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Gliomas and meningiomas are the most common brain neoplasms affecting both humans and canines, and identifying druggable targets conserved across multiple brain cancer histologies and comparative species could broadly improve treatment outcomes. While satisfactory cure rates for low grade, non-invasive brain cancers are achievable with conventional therapies including surgery and radiation, the management of non-resectable or recurrent brain tumors remains problematic and necessitates the discovery of novel therapies that could be accelerated through a comparative approach, such as the inclusion of pet dogs with naturally-occurring brain cancers. Evidence supports procaspase-3 as a druggable brain cancer target with PAC-1, a pro-apoptotic, small molecule activator of procaspase-3 that crosses the blood-brain barrier. Procaspase-3 is frequently overexpressed in malignantly transformed tissues and provides a preferential target for inducing cancer cell apoptosis. While preliminary evidence supports procaspase-3 as a viable target in preclinical models, with PAC-1 demonstrating activity in rodent models and dogs with spontaneous brain tumors, the broader applicability of procaspase-3 as a target in human brain cancers, as well as the comparability of procaspase-3 expressions between differing species, requires further investigation. As such, a large-scale validation of procaspase-3 as a druggable target was undertaken across 651 human and canine brain tumors. Relative to normal brain tissues, procaspase-3 was overexpressed in histologically diverse cancerous brain tissues, supporting procaspase-3 as a broad and conserved therapeutic target. Additionally, procaspase-3 expressing glioma and meningioma cell lines were sensitive to the apoptotic effects of PAC-1 at biologically relevant exposures achievable in cancer patients. Importantly, the clinical relevance of procaspase-3 as a potential prognostic variable was demonstrated in human astrocytomas of variable histologic grades and associated clinical outcomes, whereby tumoral procaspase-3 expression was negatively correlated with survival; findings which suggest that PAC-1 might provide the greatest benefit for patients with the most guarded prognoses.

Gerrit Kann,1,* Junaid Owasil,1,* Karina Kuczka,2 Annette Haberl,1 Timo Wolf,1 Pavel Khaykin,1 Sebastian Harder,2 Christoph Stephan,1 Nils von Hentig1 1HIVCENTER, Medical HIV Treatment and Research Unit, Johann Wolfgang Goethe University Frankfurt, Frankfurt am Main, Germany; 2Pharmazentrum Frankfurt, Institute of Clinical Pharmacology, Johann Wolfgang Goethe University Frankfurt, Frankfurt am Main, Germany*These authors contributed equally to this workCorrespondence: Nils von HentigHIVCENTER, Medical HIV Treatment and Research Unit, Johann Wolfgang Goethe University, Theodor-Stern-Kai 7, Frankfurt am Main, 60590, GermanyTel +49-69-63017680Fax +49-69-630183442Email Hentig@em.uni-frankfurt.deBackground: In the past, protease inhibitors (PIs) and the reverse transcriptase inhibitor abacavir were identified increasing the risk for thromboembolic complications and cardiovascular events (CVE) of HIV infected patients taking a combination antiretroviral therapy (cART). Results of the previous HIV-PLA I-study lead to the assumption that platelet activation could play a substantial role in increasing CVE risks.Methods: The open label, monocentric HIV-PLA II-study investigated HIV-1-infected, therapy-naïve adults (n=45) starting with cART, consisting either of boosted PI (atazanavir, n= 6, darunavir, n=11), NNRTI (efavirenz, n=14) or integrase inhibitor (raltegravir, n=14), each plus tenofovir/emtricitabine co-medication. Main exclusion criteria were tobacco smoking, the intake of NSAIDs or abacavir or past CVE. Platelet adhesive molecule p-selectin (CD62P) and FITC anti-human Integrin α-IIb/Integrin β-3 (CD41/CD61) antibody (PAC-1) binding, monocyte CD11b/monocyte-associated CD41 expression and the endogenous thrombin potential (ETP) were assessed ex vivo-in vitro at baseline, weeks 4, 12 and 24. Therapy regimens were blinded to the investigators for laboratory and statistical analyses.Results: CD11b and ETP showed no significant changes or differences between all study groups. In contrast, the mean + SD mean fluorescence units (MFI) of CD62P and PAC-1 increased significantly in patients taking PI, indicating an enhanced potential for thrombocyte activation and aggregation.Conclusion: CD62P expression, detecting the ɑ-platelet degranulation of pro-inflammatory and pro-thrombotic factors and adhesive proteins, and PAC-1 expression, representing a marker for conformation changes of the GIIb/IIIa receptor, increased significantly in patients taking HIV protease inhibitors. The findings of this study revealed a yet unknown pathway of platelet activation, possibly contributing to the increased risk for CVE under HIV protease inhibitor containing cART.Clinical Trial Registration No.: DRKS00000288.Keywords: HIV protease inhibitors, platelets, leucocytes, PAC-1, GIIb/IIIa-receptor

Abstract Ovarian cancer is commonly diagnosed in its late stages, and new treatment modalities are needed to improve patient outcomes and survival. We have recently established the synergistic effects of combination tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) and procaspase activating compound (PAC‐1) therapies in granulosa cell tumors (GCT) of the ovary, a rare form of ovarian cancer, using a mathematical model of the effects of both drugs in a GCT cell line. Here, to understand the mechanisms of combined TRAIL and PAC‐1 therapy, study the viability of this treatment strategy, and accelerate preclinical translation, we leveraged our mathematical model in combination with population pharmacokinetics (PKs) models of both TRAIL and PAC‐1 to expand a realistic heterogeneous cohort of virtual patients and optimize treatment schedules. Using this approach, we investigated treatment responses in this virtual cohort and determined optimal therapeutic schedules based on patient‐specific PK characteristics. Our results showed that schedules with high initial doses of PAC‐1 were required for therapeutic efficacy. Further analysis of individualized regimens revealed two distinct groups of virtual patients within our cohort: one with high PAC‐1 elimination and one with normal PAC‐1 elimination. In the high elimination group, high weekly doses of both PAC‐1 and TRAIL were necessary for therapeutic efficacy; however, virtual patients in this group were predicted to have a worse prognosis when compared to those in the normal elimination group. Thus, PAC‐1 PK characteristics, particularly clearance, can be used to identify patients most likely to respond to combined PAC‐1 and TRAIL therapy. This work underlines the importance of quantitative approaches in preclinical oncology.

