Phase I Trial of the Human Double Minute 2 Inhibitor MK-8242 in Patients With Advanced Solid Tumors.

Purpose To evaluate MK-8242 in patients with wild-type TP53 advanced solid tumors. Patients and Methods MK-8242 was administered orally twice a day on days 1 to 7 in 21-day cycles. The recommended phase II dose (RP2D) was determined on the basis of safety, tolerability, pharmacokinetics (PK), and by mRNA expression of the p53 target gene pleckstrin homology-like domain, family A, member 3 ( PHLDA3). Other objectives were to characterize the PK/pharmacodynamic (PD) relationship, correlate biomarkers with response, and assess tumor response. Results Forty-seven patients received MK-8242 across eight doses that ranged from 60 to 500 mg. Initially, six patients developed dose-limiting toxicities (DLTs): grade (G) 2 nausea at 120 mg; G3 fatigue at 250 mg; G2 nausea and G4 thrombocytopenia at 350 mg; and G3 vomiting and G3 diarrhea at 500 mg. DLT criteria were revised to permit management of GI toxicities. Dosing was resumed at 400 mg, and four additional DLTs were observed: G4 neutropenia and G4 thrombocytopenia at 400 mg and G4 thrombocytopenia (two patients) at 500 mg. Other drug-related G3 and G4 events included anemia, leukopenia, pancytopenia, nausea, hyperbilirubinemia, hypophosphatemia, and anorexia. On the basis of safety, tolerability, PK, and PD, the RP2D was established at 400 mg (15 evaluable patients experienced two DLTs). PK for 400 mg (day 7) showed Cmax 3.07 µM, Tmax 3.0 hours, t1/2 (half-life) 6.6 hours, CL/F (apparent clearance) 28.9 L/h, and Vd/F (apparent volume) 274 L. Blood PHLDA3 mRNA expression correlated with drug exposure ( R2 = 0.68; P < .001). In 41 patients with postbaseline scans, three patients with liposarcoma achieved a partial response (at 250, 400, and 500 mg), 31 showed stable disease, and eight had progressive disease. In total, 27 patients with liposarcoma had a median progression-free survival of 237 days. Conclusion At the RP2D of 400 mg twice a day, MK-8242 activated the p53 pathway with an acceptable safety and tolerability profile. The observed clinical activity (partial response and prolonged progression-free survival) provides an impetus for further study of HDM2 inhibitors in liposarcoma.

Discovery and Clinical Evaluation of MK-8150, A Novel Nitric Oxide Donor With a Unique Mechanism of Nitric Oxide Release.

BACKGROUND: Nitric oxide donors are widely used to treat cardiovascular disease, but their major limitation is the development of tolerance, a multifactorial process to which the in vivo release of nitric oxide is thought to contribute. Here we describe the preclinical and clinical results of a translational drug development effort to create a next-generation nitric oxide donor with improved pharmacokinetic properties and a unique mechanism of nitric oxide release through CYP3A4 metabolism that was designed to circumvent the development of tolerance. METHODS AND RESULTS: Single- and multiple-dose studies in telemetered dogs showed that MK-8150 induced robust blood-pressure lowering that was sustained over 14 days. The molecule was safe and well tolerated in humans, and single doses reduced systolic blood pressure by 5 to 20 mm Hg in hypertensive patients. Multiple-dose studies in hypertensive patients showed that the blood-pressure-lowering effect diminished after 10 days, and 28-day studies showed that the hemodynamic effects were completely lost by day 28, even when the dose of MK-8150 was increased during the dosing period. CONCLUSIONS: The novel nitric oxide donor MK-8150 induced significant blood-pressure lowering in dogs and humans for up to 14 days. However, despite a unique mechanism of nitric oxide release mediated by CYP3A4 metabolism, tolerance developed over 28 days, suggesting that tolerance to nitric oxide donors is multifactorial and cannot be overcome solely through altered in vivo release of nitric oxide. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01590810 and NCT01656408.

A phase I trial of the human double minute 2 inhibitor (MK-8242) in patients with refractory/recurrent acute myelogenous leukemia (AML).

