Regulation of pancreatic beta-cell growth and survival by the serine/threonine protein kinase Akt1/PKBalpha.

The physiological performance of an organ depends on an interplay between changes in cellular function and organ size, determined by cell growth, proliferation and death. Nowhere is this more evident than in the endocrine pancreas, where disturbances in function or mass result in severe disease. Recently, the insulin signal-transduction pathway has been implicated in both the regulation of hormone secretion from beta cells in mammals as well as the determination of cell and organ size in Drosophila melanogaster. A prominent mediator of the actions of insulin and insulin-like growth factor 1 (IGF-1) is the 3'-phosphoinositide-dependent protein kinase Akt, also known as protein kinase B (PKB). Here we report that overexpression of active Akt1 in the mouse beta cell substantially affects compartment size and function. There was a significant increase in both beta-cell size and total islet mass, accompanied by improved glucose tolerance and complete resistance to experimental diabetes.

Signaling pathways mediating insulin-stimulated glucose transport.

A major action of insulin is to accelerate the rate of uptake of sugar into muscle and adipose cells following a meal. The biochemical mechanism by which this is accomplished has been a subject of intense experimentation, although elucidation of the pathways has remained elusive. In recent years, numerous signaling molecules and cascades modulated by insulin have been identified, although few have been definitively established as important to the metabolic actions of the hormone. An exception to this is the lipid kinase phosphatidylinositide 3'-kinase, which, under many conditions, appears absolutely required for insulin to stimulate hexose uptake into adipocytes. Akt/PKB, a serine/threonine protein kinase activated by insulin in a phosphatidylinositide 3'-kinase-dependent manner, has been implicated as a critical mediator of insulin's actions on metabolism and cell survival. Nonetheless, Akt/PKB's role in many insulin effects, particularly accelerated glucose transport, remains controversial. Interestingly, soluble analogues of ceramide antagonize both insulin's activation of Akt/PKB as well as its stimulation of glucose transport, consistent with a causal relationship between the two.

Phase I clinical and pharmacology study of 502U83 given as a 24-h continuous intravenous infusion.

502U83 is an arylmethylaminopropanediol that displays significant antitumor activity in a number of murine and human tumor-model systems. In the present phase I study, a 24-h continuous intravenous infusion of this agent was given every 28 days to patients with advanced or refractory solid tumors. In all, 46 patients received a total of 96 cycles of 502U83 at doses ranging from 25 to 8,000 mg/m2. No significant hematologic, gastrointestinal, or neurologic toxicity was observed. At doses of 2,000 mg/m2 and higher, prolongation of the corrected QT interval on ECG was evident in most patients but was completely reversible, was not associated with arrhythmias, and was not dose-limiting. Dose-limiting pulmonary toxicity characterized by acute onset of dyspnea, severe hypoxemia, interstitial pulmonary edema, and death occurred in three patients treated at the highest dose levels. Plasma concentrations of 502U83 and its metabolites were measured by high-performance liquid chromatography. The 502U83 maximal concentration (Cmax) and area under the concentration-time curve (AUC) were proportional to the delivered dose; however, substantial interpatient variability in total body clearance was noted at all dose levels. Significant conversion of 502U83 to two glucuronide metabolites was detected. Metabolite concentrations were highest in the three patients who succumbed to pulmonary toxicity, although the precise contribution of these metabolites to the observed toxic effects is unknown. In view of the unfavorable clinical profile of QTc prolongation and pulmonary toxicity produced by 502U83, further clinical development of this agent has been suspended.

Splenectomy vs. alpha interferon: a randomized study in patients with previously untreated hairy cell leukemia.

