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Background and Objectives: SM-1 is a new synthetic small molecule compound with antitumor activity. The metabolism of SM-1 is a key parameter that needs to be evaluated to provide further insight into drug safety and efficacy in the early phases of drug development. Methods and Results: In this study, the biotransformation process of SM-1, including the metabolic pathways and major metabolites, was investigated based on a liquid chromatography-mass spectrometry method. Upon incubation of SM-1 with human liver microsomes, five metabolites were identified, namely dihydrodiol formation (R1), hydroxylation (R2, R3, and R5), and debenzylation (R4) of SM-1, with R1 and R4 being the major metabolites. The enzyme kinetic parameters of SM-1 were determined by a liquid chromatography-tandem mass spectrometry method. The enzyme kinetics of SM-1 obeyed the Michaelis-Menten equation. The Vmax, Km, and CLint of SM-1 in HLMs were 14.5 nmol/mg protein/h, 6.32 μM, and 2.29 mL/mg protein/h, respectively. Results: The chemical inhibition studies showed that CYP450 isoenzymes were responsible for SM-1 metabolism in HLMs, and CYP3A4 was the major CYP450 isoenzyme involved in the metabolism of SM-1; these findings were confirmed by using the human recombinant CYP3A4. Conclusion: Through the identification of the biotransformation pathways and enzyme kinetics of SM-1, the metabolic enzymes for SM-1 in HLMs are characterized.

BackgroundHigh-grade meningioma is an aggressive type of brain cancer that is often recalcitrant to surgery and radiotherapy, leading to poor overall survival. Currently, there are no FDA-approved drugs for meningioma, highlighting the need for new therapeutic options, but development is challenging due to the lack of predictive preclinical models.MethodsTo leverage the known overexpression of procaspase-3 in meningioma, PAC-1, a blood-brain barrier penetrant procaspase-3 activator, was evaluated for its ability to induce apoptosis in meningioma cells. To enhance the effects of PAC-1, combinations with either hydroxyurea or temozolomide were explored in cell culture. Both combinations were further investigated in small groups of canine meningioma patients and assessed by MRI, and the novel apoptosis tracer, [18F]C-SNAT4, was evaluated in patients treated with PAC-1 + HU.ResultsIn meningioma cell lines in culture, PAC-1 + HU are synergistic while PAC-1 + TMZ show additive-to-synergistic effects. In canine meningioma patients, PAC-1 + HU led to stabilization of disease and no change in apoptosis within the tumor, whereas PAC-1 + TMZ reduced tumor burden in all three canine patients treated.ConclusionsOur results suggest PAC-1 + TMZ as a potentially efficacious combination for the treatment of human meningioma, and also demonstrate the utility of including pet dogs with meningioma as a means to assess anticancer strategies for this common brain tumor.

WOS: 000468954500005, Background: In this study, novel ortho-hydroxy N-acyl hydrazone moiety including compounds (3a-l) were designed, based on procaspase activating compound (PAC-1) which is a small molecule known with antitumor activity. The antitumor activity was evaluated on A549 (human lung cancer cell line) and CCD 19Lu (human lung normal cell line). Methods: Twelve N'-arylidene-2-[4-(methylsulfonyl) piperazin-1-yl] acetohydrazide derivatives (3a-l) were synthesized starting from ethyl 1-piperazinylacetate. All compounds were tested using MTT method and Xcelligence-Real time cell analysis system (RTCA DP) to determine their antitumor activity. Results: Some physicochemical properties of four active compounds were also predicted using MolSoft, PreADMET and PROTOX software. Four of them, 3h, 3j, 3k and 3l bearing 3-hydroxy, 4-dimethylamino, 2,6-dichloro and 3,4-dichloro substituents in order exhibited selective cytotoxicity. Conclusion: Eligible values were obtained in the specified ranges as to be an oral/intravenous drug considering the physicochemical calculations., Anadolu University within the Anadolu University Scientific Research Project, Eskisehir, Turkey [1505S388], The authors present their thanks to Anadolu University for financial support within the Anadolu University Scientific Research Project, Eskisehir, Turkey (Project no: 1505S388).

Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), which infects over 200,000 people worldwide. Sin Nombre virus (SNV) and Andes virus (ANDV) cause the most severe form of HCPS with case fatality ratios of 30–40%. There are no specific therapies or vaccines for SNV. Using high throughput flow cytometry, we screened the Prestwick Compound Library for small molecule inhibitors of the binding interaction between UV-inactivated and fluorescently labeled Sin Nombre virus (SNV(R18)) particles, and decay accelerating factor (DAF) expressed on Tanoue B cells. Eight confirmed hit compounds from the primary screen were investigated further in secondary screens that included infection inhibition, cytotoxicity, and probe interference. Antimycin emerged as a bona fide hit compound that inhibited cellular infection of the major HCPS (SNV) and HCPS (Hantaan) causing viruses. Confirming our assay’s ability to detect active compounds, orthogonal testing of the hit compound showed that antimycin binds directly to the virus particle and blocks recapitulation of physiologic integrin activation caused by SNV binding to the integrin PSI domain.

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 IC50 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 IC50 (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.