Abstract Background Platelets in patients with type 2 diabetes mellitus (DM2) are characterized by increased activation and aggregation, which tends to be associated with a high morbidity and mortality due to cardiovascular disease (CVD). Moreover, a large proportion of DM2 patients show an inadequate response to standard antiplatelet treatments, contributing to recurrent cardiovascular events. In our previous study, we indicated that Salvianolic acid A (SAA) presents an antiplatelet effect in healthy volunteers. However, whether it can inhibit “activated platelets” with a pathologic status has not been explored. Therefore, this study was designed to investigate the antiplatelet effect of SAA and its diabetic complication-related difference in DM2. Methods Forty patients diagnosed with DM2 from January 2018 to April 2018 were recruited. Fibrinogen-binding (PAC-1) and P-selectin (CD62p) flow cytometry reagents were measured under resting and stimulated conditions by flow cytometry, while agonist-induced platelet aggregation was conducted by light transmission aggregometry. Before all these measurements were conducted, all platelet samples were preincubated with a vehicle or SAA for 10 min. Additionally, the diabetic complication-related difference in the antiplatelet effect of SAA was further studied in enrolled patients. Results The expressions of PAC-1 and CD62p were elevated in DM2, as well as the maximal platelet aggregation. In addition, SAA decreased the expressions of PAC-1 and CD62p, which were enhanced by ADP and thrombin (all P 0.05). Conclusions The present study demonstrated that SAA can inhibit platelet activation and aggregation in patients with DM2, and the inhibition did not abate for the existence of diabetic complications.

Caspases, a group of protease enzymes (cysteine proteases), exist as inactive zymogens in the cells and execute apoptosis (programmed cell death). Caspase‐3, an executioner caspase, plays an imperative role in apoptosis and becomes a primary target for cancer treatment. A number of analogues of quinazoline, quinazolinone, indoloquinazolines, quinone, naphthoquinones, pyrroloiminoquinones, styrylquinolines, tetheredtetrahydroquinoline, fluoroquinolone, thiosemicarbazones, benzotriazole, pyrimidines, chalcone, and carbazoles have been reported till date, representing caspase‐3 mediated apoptosis for cancer therapy. Simultaneously, plant isolates, including lysicamine, podophyllotoxin, and majoranolide, have also been claimed for caspase‐3‐mediated apoptosis‐induced cytotoxicity. Procaspase‐activating compound‐1 (PAC‐1) is the first FDA approved orphan drug, and its synthetic derivative WF‐208 also showed fascinating caspase‐3 mediated anticancer activity. Till date, a large number of compounds have been reported and patented for their caspase‐3‐mediated cytotoxicity and now scientist is also focusing to introduce new compounds in market to encompass anticancer activity. [ABSTRACT FROM AUTHOR]

Summary: This study was conducted to evaluate the expression of fibrinogen receptors on platelets of Philadelphia‐negative chronic myeloproliferative neoplasm (MPN) patients. We collected blood samples from 40 consecutive MPN patients and healthy volunteers. We performed flow cytometry analysis of P‐selectin expression and integrin beta‐3, activation of glycoprotein (GP) IIb/IIIa and fibrinogen receptor exposure (PAC‐1 binding). Surprisingly, we found a very low PAC‐1 binding capacity in MPN patients; however, the expression of PAC‐1 was almost completely recovered with aspirin intake. We hypothesize that the hypercoagulable states observed in MPN patients could depend on a primarily plasma‐driven impairment of fibrin turnover and thrombin generation. [ABSTRACT FROM AUTHOR]

Gliomas and meningiomas are the most common brain neoplasms affecting both humans and canines, and identifying druggable targets conserved across multiple brain cancer histologies and comparative species could broadly improve treatment outcomes. While satisfactory cure rates for low grade, non-invasive brain cancers are achievable with conventional therapies including surgery and radiation, the management of non-resectable or recurrent brain tumors remains problematic and necessitates the discovery of novel therapies that could be accelerated through a comparative approach, such as the inclusion of pet dogs with naturally-occurring brain cancers. Evidence supports procaspase-3 as a druggable brain cancer target with PAC-1, a pro-apoptotic, small molecule activator of procaspase-3 that crosses the blood-brain barrier. Procaspase-3 is frequently overexpressed in malignantly transformed tissues and provides a preferential target for inducing cancer cell apoptosis. While preliminary evidence supports procaspase-3 as a viable target in preclinical models, with PAC-1 demonstrating activity in rodent models and dogs with spontaneous brain tumors, the broader applicability of procaspase-3 as a target in human brain cancers, as well as the comparability of procaspase-3 expressions between differing species, requires further investigation. As such, a large-scale validation of procaspase-3 as a druggable target was undertaken across 651 human and canine brain tumors. Relative to normal brain tissues, procaspase-3 was overexpressed in histologically diverse cancerous brain tissues, supporting procaspase-3 as a broad and conserved therapeutic target. Additionally, procaspase-3 expressing glioma and meningioma cell lines were sensitive to the apoptotic effects of PAC-1 at biologically relevant exposures achievable in cancer patients. Importantly, the clinical relevance of procaspase-3 as a potential prognostic variable was demonstrated in human astrocytomas of variable histologic grades and associated clinical outcomes, whereby tumoral procaspase-3 expression was negatively correlated with survival; findings which suggest that PAC-1 might provide the greatest benefit for patients with the most guarded prognoses.