Twenty patients with previously untreated hairy cell leukemia were randomized to undergo either splenectomy or to receive interferon alfa-N1, a highly purified natural alpha interferon, as primary therapy. A response in the peripheral blood elements to a hemoglobin greater than 110 gm/l, a granulocyte count greater than 1 x 10(9)/l, and a platelet count greater than 100 x 10(9)/l (Catovsky criteria) was noted in all ten patients receiving alpha interferon but in only three of the patients undergoing splenectomy (P = less than .01). Median time to response was longer in the ten interferon patients (153 days) than in the three splenectomy responders (20 days). Median time to treatment failure was significantly greater in the alpha interferon patients (greater than 18 months) than in the splenectomy patients (less than 1 month). Survival was no different since patients relapsing following splenectomy subsequently responded to alpha interferon. A significant decrease in leukemic bone marrow infiltration was observed in seven of ten patients receiving alpha interferon and in none of the patients undergoing splenectomy. Side effects, primarily infections, were more frequent in patients receiving interferon. Alpha interferon is preferable to splenectomy as initial treatment for hairy cell leukemia.

A phase II study of crisnatol mesylate in patients with ovarian carcinoma.

Fourteen patients with advanced ovarian cancer received a 72 hour infusion of a new DNA intercalator, crisnatol mesylate, administered intravenously. There was no evidence of antitumor efficacy. A syndrome of nausea and vomiting associated with vertigo, dizziness and ataxia was observed in nearly all patients. Two of the patients developed severe CNS toxicity manifested in one by a grand-mal seizure and in the other by peripheral neuropathy. Further explorations into the potential efficacy of crisnatol mesylate administered intraperitoneally are underway.

A randomized comparison of two doses of human lymphoblastoid interferon-alpha in hairy cell leukemia. Wellcome HCL Study Group.

One hundred thirty-eight patients with hairy cell leukemia were randomized to receive either a dose of 2.0 megaunits (MU)/m2 or a 10-fold lower dose of 0.2 MU/m2 of a highly purified natural alpha-interferon, administered daily for 28 days followed by a three times a week schedule. Ninety-seven of these patients had previously undergone splenectomy, but otherwise none of the patients had received prior therapy for their leukemia. The two doses were comparable in their effect on improving the neutrophil and platelet count, whereas the higher dose had a greater beneficial effect on the hemoglobin level and a greater antileukemic effect on the marrow. Acute toxicity in the form of a flu-like syndrome, neurologic side effects, neutropenia, and the need for platelet transfusions was observed less frequently in the low-dose group, as was the chronic fatigue syndrome. No neutralizing antibody activity was seen in the sera from 61 patients examined. Because of its beneficial effect on the neutrophil and platelet count and a lower degree of toxicity (ie, a superior therapeutic/toxicity ratio), the low dose is recommended as initial therapy in patients with hairy cell leukemia. This therapy may be followed by dose escalation once clinical improvement is observed.

2-[(arylmethyl) amino]-2-methyl-1,3-propanediol DNA intercalators. An examination of the effects of aromatic ring variation on antitumor activity and DNA binding.

The effects of variation of aromatic ring size, shape, and side-chain position on antitumor activity and DNA binding in a series of carbocyclic 2-[(arylmethyl)amino]-2-methyl-1,3-propanediols (AMAPs) were examined. In general, the interaction of AMAPs with DNA increases as the intercalating ring system grows in area, with three distinct binding levels evident. Isomers from a specific ring system appear to bind DNA similarly. DNA binding is not the sole criterion for antitumor activity for the AMAPs studied; the magnitude of the delta Tm does not correlate with the antitumor activity observed. Significant in vivo P388 activity was seen for AMAP congeners from several tetracyclic ring systems. However, isomers from each of the specific ring systems produced a wide range of in vivo P388 activity. Thus, AMAP antitumor activity is not a function of the ring system per se, but rather appears to be related to the shape of the specific molecule. Three AMAP congeners (crisnatol (770U82, 773U82, and 502U83) are currently in clinical trials.

Vinblastine fails to improve response of renal cancer to interferon alfa-n1: high response rate in patients with pulmonary metastases.