OBJECTIVE: Evaluate safety/tolerability/efficacy of MK-8242 in subjects with refractory/recurrent AML. METHODS: MK-8242 was dosed p.o. QD (30-250mg) or BID (120-250mg) for 7on/7off in 28-day cycle. Dosing was modified to 7on/14off, in 21-day cycle (210 or 300mg BID). RESULTS: 26 subjects enrolled (24 evaluable for response); 5/26 discontinued due to AEs. There were 7 deaths; 1 (fungal pneumonia due to marrow aplasia) possibly drug-related. With the 7on/7off regimen, 2 subjects had DLTs in the 250mg BID group (both bone marrow failure and prolonged cytopenia). With the 7on/14off, no DLTs were observed in 210mg BID or 300mg BID (doses>300mg not tested). Best responses were: 1/24 PR (11 weeks;120mg QD, 7on/7off); 1/24 CRi (2 weeks;210mg BID, 7on/14off); 1/24 morphologic leukemia-free state (4 weeks; 250mg BID, 7on/7off). PK on Day7 at 210mg BID revealed AUC0-12h 8.7µM·h,Cmax 1.5µM (n=5,Tmax, 2-6h),T1/2 7.9h, CLss/F 28.8L/h, and Vss/F 317L. CONCLUSIONS: The 7on/14off regimen showed a more favorable safety profile; no MTD was established. Efficacy was seen using both regimens providing impetus for further study of HDM2 inhibitors in subjects with AML.

Mitochondrial calcium transport is regulated by P2Y1- and P2Y2-like mitochondrial receptors.

Ischemia-reperfusion injury remains a major clinical problem in liver transplantation. One contributing factor is mitochondrial calcium (mCa(2+)) overload, which triggers apoptosis; calcium also regulates mitochondrial respiration and adenosine 5'-triphosphate (ATP) production. Recently, we reported the presence of purinergic P2Y(1)- and P2Y(2)-like receptor proteins in mitochondrial membranes. Herein, we present an evaluation of the functional characteristics of these receptors. In experiments with isolated mitochondria, specific P2Y(1) and P2Y(2) receptors ligands: 2-methylthio-adenosine 5'-diphosphate (2meSADP) and uridine 5'-triphosphate (UTP), respectively, were used, and mitochondrial calcium uptake was measured. 2meSADP and UTP had a maximum effect at concentrations in the range of the known P2Y(1) and P2Y(2) receptors. The P2Y inhibitor phosphate-6-azophenyl-2',4'-disulfonate (PPADS) blocked the effects of both ligands. The phospholipase C (PLC) antagonist U73122 inhibited the effect of both ligands while its inactive analog U73343 had no effect. These data strongly support the hypothesis that mitochondrial Ca(2+) uptake is regulated in part by adenine nucleotides via a P2Y-like receptor mechanism that involves mitochondrial PLC activation.

Inhibition of phospholipase C attenuates liver mitochondrial calcium overload following cold ischemia.

BACKGROUND: Graft failure due to cold ischemia (CI) injury remains a significant problem during liver transplantation. During CI, the consumption of ATP and the increase in cellular Ca concentration may result in mitochondrial Ca (mCa) overload through the mCa uniporter, which can ultimately lead to apoptosis and graft nonfunction. We recently identified phospholipase C-dl (PLC-dl) as a novel regulator of mCa uptake in the liver, and now extend those studies to examine the role of mitochondrial PLC in liver CI injury. METHODS: Rat livers were perfused with University of Wisconsin (UW) solution. Half was homogenized immediately; the other half was cold-stored for 24 hr in UW. Mitochondria were extracted by differential centrifugation and incubated in buffer containing ATP and 0.1 or 0.2 microM Ca. The selective PLC inhibitor, U-73122, was added to determine the effects of PLC inhibition on mCa uptake following CI. Western blots and densitometry quantified mitochondrial PLC expression. Mito Tracker Red fluorescence microscopy was used to verify mitochondrial transmembrane potential. RESULTS: Twenty-four hour CI caused a significant increase in mCa uptake (P<0.001), and increasing extramitochondrial Ca potentiated this effect. The PLC inhibitor, U-73122, decreased mCa uptake in nonischemic mitochondria (P<0.001), and had a greater effect on CI mitochondria (P<0.001). Mitochondrial PLC-dl expression increased 175+/-75% following CI (P<0.05). CONCLUSIONS: These data demonstrate that PLC-dl is essential for mCa uptake following CI, and that the PLC pathway may be sensitized by CI. The CI-induced increase in mitochondrial PLC-delta1 expression represents a novel mechanism whereby mCa uptake can increase independently of cytosolic conditions.

