Evaluation of the Serum Dkk-1, Tenascin-C, Oxidative Stress Markers Levels and Wnt Signaling Pathway Genes Expression in Patients with Alzheimer's Disease.

Early diagnosis of Alzheimer's disease (AD) using potential biomarkers may help with implementing early therapeutic interventions, monitoring, and ultimately disease treatment. The current study aimed to evaluate serum levels of DKK-1, TNC, and oxidative stress markers, as well as analyzing the expression of LRP6, GSK3A, and GSK3B genes in patients with AD. Serum levels of DKK-1, TNC, TOS, TAC, and MDA were measured in 40 AD patients and 40 healthy individuals. Additionally, the relative expressions of LRP6, GSK3A, and GSK3B genes in whole blood were evaluated. Receiver operating characteristic (ROC) analysis was used to investigate the incremental diagnostic value of each factor in the study groups. Mean serum levels of DKK-1, TNC, TOS, TAC, and MDA were significantly higher in the AD group compared to the healthy group (p < 0.001). Moreover, a significant difference was observed in the expression of LRP6 and GSK3A genes (p < 0.001) between patients and healthy groups. However, the expression of GSK3B did not significantly differ between the two groups (p > 0.05). With considerable sensitivity and specificity, ROC analysis demonstrated the diagnostic efficacy of DKK-1 and TNC serum levels in AD within an area under the ROC curve of ≥ 0.98 (p ˂ 0.001). The results showed that evaluating serum levels of DKK-1 and TNC, as well as assessing the expression of LRP6, could be utilized for diagnosis and monitoring of AD patients.

Combined but not single treatment with ethinylestradiol/levonorgestrel and spironolactone reduces plasminogen activator inhibitor-1 in insulin-resistant ovariectomised rats.

OBJECTIVE: Increased circulating level of plasminogen activator inhibitor-1 (PAI-1) is associated with menopausal oestrogen deficiency. We therefore hypothesised that the combined oral contraceptive (COC) with spironolactone (SPL) improves insulin resistance (IR) in ovariectomised (OVX) rats by reducing circulating PAI-1. METHODS: Twelve-week-old female Wistar rats were divided into sham-operated (SHM), OVX, OVX+SPL (0.25 mg/kg), COC (1.0 µg ethinylestradiol and 5.0 µg levonorgestrel) and OVX+COC+SPL rats treated with COC and SPL daily for eight weeks. IR was assessed by homeostatic model assessment of IR (HOMA-IR). RESULTS: Data showed that OVX rats had a higher HOMA-IR value that is associated with increased visceral adiposity, triglycerides (TG), total cholesterol/high-density lipoprotein cholesterol (HDL-C), TG/HDL-C, plasma insulin, GSK-3, corticosterone and decreased 17ß-oestradiol. However, these effects were attenuated in OVX+COC, OVX+SPL and OVX+COC+SPL rats compared to OVX rats. OVX rats had lower PAI-1 than SHM rats, whereas the beneficial effect on IR and other parameters by COC or SPL was accompanied with increased PAI-1. Improvement of IR and other parameters with combined COC and SPL in OVX rats was accompanied with reduced PAI-1. CONCLUSION: Taken together, COC or SPL improves IR independent of PAI-1, whereas a combination of COC and SPL in OVX rats ameliorates IR in a PAI-1-dependent manner.

Ameliorative effect of nicotine exposure on insulin resistance is accompanied by decreased cardiac glycogen synthase kinase-3 and plasminogen activator inhibitor-1 during oral oestrogen-progestin therapy.

