Richard M. Lush

Phase I trial of continuous infusion flavopiridol, a novel cyclin-dependent kinase inhibitor, in patients with refractory neoplasms.

PURPOSE We conducted a phase I trial of the cyclin-dependent kinase inhibitor, flavopiridol (National Service Center [NSC] 649890), to determine the maximum-tolerated dose (MTD), toxicity profile, and pharmacology of flavopiridol given as a 72-hour infusion every 2 weeks. PATIENTS AND METHODS Seventy-six patients with refractory malignancies with prior disease progression were treated with flavopiridol, with first-cycle pharmacokinetic sampling. RESULTS Forty-nine patients defined our first MTD, 50 mg/m2/d x 3 with dose-limiting toxicity (DLT) of secretory diarrhea at 62.5 mg/kg/d x 3. Subsequent patients received antidiarrheal prophylaxis (ADP) to define a second MTD, 78 mg/m2/d x 3 with DLT of hypotension at 98 mg/m2/d x 3. Other toxicities included a proinflammatory syndrome with alterations in acute-phase reactants, particularly at doses >50 mg/ m2/d x 3, which in some patients prevented chronic therapy every 2 weeks. In some patients, ADP was not successful, requiring dose-deescalation. Although approximately 70% of patients displayed predictable flavopiridol pharmacology, we observed unexpected interpatient variability and postinfusion peaks in approximately 30% of cases. At the two MTDs, we achieved a mean plasma flavopiridol concentration of 271 nM (50 mg/m2/d x 3) and 344 nM (78 mg/m2/d x 3), respectively. One partial response in a patient with renal cancer and minor responses (n=3) in patients with non-Hodgkins lymphoma, colon, and renal cancer occurred. CONCLUSION The MTD of infusional flavopiridol is 50 mg/m2/d x 3 with dose-limiting secretory diarrhea at 62.5 mg/m2/d x 3. With ADP, 78 mg/m2/d x 3 was the MTD, with dose-limiting hypotension at 98 mg/m2/d x 3. Based on chronic tolerability, 50 mg/m2/d x 3 is the recommended phase II dose without ADP. Antitumor effect was observed in certain patients with renal, prostate, and colon cancer, and non-Hodgkins lymphoma. Concentrations of flavopiridol (200 to 400 nM) needed for cyclin-dependent kinase inhibition in preclinical models were achieved safely.

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 Histone Deacetylase Inhibition by Valproic Acid Followed by the Topoisomerase II Inhibitor Epirubicin in Advanced Solid Tumors: A Clinical and Translational Study

PURPOSE To determine the safety, toxicity, and maximum-tolerated dose of a sequence-specific combination of the histone deacetylase inhibitor (HDACi), valproic acid (VPA), and epirubicin in solid tumor malignancies and to define the clinical feasibility of VPA as an HDACi. PATIENTS AND METHODS Patients were treated with increasing doses of VPA (days 1 through 3) followed by epirubicin (day 3) in 3-week cycles. The study evaluated pharmacokinetic and pharmacodynamic end points, toxicities, and tumor response. RESULTS Forty-eight patients were enrolled, and 44 received at least one cycle of therapy. Patients (median age, 54 years; range, 39 to 78 years) received the following doses of VPA: 15, 30, 45, 60, 75, 90, 100, 120, 140, and 160 mg/kg/d. Dose-limiting toxicities were somnolence (n = 1), confusion (n = 3), and febrile neutropenia (n = 1). No exacerbation of epirubicin-related toxicities was observed. Partial responses were seen across different tumor types in nine patients (22%), and stable disease/minor responses were seen in 16 patients (39%), despite a median number of three prior regimens (range, zero to 10 prior regimens). Patients received a median number of four treatment cycles (range, one to 10 cycles), and treatment was stopped after reaching maximal epirubicin doses rather than progression in 13 (32%) of 41 patients patients. Total and free VPA plasma concentrations increased linearly with dose and correlated with histone acetylation in peripheral-blood mononuclear cells. CONCLUSION The maximum-tolerated dose and recommended phase II dose was VPA 140 mg/kg/d for 48 hours followed by epirubicin 100 mg/m2. Sustained plasma concentrations of VPA exceeding those required for in vitro synergy were achieved with acceptable toxicity. Noteworthy antitumor activity was observed in heavily pretreated patients and historically anthracycline-resistant tumors.