// Avadhut D. Joshi 1,* , Rachel C. Botham 2,* , Lisa J. Schlein 3 , Howard S. Roth 2 , Antonella Mangraviti 1 , Alexandra Borodovsky 1 , Betty Tyler 1 , Steve Joslyn 4 , Jayme S. Looper 5 , Michael Podell 6 , Timothy M. Fan 7 , Paul J. Hergenrother 2 and Gregory J. Riggins 1 1 Department of Neurosurgery, School of Medicine, Johns Hopkins University, Baltimore, MD, USA 2 Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA 3 Department of Pathobiology, University of Illinois Urbana-Champaign, Urbana, IL, USA 4 VetCT Australia, Fremantle WA, Australia 5 Department of Veterinary Clinical Sciences, Louisiana State University, Baton Rouge, LA, USA 6 Department of Neurology, MedVet Chicago, Chicago, IL, USA 7 Department of Veterinary Clinical Medicine, University of Illinois Urbana-Champaign, Urbana, IL, USA * These authors have contributed equally to this work Correspondence to: Gregory J. Riggins, email: // Paul J. Hergenrother, email: // Timothy M. Fan, email: // Keywords : PAC-1, procaspase-3 activator, glioblastoma, small molecule therapy Received : March 29, 2017 Accepted : June 09, 2017 Published : July 07, 2017 Abstract Purpose: Glioblastoma is a deadly brain cancer with a median survival time of ~15 months. Ionizing radiation plus the DNA alkylator temozolomide (TMZ) is the current standard therapy. PAC-1, a procaspase-3 activating small molecule, is blood-brain barrier penetrant and has previously demonstrated ability to synergize with diverse pro-apoptotic chemotherapeutics. We studied if PAC-1 could enhance the activity of TMZ, and whether addition of PAC-1 to standard treatment would be feasible in spontaneous canine malignant gliomas. Experimental Design: Using cell lines and online gene expression data, we identified procaspase-3 as a potential molecular target for most glioblastomas. We investigated PAC-1 as a single agent and in combination with TMZ against glioma cells in culture and in orthotopic rodent models of glioma. Three dogs with spontaneous gliomas were treated with an analogous human glioblastoma treatment protocol, with concurrent PAC-1. Results: Procaspase-3 is expressed in gliomas, with higher gene expression correlating with increased tumor grade and decreased prognosis. PAC-1 is cytotoxic to glioma cells in culture and active in orthotopic rodent glioma models. PAC-1 added to TMZ treatments in cell culture increases apoptotic death, and the combination significantly increases survival in orthotopic glioma models. Addition of PAC-1 to TMZ and radiation was well-tolerated in 3 out of 3 pet dogs with spontaneous glioma, and partial to complete tumor reductions were observed. Conclusions: Procaspase-3 is a clinically relevant target for treatment of glioblastoma. Synergistic activity of PAC-1/TMZ in rodent models and the demonstration of feasibility of the combined regime in canine patients suggest potential for PAC-1 in the treatment of glioblastoma.

Perspectives To evaluate the treatment outcome of vasodilator prostaglandin E1 (PGE1) in treating sudden sensorineural hearing loss (SSNHL) and to determine its effects on platelet activation, as reflected by changes in CD62p and PAC-1. Methods We prospectively enrolled 60 patients with confirmed SSNHL and randomly divided them into two groups: the SSNHL group received regular therapy, and the SSNHL-PGE1 group received additional intravenous injection of PGE1. After 14 days of treatment, we measured clinical improvement and CD62p-positive and PAC-1-positive platelets. 30 healthy medical staff members were included as a control group. Results The SSNHL patients had significantly higher levels of CD62p-positive or PAC-1 positive platelets than the healthy subjects. The ratios of CD62p positive or PAC-1 positive platelets significantly decreased after the two treatments. The average pure tone (PTA) hearing thresholds decreased to 26.51 ± 12.65 dB in SSNHL-PGE1 group after treatment, which was significantly lower than that of the SSNHL group (34.46 ± 10.35 dB). Patients with initial severe or profound hearing loss (PTA ≥ 71 dB) had better hearing improvement on PGE1 than on the regular treatment. Patients in the SSNHL-PGE1 treatment group had significantly lower CD62p and PAC-1 levels than those in the SSNHL group. Patients with higher initial positive CD62p and PAC-1 ratios tended to have higher potential of clinical improvement and hearing gains after PGE1 treatment. Initial CD62p and PAC-1 levels were significantly correlated with hearing thresholds in patients with SSNHL. Conclusion PGE1 application could improve treatment efficacy and suppress excessive platelet activation in patients with SSNHL.

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 The synergistic activity of temozolomide (TMZ) administered in combination with procaspase-activating compound (PAC-1) has been reported in pre-clinical mouse models and canine patients, leading to clinical trials in adults with glioblastoma. To optimize pediatric clinical trial design, a translational pharmacokinetic CSF penetration study was conducted using a pre-clinical nonhuman primate non-tumor bearing CSF access model with TMZ and PAC-1 administered alone and in combination. METHODS Four male rhesus macaques with CSF lateral ventricular reservoirs received PAC-1, 15 mg/kg orally [Human Equivalent Dose (HED) 558 mg/m2/day] or TMZ, 1-hr IV infusion, 7.5 mg/kg (HED 150 mg/m2) as single and combination agent administration. Paired plasma and CSF samples were collected for 0–96 hours. PAC-1 and TMZ were quantified by LC-MS/MS. Pharmacokinetic parameters were calculated using noncompartmental methods. Statistics were determined via Mann-Whitney test. RESULTS (Mean ± Standard Deviation): For TMZ: Plasma AUC0-24 (hr*ng/ml) single agent (n=4): 28590 ± 4888 and in combination (n=4): 32736 ± 10147. CSF AUC0-24 (hr*ng/ml) single agent (n=4):14406 ± 1279 and in combination (n=4):15614 ± 1767. CSF penetration (% AUCCSF: AUCPLASMA) single agent: 50.9% ± 2.2 and in combination:49.6% ± 8.4. For PAC-1: Plasma PAC-1 AUC0-24 (hr*ng/ml) single agent (n=4): 2556 ± 2157 and in combination (n=4): 1947 ± 1311. CSF PAC-1 AUC0-24 (hr*ng/ml) single agent (n=2): 8.7 ± 1.1 and in combination (n=3):12.9 ± 10.2. CSF penetration single agent: 0.2% ± 0.03 and in combination: 0.4% ± 0.38. CSF PAC-1 tlag and Tmax pharmacokinetic parameters decreased with concurrent TMZ administration. CONCLUSIONS In this non-tumor bearing pre-clinical nonhuman primate model the CSF penetration of PAC-1 was low and not notably affected by the concurrent administration of TMZ. TMZ CSF penetration for single administration was within previously reported ranges and also appeared unaffected when administered in combination with PAC-1.