One hundred sixty-five patients were randomized to receive either interferon alfa-n1 (Wellferon; Burroughs Wellcome Co, Research Triangle Park, NC) alone or with vinblastine. An initial six-cycle induction treatment consisted of interferon given at daily doses of 3, 5, 20, 20, and 20 x 10(6) U/m2 every 2 weeks. Vinblastine at a dose of 10 mg/m2 (later decreased to 5 mg/m2) was given on day 1 of alternate cycles. Toxicities were generally well tolerated. The overall response rate was 10% with no significant difference between treatment arms. Survival was also not significantly different for the arms. A small subset of patients (16) with metastases only to the lungs had a high complete response (CR) and partial response (PR) rate of 44%. Responses were durable, and overall survival of this group was much better than that of the other patients.

Crisnatol mesylate: phase I dose escalation by extending infusion duration.

Crisnatol mesylate is a rationally designed cytotoxic arylmethylamino-propanediol with broad spectrum cytotoxic activity. A phase I study with an unconventional escalation scheme was developed using a constant drug infusion rate (mg/m2/hr) and prolonging the infusion duration from 6 to 96 hours. Sixty-five patients received crisnatol at doses from 18 mg/m2 in 6 hrs to 3400 mg/m2 in 72 hours. The dose-limiting toxicity in two of five patients at 2700 mg/m2 and two of three patients at 3400 mg/m2 was neurologic and consisted of a syndrome of confusion, agitation, and disorientation. Phlebitis mandated the use of a central line. The mean terminal phase half-life (T1/2 beta) was 3.3 hours with a total body clearance (CL) of 22.8 L/hr/m2 and a volume of distribution (Vdss) of 53 L/m2. The median steady-state peak plasma concentration (Css) at 2700 mg/m2/72 hours was 2.7 micrograms/ml and at 3400 mg/m2/72 hours was 3.8 micrograms/ml. No responses were seen. The maximum tolerated dose (MTD) on this schedule is 2700 mg/m2/72 hours in patients with no liver disease and good performance status.

(1-Pyrenylmethyl)amino alcohols, a new class of antitumor DNA intercalators. Discovery and initial amine side chain structure-activity studies.

In the series of 1-pyrenylmethylamines studied in this work the relationships among structure, interaction with DNA, and murine antitumor activity were examined. Binding studies show that all of these 1-pyrenylmethylamine derivatives bind to some extent to DNA by intercalation. The presence of additional basic amine groups in the side chain enhances DNA binding due to electrostatic interactions. Those compounds containing only a single basic benzylic amine bind similarly to DNA. Only the presence of bulky side chains appears to decrease the DNA interactions in the compounds examined. Although antitumor activity is seen for (1-pyrenylmethyl)amino alcohols, useful antitumor activity in the series is limited to those congeners bearing the 2-amino-1,3-propanediol-type side chain. These derivatives bind moderately to DNA. DNA binding is a necessary but not sufficient criterion for antitumor activity in the series. In addition, the strength of DNA binding does not correlate with the antitumor activity in the group of active compounds. Three related 2-[(arylmethyl)amino]-1,3-propanediol derivatives (AMAPs) [crisnatol (770U82), 773U82, and 502U83] are currently in clinical trials as potential antitumor agents.

Evaluation of arylmethylaminopropanediols by a novel in vitro pharmacodynamic assay: correlation with antitumor activity in vivo.