Modulation of mitochondrial calcium management attenuates hepatic warm ischemia-reperfusion injury.

Hepatic warm ischemia and reperfusion (IR) injury occurs in many clinical situations and has an important link to subsequent hepatic failure. The pathogenesis of this injury involves numerous pathways, including mitochondrial-associated apoptosis. We studied the effect of mitochondrial calcium uptake inhibition on hepatic IR injury using the specific mitochondrial calcium uptake inhibitor, ruthenium red (RR). Rats were subjected to 1 hour of 70% warm hepatic ischemia following RR pretreatment or vehicle injection. Sham-operated animals served as controls. Analysis was performed at 15 minutes, 1 hour, 3 hours, or 6 hours after reperfusion. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations were determined. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed to assess apoptosis, and hepatocellular necrosis was semiquantitated from hematoxylin and eosin-stained tissue sections. RR pretreatment significantly decreased both AST and ALT serum levels after 6 hours of reperfusion (AST: 1,556 +/- 181 U/L vs. 597 +/- 121 U/L, P = 0.005; ALT: 1,118 +/- 187 U/L vs. 294 +/- 39 U/L, P = 0.005). Apoptosis was observed within 15 minutes of reperfusion in vehicle-pretreated animals and peaked after 3 hours of reperfusion (98 +/- 21 cells/high-power field [hpf]). Apoptosis was inhibited at all time points by RR pretreatment. Histologic evidence of necrosis was not observed prior to 3 hours of reperfusion (23% +/- 4%), and maximal necrosis was observed after 6 hours of reperfusion (26% +/- 1% percent area). RR pretreatment significantly decreased the necrotic percent area at both the 3-hour and the 6-hour time points (4.2% +/- 2%; 3.7% +/- 1%, respectively). Hepatic IR injury resulted in both apoptotic and necrotic cell death, which were attenuated by RR pretreatment. In conclusion, these observations implicate mitochondrial calcium uptake in the pathogenesis of hepatic IR injury.

Survival and functional quality of life after resection for hepatic carcinoid metastasis.

Retrospective studies suggest that resection improves 5-year survival for patients with hepatic carcinoid metastasis (HCM). The purpose of our study was to describe clinical outcomes following resection for HCM, including survival and longitudinal functional quality of life (QOL). We reviewed the records of patients undergoing resection for HCM from 1980 to 2001 at our institution. Outcome measures included tumor symptoms, biochemical tumor markers, functional QOL through Karnofsky functional scores, and survival. Thirteen patients underwent a total of 17 resections. Overall 5-year survival was 85%. Eleven patients were symptomatic, including eight with classic carcinoid syndrome. Nine experienced complete relief of symptoms and two had incomplete relief for 30 +/- 12 months. Eight patients had elevated tumor markers, and 50% of these had postoperative normalization of all tumor markers that persisted to the close of the study. For the 10 patients with longitudinal follow-up available to 54 months, significant improvement in functional QOL was observed at all follow-up time points compared to preresection functional QOL (P < 0.05). Resection of >/=90% tumor volume was significantly associated with more favorable survival and tumor marker normalization compared to resection of <90% tumor volume (P < 0.01 and P < 0.05, respectively), but trajectory of functional QOL improvement did not differ between these two groups (P=0.24). We conclude that resection for HCM is associated with significantly improved and sustained functional QOL and prolonged survival. Resection of >/=90% tumor volume is significantly associated with extended survival and normalization of tumor markers, but is not required for symptomatic or functional QOL improvement.

Mitochondrial P2Y-Like receptors link cytosolic adenosine nucleotides to mitochondrial calcium uptake.