Background: Platelets in patients with type 2 diabetes mellitus (DM2) are characterized by increased activation and aggregation, which tends to be associated with a high morbidity and mortality due to cardiovascular disease (CVD). Moreover, a large proportion of DM2 patients show an inadequate response to standard antiplatelet treatments, contributing to recurrent cardiovascular events. In our previous study, we indicated that Salvianolic acid A (SAA) presents an antiplatelet effect in healthy volunteers. However, whether it can inhibit "activated platelets" with a pathologic status has not been explored. Therefore, this study was designed to investigate the antiplatelet effect of SAA and its diabetic complication-related difference in DM2.Methods: Forty patients diagnosed with DM2 from January 2018 to April 2018 were recruited. Fibrinogen-binding (PAC-1) and P-selectin (CD62p) flow cytometry reagents were measured under resting and stimulated conditions by flow cytometry, while agonist-induced platelet aggregation was conducted by light transmission aggregometry. Before all these measurements were conducted, all platelet samples were preincubated with a vehicle or SAA for 10 min. Additionally, the diabetic complication-related difference in the antiplatelet effect of SAA was further studied in enrolled patients.Results: The expressions of PAC-1 and CD62p were elevated in DM2, as well as the maximal platelet aggregation. In addition, SAA decreased the expressions of PAC-1 and CD62p, which were enhanced by ADP and thrombin (all P < 0.01). It also reduced the platelet aggregation induced by ADP (P < 0.001) and thrombin (P < 0.05). Comparing the antiplatelet effect of SAA on DM2, with and without diabetic complications, no statistically significant difference was found (all P > 0.05).Conclusions: The present study demonstrated that SAA can inhibit platelet activation and aggregation in patients with DM2, and the inhibition did not abate for the existence of diabetic complications. [ABSTRACT FROM AUTHOR]

The venous thromboembolism, which may be caused by increased platelet activation, is a risk factor for tumor prognosis. We determined the platelet activation status for diagnosis and predicting postoperative prognosis of hepatocellular carcinoma. We conducted a prospective study of 191 patients diagnosed with HCC at Zhongshan Hospital from April 2016 to July 2016 as well as 99 healthy people. The platelet activation status was assessed by 2 platelet markers, PAC-1 and CD62p, using flow cytometry. The patients were treated with TACE or resection and monitored for ≥6 months. The diagnostic value of marker-positive platelets was determined by the receiver operating characteristic curve and the postoperative value were analyzed using the Kaplan-Meier method and COX regression model. All the 3 groups with high levels of marker-positive platelets were likely to be diagnosed with HCC and the PAC-1+ percentage had the best efficacy. The univariate analysis showed that the levels of PAC-1+ and CD62p+ platelets was risker factors for poor postoperative prognosis after both TACE and resection. Moreover, the multivariate analysis revealed that the level of PAC-1+ platelets was an independent risk factor for poor prognosis. The PAC-1+ percentage of platelets is a new indicator for diagnosis and predicting postoperative prognosis. • A new flow detection platform to assess platelet activation in peripheral blood from HCC patients. • Patients with HCC or poor prognosis had higher levels of PAC-1 and CD62p-positive platelets. • The PAC-1+ percentage of platelets had better efficacy to predict the outcome of HCC patients [ABSTRACT FROM AUTHOR]

High levels of low-density lipoprotein cholesterol (LDL-C) enhance platelet activation, whereas high levels of high-density lipoprotein cholesterol (HDL-C) exert a cardioprotective effect. However, the effects on platelet activation of high levels of LDL-C combined with low levels of HDL-C (HLC) have not yet been reported. We aimed to evaluate the platelet activation marker of HLC patients and investigate the antiplatelet effect of atorvastatin on this population. Forty-eight patients with high levels of LDL-C were enrolled. Among these, 23 had HLC and the other 25 had high levels of LDL-C combined with normal levels of HDL-C (HNC). A total of 35 normocholesterolemic (NOMC) volunteers were included as controls. Whole blood flow cytometry and platelet aggregation measurements were performed on all participants to detect the following platelet activation markers: CD62p (P-selectin), PAC-1 (GPIIb/IIIa), and maximal platelet aggregation (MPAG). A daily dose of 20 mg atorvastatin was administered to patients with high levels of LDL-C, and the above assessments were obtained at baseline and after 1 and 2 months of treatment. The expression of platelets CD62p and PAC-1 was increased in HNC patients compared to NOMC volunteers (P

Procaspase Activating Compound-1 (PAC-1) and its derivative WF-210 induce apoptosis in cancer cells by activating procaspase-3 to caspase-3. The aim of this study was to extend current knowledge about the mechanisms of PAC-1 and WF-210, particularly about their effects on tumor angiogenesis. PAC-1 and WF-210 restrained VEGF-induced human umbilical vascular endothelial cells (HUVECs) proliferation, invasion, and tube formation. PAC-1 and WF-210 abrogated VEGF-induced vessel sprouting from rat aortic rings and inhibited vascular formation in the Matrigel plug assay. PAC-1 and WF-210 suppressed phosphorylation of VEGFR2 and its downstream protein kinases c-Src, FAK, and AKT in both HUVECs and U-87 cells. When given to mice bearing subcutaneous or orthotopic xenograft, PAC-1 and WF-210 inhibited the tumor growth and tumor angiogenesis. Further tests showed that PAC-1 and WF-210 inhibited stemness and induced autophagy flux of U-87 cells. This study revealed mechanisms of PAC-1 and WF-210 other than inducing apoptosis, which provides additional support for their using in the clinic. [ABSTRACT FROM AUTHOR]

High levels of low-density lipoprotein cholesterol (LDL-C) enhance platelet activation, whereas high levels of high-density lipoprotein cholesterol (HDL-C) exert a cardioprotective effect. However, the effects on platelet activation of high levels of LDL-C combined with low levels of HDL-C (HLC) have not yet been reported. We aimed to evaluate the platelet activation marker of HLC patients and investigate the antiplatelet effect of atorvastatin on this population. Forty-eight patients with high levels of LDL-C were enrolled. Among these, 23 had HLC and the other 25 had high levels of LDL-C combined with normal levels of HDL-C (HNC). A total of 35 normocholesterolemic (NOMC) volunteers were included as controls. Whole blood flow cytometry and platelet aggregation measurements were performed on all participants to detect the following platelet activation markers: CD62p (P-selectin), PAC-1 (GPIIb/IIIa), and maximal platelet aggregation (MPAG). A daily dose of 20 mg atorvastatin was administered to patients with high levels of LDL-C, and the above assessments were obtained at baseline and after 1 and 2 months of treatment. The expression of platelets CD62p and PAC-1 was increased in HNC patients compared to NOMC volunteers (P