Diffuse Intrinsic Pontine Gliomas (DIPGs) are universally fatal tumors, resistant to the cytotoxic effects of all known therapies. We aim to better understand the mechanisms contributing to this resistance to design new combination therapies that will improve DIPG survival. DIPG's aggressive phenotype is driven by the H3K27M oncohistone mutation that leads to global genomic DNA hypomethylation and abnormal gene expression. To identify patterns of gene expression driven by the H3K27M mutation, we introduced the H3K27M mutation into murine neural stem cells and performed whole genome bisulfite sequencing (WGBS) to evaluate for genes with the greatest differential methylation in their promoter region. These studies identified an abundance of pro-survival genes such a BCL-2, BCL-xL, MCL-1, Caspase-3, and XIAP. Western blot confirmed elevated expression of these pro-survival factors and RNA-Seq of primary human DIPG confirmed increased expression compared to normal brain. PAC-1 is a compound that cleaves protective zinc bonds to activate procaspase-3, directly inducing cell death. ONC201 is a small molecule that inhibits the anti-apoptotic protein XIAP, which prevents activated caspase-3 from translocating to the nucleus to induce apoptosis. We hypothesized that PAC-1 combined with ONC201 will circumvent these protective abnormalities in the DIPG apoptotic signaling cascade to induce tumor cell death. PAC-1 induces high rates of apoptosis in DIPG (25µM concentrations increases the percent of cells undergoing apoptosis; cleaved caspase 3 immunofluorescence assay 5% to 30% (t-test p40% compared to DMSO control (ANOVA p Citation Format: Ashlyn Parkhurst, Youngran Park, Antje Arnold, Michael Koldobskiy, Charles Eberhart, Eric Raabe, Jeffery Rubens. The novel combination of PAC-1 and ONC201 circumvent H3K27M-driven pro-survival gene expression to induce DIPG cell death [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3904.

Platelet activation and functional changes in some haematological malignancies have been investigated with little or no known documentation on Burkitt lymphoma (BL). Abnormalities of platelets contribute to either haemorrhage or thrombotic episodes which are life-threatening in patients with BL. Thus, the study aimed at investigating the various platelet indices and platelet membrane glycoproteins in childhood Burkitt lymphoma. Platelet surface membrane glycoproteins (GPIIb/IIIa, P-selectin and GPIV using PAC 1, CD62p and CD36 monoclonal antibodies respectively) and platelet indices (Platelet Count [PLT], Plateletcrite [PCT], Mean Platelet Volume [MPV], Platelet Distribution Width [PDW] and Platelet Large Cell Ratio [P-LCR]) were determined in children with Burkitt lymphoma and healthy children (normal controls) based on flow cytometry and automated blood cell analysis techniques. PLT and PCT were higher in BL cases than in the normal controls with a significant difference in the PLT (P = 0.02). On the contrary, we observed a significant (p 0.05) lower levels in the other platelet indices (MPV, PDW and P-LCR) in children with BL than the controls. With the exception of CD62 P, the other platelet membrane glycoproteins examined showed a decreased level of expression before and after the addition of an Adenosine -5- diphosphate (ADP) in cases of BL. In addition, PAC-1 was probably known to be associated with Burkitt Lymphoma (Odds Ratio [OR] 6.67, Relative Risk [RR] 3.13, 95% CI 1.06-9.21; p = 0.02). Finally, oral bleeding was observed to be the commonest bleeding episodes associated with childhood BL. Flow cytometry analysis and cell counting techniques of platelet assessment has described the expression of the platelet membrane glycoproteins and parameters in children with Burkitt lymphoma. Thus, children with Burkitt lymphoma tend to show normal to increased level of circulatory platelets but decreased platelet membrane glycoprotein expressions and platelet dysfunction.

As a PAC-1 derivative, SM-1 exhibts a promising antitumour property. To better understand the relationship between the drug concentrations and pharmacological effects, both liquid chromatography coupled with tandem mass spectrometry and high performance liquid chromatography methods were developed and validated in the work. Those methods were then applied to the pharmacokinetics (PK), tissue distribution and plasma protein binding (PPB) studies of SM-1. As a results, the proposed methods were demonstrated to be accurate, precise and stable for the analysis of the SM-1 in plasma and tissue samples. Meanwhile, the PK parameters of SM-1 showed that SM-1 had good PK properties. SM-1 had good absorption in the body, with 59.01% of the absolute bioavailability in rats and 55.63% of that in dogs. SM-1 rapidly distributed to all tissues, with the highest distribution in the lung and less in the brain and muscle. The PPB rates in rat plasma, dog plasma, and human plasma were 91.1%, 91.2%, and 90.7%, respectively. These good PK properties will contribute SM-1 to be a promising anti-tumour candidate. These results also provide insights into the further pharmacological investigation of SM-1.

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.

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.

TPS3164 Background: The caspase family of cysteine proteases play key roles in the initiation and execution of apoptosis. The activation of procaspase-3 to caspase-3 is critical in both the intrinsic and extrinsic apoptotic cascades. Procaspase-3 levels are elevated in many cancers, including glioblastoma (GBM). As a result, caspase-3 levels are abnormally low in these tumors; thus they avoid apoptosis. PAC-1 is a small molecule that directly activates procaspase-3 and induces apoptosis of cancer cells. PAC-1 has activity against a wide range of cancer cell lines, and in animal models of cancer. PAC-1 crosses the blood brain barrier and has been shown to synergize with TMZ in both canine malignant glioma and meningioma that arise spontaneously. Methods: This Phase I dose escalation study uses a modified- Fibonacci 3+3 design to determine the MTD of PAC-1 when combined with TMZ in patients with recurrent malignant gliomas: anaplastic astrocytoma (AA) and GBM (open to enrollment). Here, we focus on component 2 of the study. Primary objectives: to establish MTD of PAC-1 when combined with a fixed dose of TMZ, tolerability, and toxicity using CTCAE v.4. Secondary and correlative objectives: pharmacokinetics, pharmacodynamics, preliminary anti-tumor activity correlation with procaspase-3 expression in tumor tissue, radiographic response using the Response Assessment in Neuro-Oncology (RANO) criteria, and neurocognitive function using a validated test battery. Inclusion criteria: diagnosis of recurrent high grade glioma (AA or GBM), ECOG PS 0-2, adequate organ function. Exclusion criteria: received prior cytotoxic therapy in the last 3-6 weeks (duration based on prior therapy) or uncontrolled chronic illness. Administration and design, Component 2: PAC-1, orally administered, is dosed at 375-650 mg daily (up to 3 dose levels) on days 1-21 of each 28-day cycle. A fixed dose of TMZ, (150 mg/m2), is administered orally, days 8 -12 of each cycle. The study is currently enrolling patients for Component 2. Clinical trial information: NCT02355535.

