tumor debulking procedure. Samples of both the perfusate and blood were obtained during the perfusion and 30 minutes after the perfusion. Cisplatin plasma and perfusate concentrations were determined by flameless atomic absorption spectrometry with a lower limit of detection of 2 ng/ml and a coefficient of variation (CV) < 10%. Fifty‐six patients were enrolled in the study. The mean (± SD) percentage of cisplatin present in the perfusate at the completion of perfusion was 27.8% ± 20% of the total dose. The maxi mum cisplatin concentrations in the perfusate were 10 times higher than those in plasma. The area under the concentration‐time curve (AUC) of the perfusate was 13 times higher than the AUC of plasma. A two‐compartment model with an additional peritoneal cavity compartment fits to the data best based on the Akaike information criterion. However, the interpatient variability was considerably high (CV < 100%). In conclusion, cisplatin administered by hyperthermic peritoneal perfusion resulted in a pharmacological advantage by obtaining higher and direct drug exposure to the tumor in the peritoneal cavity while limiting systemic absorption and toxicity. Using a complex two‐compartment model, the authors were able to characterize the pharmacokinetics of cisplatin given intraperitoneally via this technique.

Thalidomide up-regulates prostate-specific antigen secretion from LNCaP cells.

Abstract Thalidomide has been shown to have species- and metabolic-dependent antiangiogenic activity in vitro and in vivo, suggesting its potential in treating human angiogenesis-dependent pathologies such as solid tumors. Based on promising preclinical studies, thalidomide has entered phase II clinical trials for prostate, brain, breast cancer, and Kaposis sarcoma. However, the antiangiogenic mechanism of action is largely unresolved, as are its effects on tumor-associated gene expression, cytokine secretion, etc. We have investigated the effects of thalidomide on: 1) the secretion of prostate-specific antigen (PSA) in a human androgen-dependent prostate cell line; 2) growth and viability of human prostate cells; and 3) differential gene expression profiles of thalidomide-treated vs untreated human prostate cells. A human androgen-dependent prostate carcinoma cell line (LNCaP) and a human androgen-independent prostate carcinoma cell line (PC-3) were incubated with thalidomide 0.6, 6, or 60 μg/mL for 5–6 days. Secreted PSA from LNCaP cells was measured using a commercial enzyme-linked immunosorbant assay. Cell viability studies were conducted in both LNCaP and PC-3 cells using the same thalidomide concentrations. Furthermore, the differential gene expression of thalidomide-treated LNCaP cells was compared to that of untreated control cells using a commercially available human cancer cDNA expression array system. Thalidomide-treated LNCaP cells demonstrated increased PSA/cell levels at all concentrations tested compared to untreated control cells. Thalidomide demonstrated a cytostatic effect in LNCaP cells but had no appreciable effect on PC-3 cell viability compared to untreated control cells. Comparison of cDNA expression arrays hybridized with thalidomide-treated LNCaP cDNA probes suggests that thalidomide may up- or downregulate expression of angiogenesis-related genes, i.e., vitronectin, but these differential effects require further verification. Thalidomide over a range of doses has demonstrated nontoxic, cytostatic activity in LNCaP cells and significant upregulation of LNCaP cell PSA secretion in vitro. Furthermore, preliminary data from cDNA nucleic acid arrays of thalidomide-treated LNCaP cells suggest that thalidomide upregulates a potential angiogenic modulatory protein, the vitronectin precursor, which may eventually link thalidomides antiangiogenic activity with modulation of angiogenic vascular integrin pathways.

Phase I clinical trial of the inosine monophosphate dehydrogenase inhibitor mycophenolate mofetil (cellcept) in advanced multiple myeloma patients.

Purpose: Inosine monophosphate dehydrogenase (IMPDH) inhibitors have been used to induce leukemia blast cell differentiation but have not been tested in multiple myeloma for activity. Currently, available IMPDH inhibitor, mycophenolate mofetil (MMF), which is known as an immunosuppressant, was shown to induce apoptosis in myeloma cell lines. On the basis of our preclinical studies, we designed a clinical study to test our hypothesis that MMF has antimyeloma activity. Experimental Design: A Phase I MMF dose escalation study was conducted in relapsed and refractory myeloma patients who had documented disease progression by myeloma markers or bone marrow plasmacytosis to determine the maximum tolerated dose, toxicities, and efficacy of the drug. To assess the activity of IMPDH inhibition in the myeloma cells of patients, we measured intracellular nucleotide triphosphate levels by high-performance liquid chromatography-based analysis and examined the correlation with clinical response. Results: Among the 11 study patients, MMF was generally well tolerated and was administered up to a maximum dose of 5g/day. The most common toxicity was grade 1 fatigue (n = 4, 36%). One patient had a partial response (3g/day), four patients had stable disease, and six patients had progression of disease. There was a statistically significant difference in the intracellular dGTP level changes between the stable disease/partial response group versus progression of disease. Conclusions: MMF at 1 to 5 g/day daily dose is well tolerated by patients with relapsed and refractory multiple myeloma patients. Positive correlation between clinical response and depletion of intracellular dGTP level was shown. Future drug development to target this enzyme maybe useful in treating myelomas.

A high-performance liquid chromatography method using ultraviolet and fluorescence detection for the quantitation of UCN-01, 7-hydroxystaurosporine, from human plasma and saliva†

UCN-01, 7-hydroxystaurosporine, is an antagonist of protein kinase C, as well as causing cell cycle arrest. We developed and validated an HPLC assay method for the quantitation of UCN-01. Plasma and saliva standard curves were prepared at concentrations ranging from 0.2 to 20.0 microgram/mL and 4.0 to 200.0 ng/mL, respectively. The sample preparation consisted of acetonitrile precipitation. Separation was accomplished on a phenyl column and a C-18 precolumn insert utilizing a gradient-profile consisting of ammonium acetate and acetonitrile. UV detection was set at 295 nm for UCN-01 and 323 nm for umbelliferone, the internal standard. For fluorescence detection, excitation occurred at 290 nm, while emission was at 400 nm. The retention times were around 4 min for umbelliferone and 9.1 for UCN-01. Inter- and intra-assay errors of accuracy were less than 7. 0% and 10.7%, respectively, for the plasma standard curve and less than 7.1% and 6.7%, respectively, for the saliva standard curve. The recoveries of UCN-01 and umbelliferone from saliva were 81.4 +/- 0. 9% and 106.3 +/- 10.2%, respectively. The recovery of UCN-01 from plasma was 97.9 +/- 7.1% and for umbelliferone was 103.3 +/- 2.3%. This method is suitable for quantifying UCN-01 in patient samples and further characterizing the clinical pharmacology of this compound. Published in 2000 by John Wiley & Sons, Ltd.