Some therapeutics for parasitic, cardiac and neurological diseases activate apoptosis. Therefore, the study of apoptotic proteins in pathogenic organisms is relevant. However, the molecular mechanism of apoptosis in unicellular organisms remain elusive, despite morphological evidence of its occurrence. In Trypanosoma cruzi, the causative agent of Chagas disease, metacaspase 3 (TcMCA3), seems to have a key role in parasite apoptosis. Accordingly, this work provides data concerning TcMCA3 regulation through its interaction with procaspase-activating compound 1 (PAC-1), a procaspase 3 activator. Indeed, PAC-1 reduced T. cruzi epimastigote viability with an IC of 14.12 µM and induced loss of mitochondrial potential and exposure of phosphatidylserine, features of the apoptotic process. Notwithstanding, those PAC-1-inducible effects were not conserved in metacyclic trypomastigotes. Moreover, PAC-1 reduced the viability of mammalian cells with a greater IC (25.70 µM) compared to T. cruzi epimastigotes, indicating distinct modes of binding between caspases and metacaspases. To shed light on the selectivity of metacaspases and caspases, we determined the structural features related to the PAC-1 binding sites in both types of proteins. These data are important for improving the understanding of the apoptosis pathway in T. cruzi so that TcMCA3 could be better targeted with future pharmaceuticals. [ABSTRACT FROM AUTHOR]

Background: Procaspase-3 (PC-3) is overexpressed in various tumor types, including gliomas. Targeted PC-3 activation combined with chemotherapy is a novel strategy for treating patients with high-grade gliomas, with promising preclinical activity. This study aimed to define safety and tolerability of procaspase-activating compound-1 (PAC-1) in combination with temozolomide (TMZ) for patients with recurrent high-grade astrocytomas., Methods: A modified-Fibonacci dose-escalation 3 + 3 design was used. PAC-1 was administered at increasing dose levels (DL; DL1 = 375 mg) on days 1-21, in combination with TMZ 150 mg/m 2 /5 days, per 28-day cycle. Dose-limiting toxicity was assessed during the first 2 cycles. Neurocognitive function (NCF) testing was conducted throughout the study., Results: Eighteen patients were enrolled (13 GBM, IDH-wild type; 2 astrocytoma, IDH-mutant, grade 3; 3 astrocytoma, IDH-mutant, grade 4). Dose escalation was discontinued after DL3 (ie, PAC-1, 625 mg) due to lack of additional funding. Grade 3 toxicity was observed in 1 patient at DL1 (elevated liver transaminases) and 1 at DL 2 (headache). Two partial responses were observed at DL1 in patients with GBM, O 6 -methylguanine-DNA methyltransferase (MGMT) promoter methylated. Two patients had stable disease, and 11 experienced progression. NCF testing did not show a clear relationship between PAC-1 dose, treatment duration, and declines in NCF., Conclusions: Combination of PAC-1 and TMZ was well tolerated up to 625 mg orally daily and TMZ orally 150 mg/m 2 /5 days per 28-day cycle. The maximum tolerated dose was not reached. Further dose escalation of PAC-1 in combination with TMZ is advised before conducting a formal prospective efficacy study in this patient population., Competing Interests: The University of Illinois has filed a patent on PAC-1 in which T.M.T., T.F.M. and P.J.H. are inventors. The University of Illinois has licensed the patents on PAC-1 to Vanquish Oncology, Inc, and P.J.H. and T.M.F. serve as consultants for Vanquish Oncology, Inc. P.J.H. also serves as Chief Scientific Officer and has equity in Vanquish Oncology, Inc. T.M.T. serves as Chief Executive Officer and has equity in Vanquish Oncology, Inc. A.Z.D served as Chief Medical Officer in Vanquish Oncology. J.S.W. was a paid consultant to Vanquish Oncology, Inc. during this study. M.H. serves on a data safety monitoring board of Parexel and Advarra and a steering committee for Novartis., (© The Author(s) 2023. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

A sensitive and simple HPLC method for simultaneous determination of PAC-1 (first procaspase-activating compound), phenol red, and permeability markers (carbamazepine and furosemide) in perfusion samples was developed and validated to assess intestinal absorption of PAC-1 using single-pass intestinal perfusion technique (SPIP) in rats. The chromatographic separation was carried out on a Kromasil C column (150 mm × 4.6 mm, 5 μm) with acetonitrile-methanol-30 mmol L phosphate buffer (pH 3.0, 25:10:65, v/v/v) as mobile phase at a flow rate of 1.0 mL min, and the wavelength of the UV detector was set at 281 nm. The calibration curves were linear in the ranges of 2.40-48.0 μg mL for PAC-1; 3.60-72.0 μg mL for carbamazepine; 3.20-64.0 μg mL for furosemide, and 4.80-96.0 μg mL for phenol red ( r > 0.999). Both the intra- and inter-day precisions (RSD%) of all analytes were less than 6.8 % at three concentration levels, while accuracy ranged from 95.4 to 104.5 %. Data obtained in all method validation studies indicated that the method was suitable for the intended purpose. The effective permeability values ( P ) considering water flux with the help of non-permeable marker phenol red was calculated to be 0.42 × 10, 0.62 × 10, 0.32 × 10 cm s for PAC-1; 0.72 × 10, 0.77 × 10, 0.52 × 10 cm s for carbamazepine; 0.20 × 10, 0.16 × 10, 0.12 × 10 cm s for furosemide in duodenum, jejunum and ileum, respectively. The P value can be increased by co-perfusion with verapamil, indicating that absorption of PAC-1 is efficiently transported by P-glycoprotein (P-gp) in the gut wall. [ABSTRACT FROM AUTHOR]

Purpose: To develop more potent procaspase-3 activator, 7 novel derivatives of PAC-1 were synthesized and evaluated. Among them, SM-1 stood out for its promising activity and good pharmacokinetics properties. The purpose of this study is to elucidate the pharmacological mechanism of SM-1 and evaluate its efficacy and toxicity in-depth.Methods: To reveal the effects of SM-1 on caspase-3 activity, both in vitro activation assay and in cells fluorometric assay were tested. The protein levels and distributions of procaspase-3 and cleaved caspase-3 were also measured by western blot and immunostaining. MTT assay, apoptosis assay and mouse xenograft model were applied to evaluate the efficacy of SM-1. Preliminary safety assessments also tested the acute toxicity and tissue distribution of SM-1.Results: Compared to PAC-1, SM-1 showed higher cytotoxicity in cancer cells. Further investigation demonstrated that SM-1 relieved zinc-mediated inhibition of procaspase-3 and activated the caspase-3 activity both in tube test and in cells. Efficacy evaluation showed SM-1-induced cell apoptosis mainly via activation of caspase-3 and reduced tumor size in mouse xenograft model. Its apoptosis induction efficacy was higher than PAC-1. The preliminary safety assessment demonstrated that the overall LD50 of SM-1 lied between 500 and 1000 mg/kg and the distribution of SM-1 in brain was low.Conclusions: We identified SM-1 as a promising antitumor candidate, which displayed enhanced procaspase-3 activating activity and potent cytotoxicity for cancer cells but low toxicity for normal cells. [ABSTRACT FROM AUTHOR]