A novel series of first procaspase activating compound(PAC-1) analogues was designed, synthesized and evaluated for antitumor activity towards two cell lines[human promyelocytic leukemia cell line(HL60) and human embryonic lung fibroblast cell line(HLF)] by the MTT[3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazo-liumromide]_method in vitro. The structures of all the compounds were confirmed by 1H NMR, MS and elemental analysis. Among the compounds synthesized,(E)-2-[(3-{[4-(tert-butyl)benzyl](methyl)amino}propyl)(methyl)amino]-N′-[4-(diethylamino)-2-hydroxybenzylidene]acetohydrazide(compound 6n) exhibits a good anti-proliferative activity to the majority of tumor cells tested, and selectively cleaves cancer cells. Thus, compound 6n was identified as promising lead compound for further structural modification.

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.

Procaspase-Activating Compound 1 (PAC-1) is an ortho-hydroxy N-acyl hydrazone that enhances the enzymatic activity of procaspase-3 in vitro and induces apoptosis in cancer cells. An analogue of PAC-1, called S-PAC-1, was evaluated in a veterinary clinical trial in pet dogs with lymphoma and found to have considerable potential as an anticancer agent. With the goal of identifying more potent compounds in this promising class of experimental therapeutics, a combinatorial library based on PAC-1 was created, and the compounds were evaluated for their ability to induce death of cancer cells in culture. For library construction, 31 hydrazides were condensed in parallel with 27 aldehydes to create 837 PAC-1 analogues, with an average purity of 91%. The compounds were evaluated for their ability to induce apoptosis in cancer cells, and through this work, six compounds were discovered to be substantially more potent than PAC-1 and S-PAC-1. These six hits were further evaluated for their ability to relieve zinc-mediated inhibition of procaspase-3 in vitro. In general, the newly identified hit compounds are two- to four-fold more potent than PAC-1 and S-PAC-1 in cell culture, and thus have promise as experimental therapeutics for treatment of the many cancers that have elevated expression levels of procaspase-3.

BACKGROUND: PAC-1 is a novel compound which induces apoptosis via activation of the procaspase-3 pathway in cancer cells. Human clinical trials are ongoing with PAC-1 as a single agent (late-stage cancers) and in combination therapies (malignant glioma), in addition to studies in pet canines (glioma). This study evaluated the CSF penetration of PAC-1 via plasma and CSF pharmacokinetics in a nonhuman primate CSF Ventricular Reservoir or Lumbar Port model that allows serial CSF collection. METHODS: 4 male rhesus macaques received 15 mg/kg (Human Equivalent Dose of 558 mg/m(2)/day) of PAC-1 in 6 studies with two formulations; liquid (n=1) or pill (n=5). 1 animal received both formulations. Paired plasma and CSF (lumbar n=2 and ventricular n=4) samples were collected for 0–72 hours. PAC-1 was quantified by LC-MC/MS. The lower limit of quantitation for plasma was 10.0 ng/ml and CSF 0.2 ng/ml. PK parameters were calculated via noncompartmental methods. RESULTS: 5 studies were evaluable (n=1 liquid and n=4 pill). Plasma-Quanitifible (n=5). Liquid Formulation (n=1): AUC(0-∞) 5299.91 hr*ng/ml, Half-Life 4.99 hr, and Clearance 57.43 L/hr/m(2). Pill Formulation PK range (n=4): AUC(0-∞) 1527.7–8036.08 hr*ng/ml, Half-Life 10.2–31.5 hr, and Clearance 37.65–199.49 L/hr/m(2). CSF-Quanitifible (n=3; 2 lumbar & 1 ventricular) or undectable (n=2 ventricular). Liquid Formulation (lumbar n=1): AUC(0-∞) 50.90 hr*ng/ml, Half-Life 4.59 hr, and Clearance 5979.56 L/hr/m(2) Pill Formulation PK range (lumbar n=1 and ventricular n=1): AUC(0-∞) 7.53–31.25 hr*ng/ml, Half-Life 22.35–28.88 hr, and Clearance 9955.96–40178.44 L/hr/m(2). CSF: Plasma Ratio formulation comparison: 0.96% liquid and 0.46% pill. CONCLUSIONS: In this animal model, the CSF penetration of PAC-1 is low and formulation-dependent, with CSF: Plasma ratio 2 fold greater with liquid verses pill formulation. The CSF clearance is rapid. FUTURE STUDIES: 10.08 and 12.1 mg/kg dosages (Human Equivalent Dose 375 and 450, mg/m(2)/day, respectively) are currently undergoing evaluation in this NHP model.

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.

PAC-1 is a preferential small molecule activator of procaspase-3 and has potential to become a novel and effective anticancer agent. The rational development of PAC-1 for translational oncologic applications would be advanced by coupling relevant in vitro cytotoxicity studies with pharmacokinetic investigations conducted in large mammalian models possessing similar metabolism and physiology as people. In the present study, we investigated whether concentrations and exposure durations of PAC-1 that induce cytotoxicity in lymphoma cell lines in vitro can be achievable in healthy dogs through a constant rate infusion (CRI) intravenous delivery strategy. Time- and dose-dependent procaspase-3 activation by PAC-1 with subsequent cytotoxicity was determined in a panel of B-cell lymphoma cells in vitro. The pharmacokinetics of PAC-1 administered orally or intravenously was studied in 6 healthy dogs using a crossover design. The feasibility of maintaining steady state plasma concentration of PAC-1 for 24 or 48 h that paralleled in vitro cytotoxic concentrations was investigated in 4 healthy dogs. In vitro, PAC-1 induced apoptosis in lymphoma cell lines in a time- and dose-dependent manner. The oral bioavailability of PAC-1 was relatively low and highly variable (17.8 ± 9.5%). The achievement and maintenance of predicted PAC-1 cytotoxic concentrations in normal dogs was safely attained via intravenous CRI lasting for 24 or 48 h in duration. Using the dog as a large mammalian model, PAC-1 can be safely administered as an intravenous CRI while achieving predicted in vitro cytotoxic concentrations.