The pharmacodynamics of a new series of antitumor DNA intercalators, known as arylmethylaminopropanediols (AMAPs), has been evaluated in vitro against adherent (MCF-7 human breast cancer) and nonadherent (P388 murine leukemia) cell lines. Previous work had shown that the in vitro antitumor activity of the model AMAP crisnatol was a function of exposure (Cn x T), rather than concentration alone. A unique exposure parameter, the minimum C x T, was proposed as an end point for antitumor activity in cell culture. Comparison of crisnatol to several established agents by the minimum C x T versus the standard concentration producing 10% survival indicated that these end points were not equivalent. The current work examined the validity of the pharmacodynamic approach using AMAP isomers from three different ring systems that were known to exhibit a spectrum of activity against the P388 tumor in vivo. The results indicated that antiproliferative, but not cytotoxic, activity of AMAPs in the pharmacodynamic assay correlated with their differential activity in vivo, expressed as percentage of increase in life span. In contrast, the concentration producing 10% survival either at 1 h or after continuous exposure did not show a similar correlation. The pharmacodynamic assay also revealed that certain AMAPs, while equipotent by concentration alone, required significantly less time and therefore less overall exposure for efficacy. Finally, the activity of AMAP isomers in P388 cells differed from that in MCF-7 cells, which may indicate AMAP selectivity for certain tumor types. Since AMAP action was a function of exposure, drug effects on cellular targets could likewise depend on exposure rather than concentration. These findings emphasize the importance of relating drug mechanisms to the pharmacodynamics of anticancer agents.

Phase I and clinical pharmacology trial of crisnatol (BWA770U mesylate) using a monthly single-dose schedule.

Crisnatol is a novel lipophilic arylmethylaminopropanediol with significant antineoplastic activity in a variety of murine and human tumor models which functions as a DNA intercalator. In this Phase I trial, a 6-h infusion of the drug was administered i.v. in 700 to 1500 ml of 5% dextrose in water every 28 days. Eighty-five courses at doses of 7.5 to 516 mg/m2 were administered to 43 patients with refractory solid tumors. Reversible neurological toxicity was dose limiting at 516 mg/m2 and was manifested as somnolence, dizziness, blurred vision, unsteady gait, and alpha-slowing on electroencephalogram at the end of infusion. All neurological signs and symptoms were reversible. No hematological toxicity was observed. Other toxicities included phlebitis, mild to moderate nausea and vomiting, reversible sinus node arrest in one patient, and hypertension. Crisnatol plasma concentrations were determined by high-pressure liquid chromatography. After infusion, plasma concentrations declined biexponentially with a terminal t1/2 of 2.9 h. Using a two-compartment model, the mean apparent volume of distribution at steady state and total-body clearance were 58.8 liters/m2 and 18.3 liters/h/m2, respectively, indicative of extensive tissue distribution and rapid hepatic clearance. Peak plasma levels occurred at the end of infusion and correlated with the onset of neurological toxicity. The recommended Phase II dose for this schedule is 388 mg/m2.

High-dose lymphoblastoid interferon in advanced renal cell carcinoma: an Eastern Cooperative Oncology Group Study.

This report describes a phase II Eastern Cooperative Oncology Group (ECOG) study of high-dose lymphoblastoid interferon (IFN alpha-n1) in 39 patients with measurable, advanced renal cell carcinoma. The original treatment plan was 30 X 10(6) units/m2 (30 mU) of IFN alpha-n1 im daily X 10 days; treatments were repeated every 21 days. This dose and schedule proved intolerable, with none of seven patients able to complete greater than 10 days of therapy because of fever, lassitude, hepatic dysfunction, and myelosuppression. Patients were subsequently treated with the following regimen: 3 mU/m2 on Day 1; 5 mU/m2 on Day 2; 10 mU/m2 on Day 3; and 20 mU/m2 on Days 4-10. Thirty-three new patients received this regimen and two patients received this schedule after having received 30 mU/m2. Using this second regimen, 30% of the patients were able to complete two cycles of treatment without dose reduction or interruption. Five patients (13%) had partial response (ECOG criteria) of measurable tumor. Median time to response was 140 days. Responses were documented in lung metastases in four patients and in a pelvic soft tissue mass in the fifth. Response rates and survival times are similar to those seen in prior ECOG phase II trials in advanced renal cell carcinoma. Eight of 39 patients are still alive greater than 2 years after beginning therapy. Only two of these patients had had an objective response, however. Such prolonged survival is more frequent than has been seen in previous ECOG studies. The relative contribution of patient selection and interferon therapy to this survival is uncertain.

In vivo and in vitro antitumor activity expressed by cells of concomitantly immune mice.