Aim Evasion of apoptosis is a hallmark of human cancer cells. We sought to explore the potential synergistic antitumor activity and underlying mechanisms of the pro-apoptotic agent PAC-1 plus cisplatinum (Cis) in non-small cell lung cancer ( NSCLC) cell lines. Methods The adenocarcinoma cell lines H1299, A549, PC9, H1650 and H1975 were used as in vitro models. Colorimetric MTT assays, Western blotting and flow cytometry were used to evaluate the anti-growth effects of PAC-1 and/or Cis and apoptosis status. The activated form of CASP3 ( C-CASP3) was assessed by immunofluorescent staining. Results Single-agent Cis and PAC-1 were able to inhibit the cancer cell growth in certain dose ranges, with IC50 values of 1.9-11.7 and 5.6-14.8 μM, respectively. Sequential Cis→ PAC-1 or concurrent Cis + PAC-1, but not PAC-1→Cis combinations showed synergistic effects on cell growth inhibition in H1299 cells (combination index, CI ≤ 0.6). In contrast, other combination modes mostly showed seemingly antagonistic effects (CI > 1.0). Flow cytometric analysis showed that Cis→ PAC-1 sequential combination showed strong pro-apoptotic effects in H1299 cells. Western blots showed that in H1299, PC9 and H1975 cells, PAC-1 promoted the C-CASP3, but only in H1299 cells was there a synergistic effect with Cis on the CASP3 activation. Conclusions PAC-1 showed anti-tumor activity in NSCLCs in vitro and a synergistic effect with cisplatin in EGFRwt KRASwt H1299 cells. Our data suggest a potential treatment approach using cisplatin plus a pro-apoptotic agent acting via CASP3 activation for this subgroup of pulmonary adenocarcinomas. [ABSTRACT FROM AUTHOR]

Caspase-3 is a critical effector caspase in apoptosis cascade, and is often over-expressed in many cancer tissues. The first synthesized procaspase-3 activator, PAC-1, induces cancer cell apoptosis and exhibits antitumour activity in murine xenograft models. To identify more potent procaspase-3 activators, a series of compounds were designed, synthesized and evaluated for their ability of inducing cancer cell death in culture. Among these compounds, WF-208 stood out by its high cytotoxicity against procaspase-3 overexpressed HL-60 cells. Compared with PAC-1, WF-208 showed higher cytotoxicity in cancer cells and lower toxicity in normal cells. The further investigation described herein showed that WF-208 activated procaspase-3, degraded IAPs (The Inhibitors of apoptosis proteins) and leaded to caspase-3-dependent cell death in tumour cells, which possibly because of the zinc-chelating properties. WF-208 also showed greater antitumour activity than PAC-1 in murine xenograft model. In conclusion, we have discovered WF-208 as a promising procaspase-3 activating compound, with higher activity and higher cell selectivity than PAC-1. [ABSTRACT FROM AUTHOR]

The activation status of platelets in Immune Thrombocytopenia (ITP) patients - which is still somewhat controversial - is of potential interest, because activated platelets tend to aggregate (leading to excessive clotting or thromboembolic events) but cannot do so when platelet numbers are low, as in ITP. Although corticosteroids are the first line of therapy in ITP, the effect of steroids on activation of platelets has not been evaluated so far. We examined the status of platelet activation (with and without stimulation with ADP) in ITP patients, at the start of therapy (pre-steroid treatment, naive) and post-steroid treatment (classified on the basis of steroid responsiveness). We used flow cytometry to evaluate the levels of expression of P-selectin, and PAC-1 binding to platelets of 55 ITP patients and a similar number of healthy controls, treated with and without ADP. We found that platelets in ITP patients exist in an activated state. In patients who are responsive to steroids, the treatment reverses this situation. Also, the fold activation of platelets upon treatment with ADP is more in healthy controls than in ITP patients; treatment with steroids causes platelets in steroid-responsive patients to become more responsive to ADP-activation, similar to healthy controls. Thus steroids may cause changes in the ability of platelets to get activated with an agonist like ADP. Our results provide new insights into how, and why, steroid therapy helps in the treatment of ITP. [ABSTRACT FROM AUTHOR]