A phase II study of 5-aza-2'deoxycytidine (decitabine) in hormone independent metastatic (D2) prostate cancer.

Aims and Background Decitabine (5-aza-2′-deoxycytidine) is an S-phase-specific pyrimidine analog with hypomethylation properties. In laboratory models of prostate cancer (PC-3 and DU-145), decitabine induces cellular differentiation and enhanced expression of genes involved in tumor suppression, immunogenicity, and programmed cell death. Methods We conducted a phase II study of decitabine in 14 men with progressive, metastatic prostate cancer recurrent after total androgen blockade and flutamide withdrawal. Decitabine was administered at a dose of 75 mg/m2/dose IV as a 1 hour infusion every 8 hours for three doses. Cycles of therapy were repeated every 5 to 8 weeks to allow for resolution of toxicity. Results Two of 12 patients evaluable for response had stable disease with a time to progression of more than 10 weeks. This activity was seen in 2 of 3 African-American patients. Toxicity was similar to previously reported experience. No significant changes in urinary concentrations of the angiogenic factor bFGF, a potential biomarker of tumor activity, were identified over time in 7 unselected patients with progressive disease. Conclusions We conclude that decitabine is a well tolerated regimen with modest clinical activity against hormone-independent prostate cancer. Further investigations in patients of African-American origin may be warranted.

Phase II Study of the Mitogen-Activated Protein Kinase 1/2 Inhibitor Selumetinib in Patients With Advanced Hepatocellular Carcinoma

PURPOSE Hepatocellular carcinoma (HCC) is a common and deadly malignancy with few systemic therapy options. The RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-related kinase (ERK) pathway is activated in approximately 50% to 60% of HCCs and represents a potential target for therapy. Selumetinib is an orally available inhibitor of MEK tyrosine kinase activity. PATIENTS AND METHODS Patients with locally advanced or metastatic HCC who had not been treated with prior systemic therapy were enrolled on to the study. Patients were treated with selumetinib at its recommended phase II dose of 100 mg twice per day continuously. Cycle length was 21 days. Imaging was performed every two cycles. Biopsies were obtained at baseline and at steady-state in a subset of patients, and pharmacokinetic (PK) analysis was performed on all patients. Results Nineteen patients were enrolled, 17 of whom were evaluable for response. Most (82%) had Child-Pugh A cirrhosis. Toxicity was in line with other studies of selumetinib in noncirrhotic patients. PK parameters were also comparable to those in noncirrhotic patients. No radiographic response was observed in this group, and the study was stopped at the interim analysis. Of 11 patients with elevated α-fetoprotein, three (27%) had decreases of 50% or more. Median time to progression was 8 weeks. Inhibition of ERK phosphorylation was demonstrated by Western blotting. CONCLUSION In this study of selumetinib for patients with HCC, no radiographic responses were seen and time to progression was short, which suggests minimal single-agent activity despite evidence of suppression of target activation.

Apoptosis: Its Role in the Development of Malignancies and its Potential as a Novel Therapeutic Target

OBJECTIVE: To review the current literature regarding the role of apoptosis in the development of malignant cells and how the induction of this pathway could be used in cancer therapy. DATA SOURCE: A MEDLINE search of basic science articles pertinent to the understanding of the normal physiologic process of apoptosis was conducted. STUDY SELECTION: Because of the rapidly growing literature regarding apoptosis, only articles describing key processes in the biology of the cell and the genetic control of apoptosis were included. DATA SYNTHESIS: Apoptosis is imperative for host survival since it discards unwanted, damaged, and atypical cells. The process is therefore implicated in the continuous regulation of development, differentiation, and homeostasis. Furthermore, apoptosis is a response to physiologic and pathologic stresses that disrupt the balanced rates of cell generation and elimination. In a disease such as cancer, there is a lack of equilibrium between the rates of cell division and cell death; agents that promote or suppress apoptosis can manipulate these rates, influencing the anomalous accumulation of neoplastic cells. Pharmacologic manipulation of apoptosis represents a novel approach in targeting malignant cells and has far-reaching implications for new directions in cancer therapy. CONCLUSIONS: Apoptosis is a highly organized physiologic mechanism of destroying injured and abnormal cells as well as maintaining homeostasis in multicellular organisms. Both the activation and inhibition of apoptosis are tightly controlled. Pharmacologic manipulation of this pathway is a novel therapeutic target in cancer therapy.