Objectives This study was designed to evaluate the effects of long-term clopidogrel and aspirin administration on platelet aggregation, activation, and inflammation. Background Clopidogrel resistance was described in 15% to 30% of patients with short-term therapy, but its antiplatelet effects with long-term therapy is unknown. Methods We performed a prospective study of patients undergoing coronary stenting who were on aspirin for ≥5 days but not previously on clopidogrel. Clopidogrel 600 mg was given before stenting. Clopidogrel 75 mg/day and aspirin 325 mg/day were continued for 1 year. Light-transmittance aggregometry with 5-μmol/l adenosine diphosphate and 1-mmol/l arachidonic acid stimulation; VerifyNow clopidogrel and aspirin assays; platelet activation receptor expression of CD40L, CD62P, and PAC-1 (antibody against activated glycoprotein IIb/IIIa); and inflammatory markers of soluble CD40L and P-selectin, high-sensitivity C-reactive protein, interleukin-10, and interleukin-18 were measured at baseline; 1 day; and 1, 6, and 12 months. Our primary analysis compared light-transmittance aggregometry aggregation at 1 versus 12 months. Results We enrolled 26 patients who completed a 1-year follow-up. Maximal platelet adenosine diphosphate-stimulated aggregation was 61.8 ± 25.9% at baseline, 22.1 ± 18.3% at 1 day, 30.6 ± 16.8% at 1 month, 29.0 ± 13.3% at 6 months, and 26.7 ± 13.6% at 12 months (p = 0.099 for 12 months vs. 1 month). VerifyNow clopidogrel platelet inhibition was similar at 12 months versus 1 month (38.9 ± 19.7% vs. 45.6 ± 26.7%, p = 0.578). Likewise, there was no difference in aspirin9s effects on platelet aggregation at 12 months versus 1 month. In contrast, platelet activation receptor expression of CD40L, CD62P, and PAC-1 were higher at 12 months versus 1 month. Conclusions Our pilot study showed no attenuation of clopidogrel9s effects on platelet aggregation with long-term administration. However, platelet activation receptor expression increased with time and should be further evaluated.

Although there is no amino acid sequence similarity between maxadilan (Maxa) and pituitary adenylate cyclase activating polypeptide (PACAP), our synthetic Maxa was found to bind PACAP specific receptors (PAC-1 receptors) with a high affinity, but low potency for the accumulation of cAMP in PC12 cells. Competitive binding studies of 125I-PACAP-27 to rat cortical membranes allowed exploration of the structural requirements for this interaction using mini-libraries constructed by solid-phase peptided synthesis, that include disulfide isomers, N-, C- and middle segment deleted peptides and analogs. Maxa as well as PACAP38 inhibited the specific binding of 125I-PACAP-27 with IC50 values of 3.89 and 4.90 nM, respectively. The most potent derivative of our synthetic Maxa-analogs with an IC50 value of 1.99 nM was Maxa[1–23 + 43–61, S–S14–51 Ala1,5] which consists of N- (position 1–23) and C- (position 43-61) terminal linear fragments cross-linked by a disulfide bridge between positions 14 and 51. This peptide did not increase intracellular cAMP, at a concentration of 100 nM, but inhibited cAMP accumulation induced by 1 nM PACAP-27 in PC12 cells, whereas wild Maxa increased intracellular cAMP although it was weaker than PACAP-27. Our data suggest deletion of the middle segment between residues 24–42 affords some derivatives that behave as low affinity antagonists.

Chronic lymphocytic leukemia (CLL) is a B‐cell malignancy in which apoptotic‐resistant mature lymphocytes relentlessly accumulate in bone‐marrow, lymph nodes and peripheral blood. In these body com...

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

Zusammenfassung Thrombozyten haben eine zentrale Bedeutung bei der Arteriosklerose und auch bei akuten thrombotischen Ereignissen. Körperliche Aktivität besitzt ein hohes Potential zur Vermeidung von Gefäßerkrankungen, dagegen wird intensiven Belastungen ein mögliches Risikopotential bei der Auslösung kardiovaskulärer Ereignisse zugeschrieben. Bei den notwendigen Untersuchungen thrombozytärer Funktionszustände nach körperlicher Belastung und Training werden zunehmend flowzytometrische Methoden eingesetzt, weil diese Technik die Möglichkeit impliziert, Veränderungen direkt auf der Oberfläche von Thrombozyten zu verifizieren. Da diese Veränderungen nach körperlichen Belastungen recht moderat ausfallen, sind Verfahren mit hoher Sensitivität notwendig. Zur Untersuchung dieser Veränderungen wurden vier Assays aufgebaut: a) Spontanexpression von Aktivierungsantigenen, b) Stimulationsassay, c) Thrombozyten-Leukozyten-Konjugate spontan und d) stimuliert. In einigen Untersuchungen konnte eine erhöhte CD62P- und PAC-1-Expression auf der Oberfläche von Thrombozyten nach körperlicher Belastung bei Gesunden nachgewiesen werden. Während die Untersuchungen der CD62P-Expression auf unstimulierten Thrombozyten nach körperlicher Belastung bei geringen Veränderungen uneinheitliche Resultate zeigen können, führen körperliche Belastungen im Stimulationsversuch regelhaft zu einer erhöhten Thrombozytenreaktivität. Obwohl der Bildung von Thrombozyten-Leukozyten-Konjugaten zunehmende analytische Bedeutung zukommt, wurden belastungsinduzierte Veränderungen der Konjugate bisher kaum untersucht. In den wenigen vorliegenden Studien konnte aber gezeigt werden, dass es nach körperlichen Belastungen zu einer Zunahme an Thrombozyten-Leukozyten-Konjugaten sowie auch ihrer Subpopulationen kommt, welche sich aber sehr schnell wieder auflösen. Die Ergebnisse zeigen, dass ein Stimulationsassay sowie die Bestimmung der Thrombozyten-Leukozyten-Konjugate sensitivere Verfahren darstellen als die Bestimmung von spontanen Aktivitätsmarkern wie z.B. das CD62P. Zusammenfassend sind die beschriebenen Methoden nicht nur für Untersuchungen von physiologischen Veränderungen bei Thrombozyten einsetzbar, sondern können auch ohne weiteres bei der Untersuchung von pathologischen Zuständen verwendet werden.