Background Dysregulation of apoptotic cell death is observed in a large number of pathological conditions. As caspases are central enzymes in the regulation of apoptosis, a large number of procaspase-activating compounds (PAC-1 derivatives) and inhibitors (isatin derivatives) have been developed. Matrix metalloproteinases (MMPs) have been shown to have a dual role in apoptosis. Hence compounds that either activate or inhibit caspases should ideally not affect MMPs. As many PAC-1 derivatives contain a zinc chelating ortho -hydroxy N -acyl hydrazone moiety and isatin derivatives has two carbonyl groups on the indole core, it was of interest to determine to which extent these compounds can inhibit MMPs. Methods Eight PAC-1 and five isatin derivatives were docked into MMP-9 and MMP-14. The same compounds were synthesized, characterized, purified and tested as inhibitors of MMP-9 and MMP-14, using fluorescence quenched peptide and biological substrates. Some of the compounds were also tested for fluorescence quenching. Results Molecular docking suggested that the different compounds can bind to the MMP active sites. However, kinetic studies showed that neither of these compounds was a strong MMP inhibitor. IC 50 values over 100 μM were obtained after the enzyme activities were corrected for quenching. These IC 50 values are far above the concentrations needed to activate or inhibit the caspases. Conclusion The use of PAC-1 and isatin derivatives against caspases should have little or no effect on the activity of MMPs. General significance Activators and inhibitors of caspases are important potential therapeutic agents for several diseases such as cancer, diabetes and neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Background: The Raynaud phenomenon (RP) is an exaggerated and reversible vasospasm of small arteries triggered by cold or emotional stress. Primary RP (PRP) term is used when the underlying condition is unknown. An altered regulation in vascular tone and/or release of soluble mediators from activated platelets plays a role in PRP through an increased oxidative stress. We assessed platelet activation and oxidative stress in patients with PRP by measuring platelet PAC-1, an index of glycoprotein (Gp) IIb/IIIa receptor activation, thromboxane A2 (TXA2), an index of platelet activation and 8-epi-prostaglandin F2α (8-epi-PGF2α), a marker of endogenous in vivo peroxidation. Methods: Eighteen asymptomatic patients with PRP (age 41.37 ± 16.94 years; 17 women, 1 man) and 18 healthy subjects (age of 35.11 ± 13.16 years; 16 women, 2 men) were studied. PAC-1 was analysed by flow cytometry while circulating TXB2, a stable metabolite of TXA2 and 8-epi-PGF2α levels were assessed by ELISA kit. Results: Our results show a significant platelet activation in PRP patients as indicated by increased PAC-1 expression (65.29 ± 15.24%; P < 0.001), TXB2 (1477.83 ± 454.04 pg/mL; P= 0.003) and 8-epi-PGF2α circulating levels (42.50 ± 14.14 ng/mL; P < 0.001). An inverse correlation between the degree of PAC-1 expression and TXB2 levels ( r=−0.527; P= 0.02) was also found in PRP patients, suggesting that downregulation of GpIIb/IIIa receptor expression may occur during thrombocytopoiesis, as a consequence of the chronic exposure to increased TXB2 concentration. Conclusions: Our study for the first time shows a marked activation of GpIIb/IIIa receptor in asymptomatic patients with PRP and supports antiplatelet therapy in PRP patients. [ABSTRACT FROM AUTHOR]

Abstract: Caspase-3 is known as the key executioner caspase, activated in both the intrinsic and extrinsic apoptotic pathway, and an effector far downstream in the apoptotic cascade. Procaspase-activating compound-1 (PAC-1) and 1541 were launched as direct activators of procaspase-3 to caspase-3, and anticipated to be promising therapeutic agents for the treatment of cancer. PAC-1 has recently been evaluated in a phase I preclinical trial. However, little is known about the effect of these substances in cells. Activation of caspase-3 in whole cells may be more complicated than thought, as it is likely that this key protease is tightly regulated both in development and apoptosis. In this study, we investigated the effect of epidermal growth factor (EGF) on PAC-1-induced caspase-3 activity and cell death. We show that EGF can block caspase-3 activity generated by PAC-1, and protect both PC12 cells and primary cerebellar granule neurons against PAC-1-induced death. Similar results were obtained with 1541. Both substances reduced cellular p-ERK levels. Crosstalk between caspase-3 and growth factor signaling pathways may present a challenge for the use of such caspase-3-activating substances in cancer therapy, since aberrant growth factor signaling is frequently seen in malignant cells. This study adds important knowledge about cellular effects of procaspase-3 activators like PAC-1 and 1541. Effects mediated by these substances may also contribute to the understanding of caspase signaling in cells. [Copyright &y& Elsevier]

Abstract: A sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of PAC-1 in rat plasma. After extraction with ethyl acetate, the chromatographic separation was carried out on an ACQUITY UPLC™ BEH C18 column, with acetonitrile and water (39:61 (v/v) both containing 0.1% formic acid) as mobile phase at a flow rate of 0.20mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The calibration curve was linear over the range of 10–1500ng/mL (r >0.99). The LOQ was evaluated to be 0.3ng/mL. The method described herein is sensitive, selective and faster than other existing method, and was successfully applied to the pharmacokinetic study and gender difference investigation of PAC-1 after oral administration in rats. [ABSTRACT FROM AUTHOR]

Abstract: Procaspase-activating compound 1, PAC-1, has been introduced as a direct activator of procaspase-3 and has been suggested as a therapeutic agent against cancer. Its activation of procaspase-3 is dependent on the chelation of zinc. We have tested PAC-1 and an analogue of PAC-1 as zinc chelators in vitro as well as their ability to activate caspase-3 and induce cell death in chicken cerebellar granule neuron cultures. These neurons are non-dividing, primary cells with normal caspase-3. The results reported herein show that PAC-1 chelates zinc, activates procaspase-3, and leads to caspase-3-dependent cell death in neurons, as the specific caspase-3-inhibitor Ac-DEVD-cmk inhibited both the caspase-3 activity and cell death. Thus, chicken cerebellar granule neurons is a suitable model to study mechanisms of interference with apoptosis of PAC-1 and similar compounds. Furthermore, the present study also raises concern about potential neurotoxicity of PAC-1 if used in cancer therapy. [Copyright &y& Elsevier]

Abstract: Blood platelets are the innate immune elements that have not been investigated in human filarial infections. Platelet activation status in the endemic normals (EN), microfilaria positive individuals (MF) and patients with chronic pathology (CP) was evaluated in whole blood, under unstimulated as well as antigen exposed (BmA, E. coli) conditions for PAC-1 expression by Flow cytometry. A diminished PAC-1 expression was observed in MF compared to CP and EN spontaneously as well as upon antigen exposure. Besides this, PAC-1 expression within the groups did not exhibit any significant difference under all the experimental conditions. However in CP patients, E. coli antigen exposure resulted in a significantly reduced PAC-1 expression compared to the spontaneous expression levels. NO release in platelet culture supernatants from EN was inversely proportional to platelet aggregation. Collagen stimulated platelets from EN, exposed to sera and immune complexes from CP and MF patients resulted in elevated Nitric Oxide (NO) release, compared to those exposed to autologous sera and fetal calf serum. In addition, under similar conditions, collagen stimulated platelets from EN, exposed to filarial antigen (BmA) exhibited increased NO compared to the E. coli antigen exposed ones and light microscopic observations of cultured platelets supported the above findings. Thus it appears from the results of the present study that filarial antigen may play a role in the loss of platelet aggregation, leading to platelet inactivation. [Copyright &y& Elsevier]