ATP is a known extracellular ligand for cell membrane purinergic receptors. Intracellular ATP can work also as a regulatory ligand via binding sites on functional proteins. We report herein the existence of P2Y(1)-like and P2Y(2)-like receptors in hepatocyte mitochondria (mP2Y(1) and mP2Y(2)), which regulate mCa(2+) uptake though the uniporter. Mitochondrial P2Y(1) activation stimulates mCa(2+) uptake; whereas, mP2Y(2) activation inhibits mCa(2+) uptake. ATP acts preferentially on mP2Y(2) receptors, while ADP and AMP-PNP stimulate both the mP2Y(1) and mP2Y(2). PPADS inhibits ADP stimulated mP2Y(1)-mediated mCa(2+) uptake. In addition, UTP, a selective P2Y(2) agonist, strongly inhibits mCa(2+) uptake. The newly discovered presence and function of these receptors is significant because it explains increased mCa(2+) uptake in the setting of low cytosolic [ATP] and, therefore, establishes a mechanism for direct feedback in which cytosolic [ATP] governs mitochondrial ATP production through regulation of mCa(2+) uptake.

Novel role of phospholipase C-delta1: regulation of liver mitochondrial Ca2+ uptake.

Mitochondrial Ca2+ (mCa2+) handling is an important regulator of liver cell function that controls events ranging from cellular respiration and signal transduction to apoptosis. Cytosolic Ca2+ enters mitochondria through the ruthenium red-sensitive mCa2+ uniporter, but the mechanisms governing uniporter activity are unknown. Activation of many Ca2+ channels in the cell membrane requires PLC. This activation commonly occurs through phosphitidylinositol-4,5-biphosphate (PIP2) hydrolysis and the production of the second messengers inositol 1,4,5-trisphosphate [I(1,4,5)P3] and 1,2-diacylglycerol (DAG). PIP2 was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa2+ uptake through the uniporter. Western blot analysis with anti-PLC antibodies demonstrated the presence of PLC-delta1 in pure preparations of mitochondrial membranes isolated from rat liver. In addition, the selective PLC inhibitor U-73122 dose-dependently blocked mCa2+ uptake when whole mitochondria were incubated at 37 degrees C with 45Ca2+. Increasing extra mCa2+ concentration significantly stimulated mCa2+ uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mCa2+ uptake, whereas U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa2+ uptake in any experimental condition. Membrane-permeable I(1,4,5)P3 receptor antagonists 2-aminoethoxydiphenylborate and xestospongin C also inhibited mCa2+ uptake. Although extra mitochondrial I(1,4,5)P3 had no effect on mCa2+ uptake, membrane-permeable DAG analogs 1-oleoyl-2-acetyl-sn-glycerol and DAG-lactone, which inhibit PLC activity, dose-dependently inhibited mCa2+ uptake. These data indicate that PLC-delta1 exists in liver mitochondria and is involved in regulating mCa2+ uptake through the uniporter.

Long-term survival after resection for primary hepatic carcinoid tumor.

BACKGROUND: Primary hepatic carcinoid tumors (PHCTs) are extremely rare, and fewer than 50 cases have been reported in the English-language literature. We report a patient with a PHCT and review the cases in the literature. METHODS: Our patient presented with symptoms and underwent liver resection for PHCT and regional lymph node metastasis. He underwent two more liver resections over the following 7 years for recurrent PHCT. Cases reported in the English-language literature were reviewed and survival analysis was performed with the Kaplan-Meier method. The survival impacts of age, gender, tumor foci, extrahepatic metastasis, unilobar versus bilobar disease, and type of preoperative treatment were determined by means of log-rank test. RESULTS: Our patient has been free of symptoms for 14 years of follow-up and free of disease for 8 years of follow-up. Forty-eight cases of PHCT were found in the literature, and 92% of these patients underwent resection. Actuarial 5- and 10-year survival for all patients was 78% and 59%, respectively, whereas for resected patients, 10-year survival was 68%. The administration of preoperative chemotherapy, radiation therapy, or chemoembolization did not impact survival, nor did age, gender, presence of extrahepatic metastasis, number of tumors, or distribution of the tumor within the liver. CONCLUSIONS: Resection is the treatment of choice for PHCT and has provided favorable outcomes. Resection for PHCT can be performed in most patients and offers long-term survival.