Hemoglobin vesicles (HbV), a type of liposome-encapsulated hemoglobin (LEH), have been recently developed as an artificial oxygen carrier. The efficacy of HbV has been demonstrated in the transfusion of HbV into rodent models of hemorrhagic shock. It is important to evaluate the compatibility of HbV with human blood cells. We examined the effects of HbV on human platelet activation in vitro by estimating the platelet release reaction and expression of platelet surface activation markers in the presence or absence of agonists. HbV concentration in the reaction volume was prepared at 20% or 40%. Preincubation of platelet-rich plasma (PRP) with HbV had no adverse effects on RANTES or serotonin release from platelets. Preincubation of whole blood with HbV also had no effects on exposure of P-selectin on platelets. However, binding of PAC-1, a monoclonal antibody that detects the activation-dependent conformational change of αIIbβ3 to platelets, was amplified by preincubation of whole blood with HbV in the presence of relatively low concentration of ADP. These results suggest that HbV enhances the binding of PAC-1 to platelets. The clinical meaning of this increased binding of PAC-1 needs to be addressed.

Severe haemophilia is a serious, haemorrhagic disorder of the plasmatic coagulation system. In this study we investigated, whether 'compensatory' activation of the platelet coagulation system occurs in this situation. Platelet function was investigated with aggregation, adhesion and flow cytometric assays. In addition, we performed clot and platelet plug formation tests and determined endogenous thrombin potentials in patients with severe haemophilia A or B; results were compared to those of healthy controls. Platelet aggregation in response to stimulation with ADP, ristocetin and epinephrine was similar in patients and controls; aggregation in response to collagen was reduced significantly in haemophiliacs. Flow cytometric analysis of P-selectin (CD 62P) and CD 63, of the conformationally changed GP IIb/IIIa with PAC 1 and of thrombospondin bound to CD 36 (GP IV) was performed at baseline and post stimulation. Baseline expression of all markers was similar in haemophiliacs and controls. After stimulation of the platelet thrombin receptors with the thrombin receptor activating peptide (TRAP) 6, the surface expression of all markers increased significantly; again, the expression was similar in haemophiliacs and controls. With thrombelastography and PFA 100 analysis, clot formation under low shear and platelet plug formation under high shear is measured. Both test results revealed a significantly reduced clot and platelet plug formation capacity in severe haemophiliacs. Our results did not reveal signs of enhanced platelet preactivation in haemophiliacs, indicating that baseline platelet reactivity in severe haemophilia remains in a neutral state, despite the severely haemorrhagic condition. As expected, both thrombin and clot formation capacities were impaired significantly in severe haemophilia. The reduced response to collagen-based platelet stimulation tests is indicative of a concomitant platelet function defect. This defect probably contributes to the intensity of bleeding events in patients with severe haemophilia.

BACKGROUND: Granulosa cell tumour (GCT) constitutes ~5% of ovarian neoplasms. Surgery remains the primary treatment modality due to a generally poor response to chemotherapy in GCT patients. Procaspase activating compound-1 (PAC1) is a small-molecule drug shown in vitro to sequester inhibitory zinc ions from the Caspase-3 (CASP3) zymogen allowing CASP3 to automature and execute apoptosis. PAC1 has demonstrated safety and efficacy in vivo against several cancers and is currently in Phase I trials for advanced malignancies. TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptosis ligand that can bind membrane-bound death receptors. This binding triggers the extrinsic apoptotic pathway resulting in activation of CASP3 to execute its proteolytic role and programmed cell death. TRAIL has been found to induce apoptosis selectively in cancer cells in vitro and in vivo, and has been well tolerated in human patients. We hypothesise that combining PAC1 activation of CASP3 with induction of apoptotic signaling by exogenous TRAIL will significantly heighten biologic effect, reduction of disease, and that these effects will be obtained at doses lower than those required by either agent alone. Here we report on in vitro experiments in support of this hypothesis, suggesting that combining PAC1 with TRAIL may be effective therapy for treatment of GCT. METHODS: The GCT cell line KGN was treated in vitro with a 6-log range of PAC1 concentration for 48 hours to establish a dose-response curve (using a real-time cell analyzer, RTCA). In parallel, KGN cells were treated with a 6-log range of TRAIL concentration to establish its dose-response curve. Calculated EC50 values were then used for both PAC1 (20 μM) and TRAIL (10 ng/mL) to evaluate the biologic response of simultaneous treatment with PAC1 and TRAIL, and TRAIL delayed 24 hours after PAC1 treatment (using resazurin viability assay and RTCA). Separately, cells from fresh primary and recurrent tumour samples were cultured in vitro for 5 days, then treated with PAC1 (20 μM), TRAIL (10 ng/mL), or the combination, and finally assayed for viability and caspase 3/7 activity 48 and 72 hours later. RESULTS: Dose-response assays indicate treatment with PAC1 strongly reduces viability of KGN cells compared to untreated control (p CONCLUSION: Combining CASP3 activator PAC1 with apoptosis-inducing agents may be an effective strategy for treatment of GCT and warrants preclinical assessment. Citation Format: Powel Crosley; Kate Agopsowicz; Marjut Pihlajoki; Markku Heikinheimo; Anniina Färkkilä; Mary Hitt. PAC–1 COMBINATION WITH TRAIL ENHANCES APOPTOSIS IN CELL–LINE AND PRIMARY CULTURED ADULT GRANULOSA CELL TUMOUR CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-086.

Treatment with thalidomide is associated with vascular thrombosis. The effect of thalidomide on platelet activation is unclear, although the use of aspirin is justified for thromboprophylaxis. A study on platelet activation markers was done among multiple myeloma patients receiving thalidomide therapy with warfarin as thromboprophylaxis. Strict criteria and procedure were set to avoid misinterpretation of platelet activation other than due to the thalidomide's effect. Blood specimen pre and post thalidomide therapy were used for flow cytometric analysis. Platelet surface P-selectin, CD62P expression and PAC-1 (antibody that recognizes conformational change of the GPIIb/IIIa complex) were examined by using three-colour flowcytometer. Increased expression marker for PAC-1 was observed after 4 weeks of thalidomide treatment (P 0.05) indicating one aspect of platelet activation activity seen in these patients. The mechanism of thrombosis by thalidomide is probably multifactorial and one of them is likely through platelet activation. Further study on the affected pathway/s in the platelet activation process would confirm the exact mechanism of thalidomide-induced thrombosis and potential extended usage of this drug in future.