Potentiation of a Topoisomerase I Inhibitor, Karenitecin, by the Histone Deacetylase Inhibitor Valproic Acid in Melanoma: Translational and Phase I/II Clinical Trial

Purpose: The novel topoisomerase I inhibitor karenitecin (KTN) shows activity against melanoma. We examined whether histone deacetylase inhibition could potentiate the DNA strand cleavage, cytotoxicity as well as the clinical toxicity, and efficacy of KTN in melanoma. Experimental Design: Apoptosis, COMET, and xenograft experiments were carried out as described previously. A phase I/II trial of valproic acid (VPA) and KTN was conducted in patients with stage IV melanoma, with any number of prior therapies, Eastern Cooperative Oncology Group performance status 0-2, and adequate organ function. Results: VPA pretreatment potentiated KTN-induced apoptosis in multiple melanoma cell lines and in mouse A375 xenografts. VPA increased KTN-induced DNA strand breaks. In the phase I/II trial, 39 patients were entered, with 37 evaluable for toxicity and 33 evaluable for response. Somnolence was the dose-limiting toxicity. The maximum tolerated dose for VPA was 75 mg/kg/d; at maximum tolerated dose, serum VPA was ∼200 μg/mL (1.28 mmol/L). At the dose expansion cohort, 47% (7 of 15) of patients had stable disease; median overall survival and time to progression were 32.8 and 10.2 weeks, respectively. Histone hyperacetylation was observed in peripheral blood mononuclear cells at maximum tolerated dose. Conclusion: VPA potentiates KTN-induced DNA strand breaks and cytotoxicity. VPA can be combined at 75 mg/kg/d for 5 days with full-dose KTN without overlapping toxicities. In metastatic poor prognosis melanoma, this combination is associated with disease stabilization in 47% of patients. Further testing of this combination appears warranted.

The pharmacokinetics of TNP-470, a new angiogenesis inhibitor.

Study Objective. To characterize the pharmacokinetic profile of TNP‐470, a synthetic analog of fumagillin that is a potent inhibitor of angiogenesis and inhibits neovascularization in several solid tumor models.

Complete Sustained Response of a Refractory, Post-Transplantation, Large B-Cell Lymphoma to an Anti-CD22 Immunotoxin