Growth of a primary tumor is often accompanied by the development of resistance to subsequent challenge implants of the same tumor, i.e., concomitant immunity. Using the P815 mastocytoma tumor, the kinetics of concomitant immunity was found to be governed by duration of exposure to the tumor and tumor mass. By implanting small "challenges" prior to the immunizing tumor, resistance to the growth of existing tumor foci was demonstrated. Winn-type assays revealed that antitumor activity was present in cell populations from the peritoneal exudate and lymph node draining the tumor. Peritoneal exudate cells, when infused systemically, were also able to confer protection against P815 mastocytoma challenge, suggesting their role as mediators of concomitant immunity. The 51Cr release technique indicated that cytolytic activity in lymph node cells, peritoneal exudate cells, and the spleen was present over a time course parallel to the kinetics of in vivo challenge. The peritoneal resident cell population was only slightly active; thus, effectors accumulated in the inflammatory exudate. Removal of specific subsets of cells from effector populations with antibody to surface markers and complement produced similar effects on both Winn and cytolytic assays. Anti-Thy 1.2 ablated measurable activity. It was substantially but not completely reduced by anti-Lyt 1.1 and only to a small degree by anti-Lyt 2.1.

In vitro cytotoxicity expressed by cells active against established tumors in vivo.

Although antitumor activity by host cells has been documented in vivo and in vitro, the cellular relationships between these two classes of studies are not clear. Cells capable of causing the regression of solid tumors are generated in lymph nodes draining sites of immunization with Corynebacterium parvum:irradiated P815 mastocytoma admixtures. These cells are active in a 51Cr release assay at a low effector:target ratio producing a characteristic low level of specific 51Cr release which required 24 hr for optimal development. The activity is immunologically specific for the immunizing tumor and is mediated by nonadherent, rapidly dividing (vinblastine-sensitive) cells. They are absent in thymectomized animals and susceptible to alpha-Thy 1.2 antibody and complement. They are present in peritoneal exudates, consistent with the systemic resistance demonstrable in the animal model. The properties and development kinetics of effector cells measured by 51Cr release correlate closely with those of cells showing in vivo activity, supporting the identity of the two populations.

Cell mediated immunity to Corynebacterium parvum and its role in tumor control.

The injection of C. parvum provokes a transient cell-mediated immune response to its own bacterial antigens. During this period a subcutaneous challenge dose results in the appearance of a characteristic delayed type hypersensitivity (DTH) skin reaction. If a suspension of syngeneic tumor cells is included in this challenge, they are destroyed in immune but not control animals. Similarly, solid tumors of limited size can be made to regress under these conditions. Adoptive transfer studies revealed that, in keeping with other DTH reactions, this effect was mediated by a population of rapidly dividing, theta-bearing lymphocytes. The ramifications of this response with respect to antitumor therapy are discussed.

Subversion of host defense mechanisms by murine tumors. I. A circulating factor that suppresses macrophage-mediated resistance to infection.

The subcutaneous injection of cells of any one of five unselected murine tumors resulted very rapidly in the liberation into the circulation of a small molecular weight factor that severely impaired the capacity of the host to resist experimental infection with Listeria monocytogenes and Yersinia enterocolitica. It was found that the factor appeared in blood within 8 h of injecting tumor cells subcutaneously. That it possessed potent physiological activity was evidenced by the demonstration that an infusion of as little as 0.015 ml of tumor-bearer serum strikingly suppressed the capacity of normal recipients to resist bacterial infection. It was reasoned on the basis of the knowledge that the only cells in mice with the capacity to destroy Listeria are macrophages, that suppression of antibacterial resistance was caused by the ability of the tumor-suppressor factor to interfere, either directly or indirectly, with the antibacterial functions of these mononuclear phagocytic cells. The results are consistent with the hypothesis that at least some malignant neoplastic cells are naturally selected to avoid destruction by native and acquired antitumor mechanisms of mononuclear phagocytes.