Integrin alphaIIbbeta3 plays a critical role in platelet aggregation through its interaction with fibrinogen. Elucidation of the mechanisms of alphaIIbbeta3-fibrinogen interaction is critical to understanding hemostasis and thrombosis. Here we report that mutations of Gly-184, Tyr-189, Tyr-190, Phe-191, and Gly-193 within the predicted turn structure of the third amino-terminal repeat of alphaIIb significantly block binding of alphaIIbbeta3 to soluble fibrinogen. These mutations also block binding of alphaIIbbeta3 to ligand-mimetic monoclonal antibodies PAC-1, OP-G2, LJ-CP3, which have an RGD-related RYD sequence in their antigen-binding sites. These mutations do not significantly affect the expression of alphaIIbbeta3, in contrast to most of the natural alphaIIb mutations occurring in Glanzmann's thrombasthenic patients. The data suggest that these residues are critically involved in alphaIIbbeta3-ligand interactions.

This investigation addressed the paradox that disintegrins and small RGD-ligands readily bind to the resting alphaIIbbeta3 integrin, while macromolecules with similar integrin recognition motifs require an activated, or primed, receptor. Three structurally similar pharmaceutical integrin antagonists (eptifibatide, tirofiban, and roxifiban) were each incubated with resting alphaIIbbeta3; after drug wash-out, the receptor's ability to recognize PAC-1, an activation-dependent IgM with an RYD integrin-targeting site was measured. Their promotion of PAC-1:alphaIIbbeta3 binding (solid phase assay), eptifibatidetirofibanroxifiban, correlated with their ability to shift the receptor to an open conformer, as measured by analytical ultracentrifugation. Surface plasmon resonance (SPR) demonstrated that PAC-1 bound rapidly (k(on) approximately 5 x 10(5) l/mol-s, 25 degrees C) and tightly (Kd approximately 1 nM) to eptifibatide-primed integrins, captured on a biosensor using an IgG specific for alphaIIb's cytoplasmic domain. Varying the interval between integrin capture and antagonist dissociation indicated that transiently primed alphaIIbbeta3 retains the ability to rapidly bind PAC-1 from 2-90 min, although the dissociation rate increased at later times, indicative of a weakening of the complex. Fluorescence anisotropy (fluorophore-tagged analogue exchange assay) demonstrated that eptifibatide dissociates rapidly from alphaIIbbeta3 (half-time2 min), consistent with the priming window determined by SPR. van't Hoff analysis of alphaIIbbeta3:PAC-1's temperature-dependent Kd indicated entropy/enthalpy compensation, similar to (resting) integrin binding to the disintegrin echistatin. Eyring analysis of k(on) yielded DeltaG degrees approximately 10 kcal/mol for PAC-1 binding to primed alphaIIbbeta3, 3 kcal/mol lower than that of echistatin. These observations suggest that priming lowers the transition-state energy barrier, enabling rapid macromolecular ligand binding to activated integrins. Recognizing the limitations in extrapolating from laboratory to pathophysiological conditions, we propose that similar priming mechanisms may contribute to the unexpected platelet-activating effects of pharmaceutical integrin antagonists.

Background and Purpose Cilostazol, a phosphodiesterase III inhibitor, is known to be a useful antiplatelet agent that inhibits the progression of atherosclerosis in ischemic stroke. This study investigated the effects of combining cilostazol with aspirin on the expressions of P-selectin and PAC-1 on activated platelets in acute ischemic stroke. Methods We analyzed 70 patients with acute ischemic stroke (

PAC-1 is an oral administered procaspase-3 activating drug developed to overcome the malignant cell's barrier to apoptosis. The promising preclinical safety and efficacy of PAC-1 has led to its consideration for a first in human phase 1 clinical trial for recurrent and advanced malignancies. We have found that because PAC-1 is able to reach intracranial brain tumors in effective concentrations, that it is particularly useful for glioblastoma (GBM), an aggressive and invasive malignancy responsible for the most deaths due to brain cancer. In glioblastoma patients a worse prognosis was correlated with high expression of procaspase-3 mRNA, indicating that procaspase-3 activation may be involved in the pathology of the tumor progression. In preclinical testing of PAC-1, it showed significant survival benefit and low systemic toxicity, in orthotopic animal models of glioblastoma. An aqueous suspension of PAC-1 delivered orally to rats with intracranial 9L glioblastoma improved survival compared to untreated controls. Additionally, when PAC-1 was administered as an oral capsule to rats with 9L glioblastoma, median survival increased over three fold. Oral PAC-1 improved survival significantly also in a human xenograft model (p = 0.04) compared to controls. PAC-1 combined effectively with standard of care, and in some animals, either alone or in combination resulted in long term survival. PAC-1 has the ability to penetrate sufficiently within intracranial tumors and its ability to overcome resistance to apoptosis and activate widespread cell death specifically in the malignant cells makes it a promising small molecule for further preclinical and clinical development. Citation Format: Avadhut D. Joshi, Rachel C. Botham, Howard S. Roth, Timothy M. Fan, Theodore M. Tarasow, Paul J. Hergenrother, Gregory J. Riggins. An oral procaspase activating drug, PAC-1, shows preclinical promise for glioblastoma therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3620. doi:10.1158/1538-7445.AM2015-3620

Mutation and aberrant expression of apoptotic proteins are hallmarks of cancer. These changes prevent proapoptotic signals from being transmitted to executioner caspases, thereby averting apoptotic death and allowing cellular proliferation. Caspase-3 is the key executioner caspase, and it exists as an inactive zymogen that is activated by upstream signals. Notably, concentrations of procaspase-3 in certain cancerous cells are significantly higher than those in noncancerous controls. Here we report the identification of a small molecule (PAC-1) that directly activates procaspase-3 to caspase-3 in vitro and induces apoptosis in cancerous cells isolated from primary colon tumors in a manner directly proportional to the concentration of procaspase-3 inside these cells. We found that PAC-1 retarded the growth of tumors in three different mouse models of cancer, including two models in which PAC-1 was administered orally. PAC-1 is the first small molecule known to directly activate procaspase-3 to caspase-3, a transformation that allows induction of apoptosis even in cells that have defective apoptotic machinery. The direct activation of executioner caspases is an anticancer strategy that may prove beneficial in treating the many cancers in which procaspase-3 concentrations are elevated.