Glossary CD19, CD20, CD21, CD22: B-cell surface markers frequently expressed on the surface of B-cell tumors. The immunotoxin used in our patient is directed against CD22. EBER-1: Epstein-Barr encoded RNA. Immunotoxin: A drug formed by chemical linkage of an antibody to a toxin. RFB4-dgA: The immunotoxin used in our patient; it consists of the murine monoclonal antibody RFB4, which is linked to the deglycosylated A-chain of ricin, a plant toxin. Vascular leak syndrome: A side effect of immunotoxin treatment that consists of nontargeted damage to endothelial cells and manifests as hypoalbuminemia and edema in its mild form. Transplant recipients have an increased risk for post-transplantation lymphoproliferative diseases associated with Epstein-Barr virus. The incidence of such diseases is 1% in recipients of kidney transplants, 2.4% in recipients of heart or heart and lung transplants, 1% in recipients of liver transplants, and 0.6% in recipients of pancreas transplants [1]. Morphologic criteria distinguish at least three types of lymphoproliferative diseases: polyclonal B-cell or plasma cell hyperplasia; polymorphic B-cell hyperplasia and lymphoma, which are usually monoclonal; and monomorphic B-cell lymphoma [2, 3]. Particularly in polyclonal forms of disease, a decrease in the intensity of immunosuppressive therapy (for example, a decrease in the cyclosporine dose) with or without antiviral treatment leads to long-term clinical improvement [2]. If decreased immunosuppression as a therapeutic strategy is not successful, patients may receive chemotherapy; these patients, however, have a poor prognosis. The optimal management of patients whose disease progresses after chemotherapy is unknown [1]. An immunotoxin is formed when an antibody directed toward a tumor cell is chemically linked with a toxin, such as ricin. The antibody targets the toxin to the neoplastic cell, where it is internalized, inhibits protein synthesis, and results in the death of tumor cells [4]. We describe a patient who developed a monomorphic large B-cell lymphoma that was refractory to decreased immunosuppression plus antiviral therapy and to four cycles of combination chemotherapy. The patient achieved a sustained complete response after three courses of treatment with RFB4-dgA, an immunotoxin directed against the CD22 B-cell surface antigen. Case Report In December 1992, a 31-year-old woman presented with biopsy-proven crescentic IgA nephropathy. Despite therapy with high-dose steroids, cyclo-phosphamide, and plasmapheresis, she developed azotemia and required hemodialysis in March 1993. In August 1993, the patient received a kidney transplant donated by her brother. The patients serologic test results were positive for varicella-zoster virus but negative for human immunodeficiency virus, cytomegalovirus, and Epstein-Barr virus. The donors test results were negative for cytomegalovirus but positive for Epstein-Barr virus. At discharge, the patients serum creatinine level was 0.9 mg/dL; at this time, the patient was receiving a regimen of prednisone, azathioprine, and cyclosporine. Two months after transplantation, herpes zoster developed on the right side of the thorax. This condition responded to discontinuation of therapy with azathioprine and acyclovir. Azathioprine treatment was restarted at a lower dose 1 month later. A right axillary mass and back pain developed 4 months after transplantation. Computed tomography of the abdomen and pelvis revealed a right para-aortic mass and a right adnexal mass that were contiguous with the graft. An excised portion of the axillary mass showed a large B-cell lymphoma [5] (categorized according to the REAL classification [revised European-American classification of lymphoid neoplasms]). Cyclosporine and azathioprine therapies were discontinued in January 1994, and treatment with prednisone (20 mg/d) and acyclovir (800 mg five times daily) was continued; despite these measures, disease was still evident. The patient received four cycles of CHOP (cyclophosphamide, doxorubicin, prednisone, and vincristine) [6]. The mass near the graft did not shrink, and the para-aortic lymphadenopathy persisted (Figure 1, arrow). The patient was referred to the National Cancer Institute in order to be considered for a clinical trial of RFB4-dgA. Figure 1. Findings in a patient with a refractory post-transplantation large B-cell lymphoma treated with an anti-CD22 immunotoxin. arrow At presentation to the National Cancer Institute, the patient reported fatigue, back and pelvic pain, and night sweats. Physical examination showed that the patient had a cushingoid appearance, but peripheral adenopathy and splenomegaly were not seen. To determine the presence of the CD22 antigen on the tumor cells, the patient underwent percutaneous biopsy of the para-aortic adenopathy. The biopsy specimen showed a malignant lymphoma with extensive necrosis (Figure 1, panel A), which was categorized as a large B-cell lymphoma according to the REAL classification [5]. A portion of the biopsy specimen was stained with a panel of monoclonal antibodies by an immunoperoxidase technique [7]. The cells were positive for B-cell antigens, including CD19, CD20, CD21, and CD22, and were negative for T-cell antigens. In situ hybridization was