CONTEXT: Cigarette smoking is considered to be a major risk factor for the development of diabetes and cardiovascular disease. Oestrogen-progestin combined oral contraceptive (COC) use has been associated with adverse cardiometabolic events. OBJECTIVE: We hypothesized that nicotine would ameliorate insulin resistance (IR) that is accompanied by decreased cardiac glycogen synthase kinase-3 (GSK-3) and plasminogen activator inhibitor-1 (PAI-1). METHODS: Female Wistar rats received (po) low-(0.1 mg/kg) or high-nicotine (1.0 mg/kg) with or without COC containing 5.0 µg levonorgestrel plus 1.0 µg ethinylestradiol daily for 8 weeks. RESULTS: Data showed that COC treatment or nicotine exposure led to IR, glucose deregulation, atherogenic dyslipidemia, increased corticosterone, aldosterone, cardiac and circulating GSK-3 values and PAI-1. However, these effects with the exception of corticosterone and aldosterone were ameliorated in COC + nicotine-exposed rats. CONCLUSION: Amelioration of IR induced by COC treatment is accompanied by decreased circulating PAI-1, cardiac PAI-1 and GSK-3 instead of circulating aldosterone and corticosterone.

Carbohydrate supplementation attenuates decrement in performance in overtrained rats.

Carbohydrate ingestion at the end of a single exercise is recognized as delaying fatigue and accelerating recovery, but whether chronic ingestion can prevent overtraining during periods of intense training has not yet been elucidated. This study aimed to determine whether carbohydrate supplementation minimizes overtraining in Wistar rats. The animals underwent 11 weeks of training (running) on a treadmill, and the last 3 weeks were designed to induce overtraining. One group was supplemented with carbohydrates (EX-CHO) (n = 13), 1 group had no supplementation (EX) (n = 10), and a third group remained inactive (C) (n = 9). Performance tests were given before training (Pr1) and at the 8th (Pr2) and 11th (Pr3) training week. Food intake, body weight, testosterone, cortisol, malondialdehyde, creatine kinase, and activities of the PI3-K, Akt-1, mTOR, and GSK-3 enzymes were measured. In the EX group, there was a significant 32.6% performance decrease at Pr3 when compared with Pr2. In addition, at protocol completion, the EX-CHO group had a greater gastrocnemius weight than did the C group (p = 0.02), which the EX group did not. Training caused anorexia, decreased testosterone (p = 0.001), and increased malondialdehyde (p = 0.009) in both exercise groups compared with the C group, with no influence of carbohydrate supplementation on these variables (p > 0.05). Compared with in the C group, the activity of Akt-1 was higher in the EX-CHO group but not in the EX group (p = 0.013). Carbohydrate supplementation promoted an attenuation in the performance decrement and maintained gastrocnemius muscle mass in animals that had undergone overtraining protocols, which was accompanied by increased activity of the Akt-1 molecular indicator.

The histone deacetylase sirtuin 2 is a new player in the regulation of platelet function.

BACKGROUND: Histone deacetylases (HDACs) play a key role in signaling in many cell types. However, little is known about the participation of HDACs, particularly sirtuins (SIRTs), in platelet reactivity. OBJECTIVE: To investigate the role of HDACs in platelets, we examined the effects of SIRT inhibition on platelet function and protein acetylation in human platelets. METHODS: We used washed platelets obtained from healthy subjects. Cambinol (SIRT1 and SIRT2 inhibitor), AGK2 (specific SIRT2 inhibitor) and EX527 (specific SIRT1 inhibitor) were used as SIRT inhibitors. Platelets were stimulated with collagen, thrombin, or U46619, and platelet responses were determined according to optical aggregometry findings, dense granule release, and cytosolic calcium levels (Fura-2AM fluorescence). Protein acetylation and phosphorylation were assessed by immunoblotting. RESULTS: SIRT inhibition remarkably reduced platelet responses (aggregation, granule release, and cytosolic calcium level; P < 0.05). SIRT2 was present in platelets at the level of mRNA and protein, and its specific inhibition reduced platelet responses. The acetylated protein pattern observed in resting platelets changed during platelet aggregation. Inhibition of SIRT2 increased the acetylation of Akt kinase, which in turn blocked agonist-induced Akt phosphorylation and glycogen synthase kinase-3ß phosphorylation, which are markers of Akt activity. Finally, collagen-induced aggregation provoked Akt acetylation. CONCLUSIONS: Regulation of protein acetylation by SIRT2 plays a central role in platelet function. The effects of SIRT2 are mediated in part by the acetylation and inhibition of Akt. These results open a new avenue for research into the control of platelet function, and may help to identify new therapeutic targets.