Our recent research has indicated that intracerebroventricular (ICV) administration of pituitary adenylate cyclase-activating polypeptide (PACAP) suppresses food intake in the goldfish. We therefore examined feeding- and fasting-induced changes in the expression of mRNAs for PACAP and its receptors in the goldfish brain, and the effect of ICV administration of synthetic PACAP on locomotor activity in the goldfish. Semiquantitative analysis revealed that the expression of mRNAs for PACAP and the PAC 1 receptor was significantly increased by excessive feeding (daily food supply at ≥5% of the body weight [BW]) for 7 days. ICV administration of PACAP at 20 pmol/g BW induced a significant decrease in locomotor activity during the 60-min post-treatment observation period. These results suggest that PACAP may have an anorexigenic action via hypomotility in goldfish.

Background/Aim: During hemodialysis, platelets and leukocytes are activated and form platelet-leukocyte coaggregates in which GPIIb/IIIa (CD41/CD61) and CD62P (P-selectin) are involved. However, it is still controversial whether platelet activation and platelet-leukocyte coaggregate formation are dependent on the dialyzer membrane material. Method: We examined the appearance of activation-dependent antibody on platelets as an index of platelet activation, and the appearance of platelet-specific antigen on leukocytes as an index of platelet-leukocyte coaggregation, during hemodialysis in 7 patients treated using regenerated cellulose (RC) membrane and next using polysulfone (PS) membrane. In order to reduce the influence of factors other than dialyzer membrane material, this study was conducted in a prospective crossover fashion using a pyrogen-free bicarbonate dialysate. Moreover, flow cytometric techniques with whole blood were employed, which reduce artificial cell activation during the cell or plasma separation procedure. The platelet-specific monoclonal antibodies used in this study were anti-CD61, PAC-1 (which recognizes only the conformationally activated GPIIb/IIIa) and anti-CD62P. Results: Changes in the percentage of PAC-1-positive platelets were significantly greater during hemodialysis with RC than with PS. However, changes in the percentage of CD62P-positive platelets were not significantly different between hemodialysis with RC and PS. Changes in the percentage of CD61- or CD62P-positive leukocytes were significantly greater during hemodialysis with RC than with PS. Although changes in percentage of PAC-1-positive platelets did not parallel those of CD62P-positive platelets during hemodialysis, there was a significant positive correlation between the percentage of CD61-positive leukocytes and the percentage of CD62P-positive leukocytes. Conclusion: This study, conducted in a prospective crossover fashion using a pyrogen-free bicarbonate dialysate in order to reduce the influence of factors other than the dialyzer membrane material, demonstrated that both the degrees of GPIIb/IIIa activation and platelet-leukocyte coaggregation were greater during hemodialysis with RC than PS.

We determined the morphological differences between NNKY5-5-induced and shear stress-induced platelet aggregates using confocal laser scanning microscopy, and investigated the effects of cytokines on these aggregates. As shear stress was increased from low to high, many aggregates were generated. Some platelets and aggregates already exhibited PAC-1 binding at low shear stress. And finally, aggregates were clearly stained by PAC-1 at high shear stress. Low shear stress-induced aggregates included fibrinogen, but not von Willebrand factor (vWF). In contrast, high shear stress-induced aggregates included both fibrinogen and vWF. NNKY5-5-induced platelet aggregates exhibited both PAC-1 binding and fibrinogen, but vWF was not found in them. The aggregate formation in NNKY5-5-treated platelet-rich plasma (PRP) at low shear stress was clearly more pronounced than that in untreated PRP. In addition, aggregates in NNKY5-5-treated PRP formed after 6 min under low shear stress were clearly stained by PAC-1 and included fibrinogen, but vWF was not found in them. In order to investigate the effects of cytokines on platelet activation under NNKY5-5 stimulation, changes in light transmission and light scatter were assessed with an AG-10. G-CSF, IL-6 and thrombopoietin (TPO) each enhanced light scatter compared to control PRP, although IL-3, GM-CSF, erythropoietin and stem cell factor did not. In particular, TPO significantly enhanced the ‘%-T’ and ‘S-Max’ of NNKY5-5-treated PRP. TPO clearly enhanced the aggregate formation with high shear stress or NNKY5-5 stimulation. These results suggest that the glycoprotein Ib (GPIb)-unmediated aggregates can be formed with only fibrinogen, but the GPIb-mediated aggregates by vWF and fibrinogen synergistically. In particular, the latter is formed more firmly, and both aggregates are enhanced by TPO.

Procaspase-activating compound-1 (PAC-1) is the first direct caspase-activating compound discovered; using an in vitro cell-free system of caspase activation. Subsequently, this compound was shown to induce apoptosis in a variety of cancer cells with promising in vivo antitumor activity in canine lymphoma model. Recently, we have reported its ability to kill drug-resistant, Bcl-2/Bcl-xL overexpressing and Bax/Bak-deficient cells despite the essential requirement of mitochondrial cytochrome c (cyt. c) release for caspase activation, indicating that the key molecular targets of PAC-1 in cancer cells are yet to be identified. Here, we have identified Ero1α-dependent endoplasmic reticulum (ER) calcium leakage to mitochondria through mitochondria-associated ER membranes (MAM) and ER luminal hyper-oxidation as the critical events of PAC-1-mediated cell death. PAC-1 treatment upregulated Ero1α in multiple cell lines, whereas silencing of Ero1α significantly inhibited calcium release from ER and cell death. Loss of ER calcium and hyper-oxidation of ER lumen by Ero1α collectively triggered ER stress. Upregulation of GRP78 and splicing of X-box-binding protein 1 (XBP1) mRNA in multiple cancer cells suggested ER stress as the general event triggered by PAC-1. XBP1 mRNA splicing and GRP78 upregulation confirmed ER stress even in Bax/Bak double knockout and PAC-1-resistant Apaf-1-knockout cells, indicating an induction of ER stress-mediated mitochondrial apoptosis by PAC-1. Furthermore, we identified BH3-only protein p53 upregulated modulator of apoptosis (PUMA) as the key molecular link that orchestrates overwhelmed ER stress to mitochondria-mediated apoptosis, involving mitochondrial reactive oxygen species, in a p53-independent manner. Silencing of PUMA in cancer cells effectively reduced cyt. c release and cell death by PAC-1.