performed for Epstein-Barr virus using the Epstein-Barr encoded RNA (EBER1) probe [8]; U6 (an abundant RNA polymerase III transcript of cellular origin) was used to control for preservation of RNA. All of the large lymphoid cells showed a positive signal (Figure 1, panel B). These data clearly indicate the relation between the occurrence of this tumor and Epstein-Barr virus infection. In paraffin-embedded sections of the biopsy specimens (not shown), the nuclei showed positive staining for p53 tumor suppressor protein. This finding strongly suggests the presence of a mutated form of the p53 tumor suppressor gene, which has been seen in patients with lymphomas who had poor prognosis with chemotherapy [9]. The patient began treatment with the immunotoxin at the previously determined maximal tolerated dose, 19.2 mg/m2 body surface area every 192 hours by continuous infusion [10]. The immunotoxin was given in three courses at monthly intervals until July 1994. Since January 1994, the patient had continued to receive the same dose of acyclovir and prednisone (5 mg alternating with 10 mg daily) to prevent renal graft rejection and symptoms of adrenal insufficiency. No further immune manipulation was done before or during administration of the immunotoxin. The first course of immunotoxin therapy was complicated by mild to moderate vascular leak syndrome (grade II) [10]; on the third day of the first course, increasing peripheral edema, moderate weight gain, resting tachycardia, and mildly decreased urine output were seen. The patient continued to receive immunotoxin therapy and was also given furosemide and 1 unit of packed red blood cells; the signs and symptoms then resolved. Azotemia did not occur. After the first course of immunotoxin therapy, the patient was considered to have had a partial response in both para-aortic and pelvic adenopathy, measured by computed tomography of the abdomen and pelvis. No evidence suggested that human antimouse or human antiricin antibodies had developed. With the second and third courses of immunotoxin therapy, the only toxic effect was a slightly decreased serum albumin level. After the third course of immunotoxin therapy, para-aortic adenopathy markedly improved but the pelvic mass did not decrease further. Computed tomography done 12 months after immunotoxin therapy began showed no evidence of disease (compare panel C with panel D of the Figure 1). A negative gallium scan also indicated that the patient had a stable, complete response, although the avidity of the tumor for gallium had not been assessed before treatment. In October 1995, biopsy of the transplanted kidney was performed because the serum creatinine level had progressively increased from 0.9 mg/dL (the level in May 1995) to 1.8 mg/dL. The biopsy specimen showed well-preserved glomeruli, mild to moderate interstitial fibrosis, and a rich cellular infiltrate that was consistent with rejection. No evidence suggested recurrent glomerulonephritis or a lymphoma. In situ hybridization studies of the biopsy specimen showed no expression of EBER-1 RNA. The patient was treated with a high, tapering dose of prednisone for 3 weeks, and her creatinine level stabilized at 1.5 mg/dL. A specimen obtained from repeated renal biopsy in December 1995 showed resolution of the cellular infiltrate. Thirty-two months after immunotoxin treatment began, the patient remains clinically well; she is in complete remission, and the transplanted kidney has been retained. Pharmacology Using methods described elsewhere [11], we measured the immunotoxin concentration during treatment. The peak concentration achieved during the first course of immunotoxin therapy (1833 ng/mL) was greater than that seen during the second (1130 ng/mL) and third (964 ng/mL) courses. These findings correlated with evidence of less toxicity (grade I vascular leak syndrome during the second and third courses compared with grade II during the first course). This result is consistent with our previous observation of a clear relation between the maximal serum concentration of RFB4-dgA and the degree of vascular leak syndrome [10]. The basis for this nonuniform pharmacologic behavior during each course is not clear but is consistent with the interpatient and intrapatient variability encountered with this immunotoxin [10, 11]. The terminal half-lives observed (19.6 to 37.3 hours) were similar to those seen previously with this immunotoxin in patients with non-Hodgkin lymphoma who did not have circulating tumor cells. Prolonged terminal half-lives of approximately this magnitude have also correlated with development of clinical response to this immunotoxin [11]. Discussion

Effects of the antiestrogen tamoxiten on low-density lipoprotein concentrations and oxidation in postmenopausal women

Our study demonstrates that tamoxifen, when administered to postmenopausal women at a conventional dosage, reduces LDL levels and protects LDL from oxidation. The protective effect of tamoxifen against the development of breast cancer in women considered at risk is being investigated in a placebo-controlled trial sponsored by the National Institutes of Health. Whether tamoxifen also protects against the development of cardiovascular disease in this trial is also of considerable interest.