Multiplex immunoassay analysis of plasma shows prominent upregulation of growth factor activity pathways linked to GSK3ß signaling in bipolar patients.

BACKGROUND: Our understanding of bipolar disorder (BD) aetiology has advanced in recent years but our ability to translate this to improve patient care in the clinic is still limited. METHODS: In this study, we have measured the concentrations of 190 different molecules using sensitive multiplex immunoassays in plasma of 17 BD patients compared to 46 matched control subjects. RESULTS: The analyses led to the identification of 26 dysregulated proteins in BD patients compared to controls. These molecules were comprised mostly of growth factors, hormones, lipid transport and inflammatory proteins. Decreased apolipoprotein A1 has previously been associated with BD patients and this was confirmed in our study. LIMITATIONS: The present pilot study was limited by its small sample size, use of multiple drug treatments and the lack of dietary restrictions at the time of sampling. CONCLUSIONS: Future studies may increase our understanding of BD which will help to pave the way for much-needed patient stratification for better treatment outcomes.

GSK3ß, CREB, and BDNF in peripheral blood of patients with Alzheimer's disease and depression.

BACKGROUND: Glycogen synthase kinase-3ß (GSK3ß), cAMP-response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) play critical roles in neuronal survival, synaptic plasticity and memory and participate in the pathophysiology of both depressive disorder and Alzheimer's disease (AD). METHODS: This study was designed to determine the association of GSK3ß activity, CREB activity and BDNF concentration in peripheral blood of patients with AD with or without depressive symptoms and in depressive patients without AD. GSK3ß activity in platelets, CREB activity in lymphocytes and BDNF concentration in plasma, platelet-rich plasma or platelets were measured in 85 AD patients (36 of whom displayed co-morbid depressive symptoms), 65 non-AD patients with depressive disorder and 96 healthy controls. AD patients were clinically assessed for stage of dementia, cognitive impairment and severity of depressive symptoms. Depressive patients were clinically assessed for severity of depression. RESULTS: We observed increased CREB activity and GSK3ß activity in AD with depressive symptoms or in AD at mild stage of dementia. Decreased BDNF concentration was found in platelet-rich plasma of AD patients at moderate to severe stages of dementia or in AD without depressive symptoms. An association was revealed of the severity of cognitive impairment with the increase of GSK3ß in the platelets of AD patients with mild dementia. In depressive patients, a lower concentration of phosphorylated GSK3ß was associated with a higher severity of depression. Association was confirmed between severity of depression, CREB activation, and BDNF concentration in drug-naïve depressive patients. CONCLUSION: Our data demonstrated that AD is accompanied by increased CREB activity in lymphocytes and a decreased concentration of BDNF in platelet-rich plasma. The decreased BDNF concentration appears to correlate with moderate to severe stages of dementia in AD. Observation of decreased phosphorylation of GSK3ß in platelets of both AD patients with depressive symptoms and depressive patients after treatment confirms the role of increased GSK3ß activity in the pathophysiology of both AD and depressive disorder. Associations were confirmed between AD and platelet GSK3ß activity, lymphocyte CREB activity and plasma BDNF. CREB activity and platelet BDNF concentration seems to be related to depressive disorder.

Higher proportion of inactive Gsk3ß in platelets of elderly patients with bipolar disorder: an effect of treatment?