Abstract: PAC-1 was subjected to acid degradation conditions at 80°C for 10h. Four unknown degradation products were isolated from PAC-1 by semi-preparative high performance liquid chromatography using isocratic elution conditions. Based on the ESI-MS and NMR spectral data the structures of these four degradation products were characterized as 2-allyl-6-((E)-((E)-(2-hydroxy-3-(2-hydroxypropyl)benzylidene)hydrazono)methyl)phenol, 2-hydroxy-3-(2-propenyl)-[[2-hydroxy-3-(2-propenyl)phenyl]methylene]hydrazone, 6,6′-(1E,1′E)-hydrazine-1,2-diylidenebis(methan-1-yl-1-ylidene)bis(2-(2-hydroxypropyl)phenol) and 2-hydroxy-3-(2-hydroxypropyl)benzaldehyde. [Copyright &y& Elsevier]

Abstract: The direct induction of apoptosis has emerged as a powerful anticancer strategy, and small molecules that either inhibit or activate certain proteins in the apoptotic pathway have great potential as novel chemotherapeutic agents. Central to apoptosis is the activation of the zymogen procaspase-3 to caspase-3. Caspase-3 is the key “executioner” caspase, catalyzing the hydrolysis of a multitude of protein substrates within the cell. Interestingly, procaspase-3 levels are often elevated in cancer cells, suggesting a compound that directly stimulates the activation of procaspase-3 to caspase-3 could selectively induce apoptosis in cancer cells. We recently reported the discovery of a compound, PAC-1, which enhances procaspase-3 activity in vitro and induces apoptotic death in cancer cells in culture and in mouse xenograft models. Described herein is the mechanism by which PAC-1 activates procaspase-3 in vitro. We show that zinc inhibits the enzymatic activity of procaspase-3 and that PAC-1 strongly activates procaspase-3 in buffers that contain zinc. PAC-1 and zinc form a tight complex with one another, with a dissociation constant of approximately 42 nM. The combined data indicate that PAC-1 activates procaspase-3 in vitro by sequestering inhibitory zinc ions, thus allowing procaspase-3 to autoactivate itself to caspase-3. The small-molecule-mediated activation of procaspases has great therapeutic potential and thus this discovery of the in vitro mechanism of action of PAC-1 is critical to the development and optimization of other procaspase-activating compounds. [Copyright &y& Elsevier]

We examined the magnitude of 20-min moderate exercise-induced platelet activation in 50 volunteers with normal ( n=31) or elevated blood pressure (EBP; n=19). Blood was drawn before, immediately after, and 25 min after exercise. Antibody-staining for platelet activation markers, P-selectin, and fibrinogen receptors was done with and without adenosine diphosphate (ADP) stimulation in whole blood for flow cytometric analyses. Exercise led to increases in percent aggregated platelets and percent platelets expressing P-selectin or PAC-1 binding ( ps≤.001). This increase in percent platelets expressing P-selectin continued even after a 25-min rest only in the EBP group ( p≤.01) accompanied by an increase in percent of aggregated platelets ( p≤.05). Although ADP stimulation led to increased platelet activation at rest, it was attenuated following exercise, even among EBP individuals. A moderate exercise challenge induced prolonged platelet activation in individuals with EBP but attenuation in activation to further stimulation by an agonist. Findings suggest that a recovery period after physical stress appears critical in individuals with high BP regarding platelet activation and aggregation, which can lead to an acute coronary syndrome in vulnerable individuals. [ABSTRACT FROM AUTHOR]

Abstract: A sensitive and specific high performance liquid chromatography method with UV detection was developed and validated for the determination of PAC-1 in rat plasma. After extraction with ethyl acetate, the chromatographic separation was carried out on a Diamonsil C18 column (150mm×4.6mm i.d., 5μm particle size, Zhonghuida) protected by a ODS guard column (10mm×4.6mm i.d., 5μm particle size), using acetonitrile–methanol–phosphate buffer (pH 3.0, 30mM) (31:3:66, v/v/v) as mobile phase at a flow rate of 1.0mL/min, and wavelength of the UV detector was set at 281nm. No interference from any endogenous substances was observed during the elution of PAC-1 and internal standard (IS, indapamide). The calibration curve was linear over the range of 0.05–20μg/mL (r >0.99). The lower limit of quantification was evaluated to be 50ng/mL. The method was successfully applied to the pharmacokinetic study of PAC-1 after intravenous and oral administration in rats, respectively. [Copyright &y& Elsevier]

Abstract: There is an epidemiological association between periodontitis and cardiovascular disease (CVD). In periodontitis, low grade systemic inflammation and bacteremia occur regularly. Such events may contribute to platelet activation and subsequent pro-coagulant state. This study aimed to investigate platelet activation in periodontitis patients. The study is composed of two parts. In the first part, plasma levels of soluble(s) P-selectin and sCD40 ligand were measured as general markers of platelet activation in periodontitis patients (n =85) and in healthy controls (n =35). In the second part, surface-exposed P-selectin and the ligand-binding conformation of the glycoprotein IIb–IIIa complex (binding of PAC-1 antibody) were determined on individual platelets in whole blood of periodontitis patients (n =18) and controls (n =16). Patients had significantly elevated plasma levels of sP-selectin (P <0.001) and increased binding of PAC-1 on isolated platelets (P =0.033). Platelet activation was more pronounced in the patients with more severe periodontal disease, showing a severity-dependence. The levels of sCD40 ligand and of platelet-bound P-selectin were not increased. Periodontitis is associated with increased platelet activation. Since platelet activation contributes to a pro-coagulant state and constitutes a risk for atherothrombosis, platelet activation in periodontitis may partly explain the epidemiological association between periodontitis and CVD. [Copyright &y& Elsevier]

Abstract: In the present study, the metabolic profile of PAC-1, a potential anticancer drug, was investigated using liquid chromatography–mass spectrometric (LC/MS) techniques. Two different types of mass spectrometers – a quadrupole time-of-flight (Q-TOF) mass spectrometer and an ion trap (IT) mass spectrometer – were employed to acquire structural information on PAC-1 metabolites. A gradient liquid chromatographic system composed of 0.2% formic acid in methanol and 0.2% formic acid in water was used for metabolite separation on an Agilent TC-C18 column. A total of 16 metabolites were detected. The corresponding product ion spectra were acquired and interpreted, and structures were proposed. Accurate mass measurement using LC-Q-TOF was used to determine the elemental composition of metabolites thereby confirming the proposed structures of these metabolites. Phase I metabolic changes were predominantly observed, including debenzylation, dihydrodiol formation, hydroxylation, and dihydroxylation. The detected phase II metabolites included PAC-1 and hydroxylated PAC-1 glucuronide conjugates. Based on metabolite analysis, several PAC-1 metabolic pathways in rat were proposed. [Copyright &y& Elsevier]