OBJECTIVE: It has been postulated that mood stabilizers inhibit glycogen synthase kinase 3-beta (Gsk3ß) activity, mainly through its phosphorylation on serine-9 (Ser9). However, in vivo studies addressing Gsk3ß activity in patients with bipolar disorder are scarce. Here, we compare Gsk3ß inactivation (as indicated by Ser9-phosphorylation) in platelets of elderly patients with bipolar disorder undergoing clinical treatment and healthy elderly adults not taking medication. METHODS: Platelet samples were obtained from 37 elderly adults (bipolar disorder = 19, controls = 18). Relative changes in Gsk3ß inactivation was estimated by comparing the ratios of phosphorylated Gsk3ß to total Gsk3ß (p-Gsk3ß Ser9/Gsk3ß) between the disease and control groups. RESULTS: Phosphorylated-Gsk3ß (p < 0.001) and the p-Gsk3ß Ser9/Gsk3ß ratio (p = 0.006) were elevated in bipolar patients. In the bipolar disorder group, p-Gsk3ß Ser9/Gsk3ß was positively correlated with serum lithium levels (r = 0.478, p = 0.039). CONCLUSIONS: Gsk3ß inactivation is higher in this group of elderly adults undergoing treatment for bipolar disorder. However, whether the treatment or the disease causes Gsk3ß inactivation was confounded by the lack of an unmedicated, bipolar control group and the non-uniform treatment regimens of the bipolar disorder group. Thus, further studies should help distinguish whether Gsk3ß inactivation is an effect of drug treatment or an intrinsic characteristic of bipolar disorder.

Utilizing glycogen synthase kinase-3ß as a marker for the diagnosis of graft-versus-host disease.

INTRODUCTION: Graft-versus-host disease (GVHD) is a deadly complication of allogeneic hematopoietic stem cell transplantation. Timely diagnosis is critical, because mortality rates for GVHD are high, increasing with disease severity. A diagnostic tool to predict GVHD before the onset of clinical symptoms could save many lives. On the cellular level, GVHD occurs when T cells from the transplant attack the tissues of the host, after perceiving them to be foreign. T-cell proliferation occurs even before clinical symptoms appear. Glycogen synthase kinase (GSK)-3ß is a protein which regulates proliferation in many cell types including T-cells. GSK-3ß has never been directly connected with GVHD and we applied GSK-3ß as a novel marker for GVHD prediction, seeking herein to determine whether GSK-3ß can be utilized as a marker for the early diagnosis of GVHD. METHODS: For the mouse model of acute GVHD, irradiated mice underwent allogeneic splenocyte transplantation and GSK-3ß expression levels and phosphorylation states were monitored in harvested spleens by western blot. FACS analysis was used to measure the number of T cells within the harvested spleens. RESULTS: Mice developed observable GVHD symptoms by day 5 post-transplantation, with severe symptoms on day 6 requiring mice to be killed for humane reasons. A significantly increased number of T cells in the allogeneic mice correlated with GVHD development. GSK-3ß protein expression levels and phosphorylation levels were significantly lower in allogeneic (GVHD) mice compared with negative (untreated) and positive (syngeneic transplant; non-GVHD) controls over time. CONCLUSION: GSK-3ß was directly connected with the onset and progression of GVHD. Therefore, it can be utilized as a marker for GVHD diagnosis in animals and potentially in humans.

Phase I study of enzastaurin and bevacizumab in patients with advanced cancer: safety, efficacy and pharmacokinetics.

PURPOSE: Given distinct mechanism of actions of enzastaurin and bevacizumab, preclinical studies suggest enhanced antitumor activity in combination. This phase I study assessed the combination's safety and efficacy. PATIENTS AND METHODS: Six advanced cancer patients could be enrolled in each of 11 cohorts. Patients received an enzastaurin loading dose. Oral enzastaurin (500 mg once daily [QD], 250 mg twice daily [BID], 375 mg BID, 500 mg BID, and 750 mg BID) was escalated in each cohort in combination with bevacizumab dosed at 5 mg/kg every 2 weeks, 10 mg/kg every 2 weeks, or 15 mg/kg every 3 weeks until a dose-limiting toxicity (DLT) occurred in 2 of 6 patients in any cohort. RESULTS: Sixty-seven patients (31, ovarian cancer [ovcar]) were evaluable for safety and efficacy. Six treatment-related DLTs occurred: grade 3 fatigue (n=4), grade 4 cerebral hemorrhage, and grade 3 elevated aspartate transaminase. Common drug-related toxicities included change in color of urine and stool, fatigue, pain, diarrhea, and nausea. The maximum tolerated dose of enzastaurin was 750 mg BID in combination with any tested bevacizumab dose/schedule. Overall response rate was 19.4 % (32.3 % ovcar). Median time to progression was 3.7 months (95 % confidence interval [CI], 2.7-5.5), with 8.3 months (95 % CI, 3.7-11.1) in ovcar. Overall, 35.9 % (50.4 % ovcar) of patients remained without disease progression after 6 months. CONCLUSION: The recommended phase II doses of enzastaurin were 500 mg QD up to 500 mg BID with any tested dose/schedule of bevacizumab. This combination demonstrated encouraging clinical activity, particularly in ovcar.