There is an urgent need of novel approaches to drugs in the cancer, HIV, and bacterial areas. Increasing resistance to conventional therapies is observed. This minireview provides novel insights for drugs in these three areas. The agents PAC-1 (anticancer), DHBNH (anti-HIV), AHL (autoinducer), and UCS1025A (anticancer) have recently attracted attention due to considerable potential based on new approaches. PAC-1 activates procaspase-3 to caspase-3, resulting in induction of apoptosis in tumor cells. DHBNH binds to a newly revealed site on HIV reverse transcriptase. The drug mainly inhibits RNase H (RNA-cleaving). AHLs comprise an important class that participates in bacterial cell communication. UCS1025A is a fungus-derived inhibitor of the enzyme telomerase, present in cancer cells, which is crucially involved in tumor cell immortality. All four agents possess chelating sites for metal binding, which has not been appreciated. In PAC-1 and DHBNH, the coordinating portion is similar to salicylaldehyde semicarbazone. For AHL and UCS1025A, the metal-binding moiety is a β -ketoamide. Metal complexes of heavier metals are well-known electron transfer (ET) functionalities that can generate reactive oxygen species. Hence, it is reasonable to hypothesize a commonality in mechanism based on metal ET. Differences in receptor binding can result, in part, in diverse physiological responses. There is considerable literature that addresses involvement of signal transduction with the various physiologically active agents discussed herein. Thus, cell communication appears to play an important role in the biochemistry of these endogenous and exogenous substances. Details of cell signaling are presented for complexes of metals (Fe, Cu, Ni, and As), telomerase, caspase-3, and RNase. In addition, practical medical aspects are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: We have studied the relationship between the ratio of activated platelets and the thickness of intima and media of the carotid artery in ischemic CVD patients in the chronic stage. Methods: Platelet activation was assessed by means of flow cytometry of whole blood using activation-dependent monoclonal antibodies (MoAb). Forty-one MRI-proven normative subjects and 55 patients with a history of ischemic CVD were examined. The intima-media thickness of the carotid artery was measured by using B-mode ultrasound in all subjects. Results: The appearance rates of PAC-1-positive and CD62P-positive platelets (%) were increased in ischemic CVD patients compared with those in controls (p <0.0001, p <0.001, respectively) The patients and controls were divided into those with atherosclerosis (Ath+), defined as intima-media thickness 1.1 mm, and those without (Ath−). There was no significant difference of PAC-1-positive platelets between the Ath− and Ath+ subgroups in either group, but there was increase in Ath− ischemic CVD patients versus Ath− control subjects (p <0.01), and in Ath+ patients versus Ath+ controls (p <0.05). CD62-positive platelets in the Ath+ subgroup were significantly increased versus the Ath− subgroup in both the controls (p <0.001) and ischemic CVD patients (p <0.05), and there was also an increase in Ath− patients versus Ath− controls (p <0.05). Conclusion: Platelet activation markers were increased in patients with ischemic CVD compared with controls. A significant relationship was found between increased CD62-P-positive platelets and carotid artery abnormalities in both controls and ischemic CVD patients, suggesting that platelet activation may be a potential marker for atherosclerosis. [Copyright &y& Elsevier]

Platelet–leukocyte aggregates are considered to play a significant role in blood coagulation and inflammatory processes. We hypothesized that hormonal changes during the menstrual cycle affect the formation of heterotypic aggregates and therefore may constitute cycle-dependent variations of the susceptibility for thromboembolic events and inflammatory disease. We therefore measured platelet–leukocyte interaction by the determination of platelet–leukocyte aggregates (PLA), platelet P-Selectin expression, and platelet fibrinogen receptor activation by PAC-1 binding in 20 healthy women during their menstrual cycle by flow cytometry. The number of platelet–granulocyte aggregates (PGA) and platelet–monocyte aggregates (PMA) was higher at ovulation compared to any other time-point of the menstrual cycle ( p &ThickSpace;=&ThickSpace;0.005, p &ThickSpace;=&ThickSpace;0.022, respectively). Likewise, P-Selectin expression peaked on day 14 ( p &ThickSpace;=&ThickSpace;0.040). The course of PLA formation during the menstrual cycle followed the course of estrogen levels, strongly suggesting direct effects of estrogen on platelet–leukocyte interaction. The susceptibility to form platelet–leukocyte aggregates that are inducible in vitro by a suboptimal concentration of thrombin receptor activating peptide-6 decreased slightly during the transition from day 1 to 14 ( p &ThickSpace;=&ThickSpace;0.040). These data indicate that platelet function varies during particular phases of the normal menstrual cycle. [ABSTRACT FROM AUTHOR]

Flow cytometric evaluation of platelet function extends our understanding of platelets’ role in various clinical conditions associated with either bleeding disorders, thrombosis, or monitoring of antiplatelet therapy. The use of suboptimal concentrations of various agonists may allow assessing the “activatability” of platelets. We determined platelet responsiveness to thrombin-receptor-activating peptide-6, arachidonic acid, adenosine 5c-diphosphate (ADP), epinephrine, collagen, and ristocetin at suboptimal concentrations by determination of P-selectin expression and binding of PAC-1 in 26 healthy male individuals. The response varied considerably from one individual to the next. However, within individuals, responses to all agonists except collagen correlated strongly ( p<0.05), suggesting a global variability of platelet responses. Moreover, P-selectin expression and PAC-1 binding were strongly correlated ( p<0.05). Interestingly, with epinephrine, PAC-1 positive events outnumbered P-selectin positive events, while this was not seen with the other agonists. Thus, epinephrine may specifically affect the conformational switch mechanism and receptor clustering. Our data indicate that the in vitro response to suboptimal concentrations of agonists varies, but individuals with selective platelet defects may still be identified based on data obtained with the various agonists. [ABSTRACT FROM AUTHOR]