Long-term sertraline treatment increases expression and decreases phosphorylation of glycogen synthase kinase-3B in platelets of patients with late-life major depression.

BACKGROUND: Abnormal regulation of glycogen synthase kinase 3-beta (GSK3B) activity has been implicated in the pathophysiology of mood disorders. Many pharmacological agents, including antidepressants, can modulate GSK3B. The aim of the present study was to investigate the effect of short-and long-term sertraline treatment on the expression and phosphorylation of GSK3B in platelets of patients with late-life major depression. METHODS: Thirty-nine unmedicated elderly adults with major depressive disorder (MDD) were initially included in this study. The comparison group comprised 18 age-matched, healthy individuals. The expression of total and Ser-9 phosphorylated GSK3B (pGSK3B) was determined by Enzyme Immunometric Assay (EIA) in platelets of patients and controls at baseline, and after 3 and 12 months of sertraline treatments for patients only. During this period, patients were continuously treated with therapeutic doses of sertraline. GSK3B activity was indirectly estimated by calculating the proportion of inactive (phosphorylated) forms (pGSK3B) in relation to the total expression of the enzyme (i.e., GSK3B ratio). RESULTS: Depressed patients had significantly higher levels of pGSK3B as compared to controls (p < 0.001). Within the MDD group, after 3 months of sertraline treatment no significant changes were observed in GSK3B expression and phosphorylation state, as compared to baseline levels. However, after 12 months of treatment we found a significant increase in the expression of total GSK3B (p = 0.05), in the absence of any significant changes in pGSK3B (p = 0.12), leading to a significant reduction in GSK3B ratio (p = 0.001). CONCLUSIONS: Our findings indicate that GSK3B expression was upregulated by the continuous treatment with sertraline, along with an increment in the proportion of active forms of the enzyme. This is compatible with an increase in overall GSK3B activity, which may have been induced by the long-term treatment of late-life depression with sertraline.

GSK-3ß negatively regulates megakaryocyte differentiation and platelet production from primary human bone marrow cells in vitro.

Glycogen synthase kinase (GSK)-3, a constitutively active serine-threonine kinase, acts as a key regulator of major signaling pathways, including the Wnt, Hedgehog, and Notch pathways. Although a number of studies have demonstrated that GSK-3 plays a critical role in several cellular processes, such as differentiation, growth, and apoptosis, the effects of GSK-3 on platelet production have not been explored. There are two GSK-3 isoforms, GSK-3α and GSK-3ß. GSK-3ß is more highly expressed in platelets. In the present study, primary human bone marrow cells were cultured for 12 days in megakaryocyte lineage induction (MKLI) media to induce their differentiation into megakaryocyte (MK) lineage cells, in the presence or absence (+/-) of TWS119, a GSK-3ß inhibitor, during MK differentiation from stem cells and subsequent platelet production. MK maturation, MK production, and subsequent platelet production were markedly enhanced in cells cultured in TWS119 (+) compared with cells cultured in TWS119 (-). These effects on MK lineage cells were thrombopoietin (TPO)-dependent. We next performed the experiment focusing on the inhibitory effect of GSK-3ß on platelet production. Bone marrow cell-derived CD41 (+)/CD42b (+)/propidium iodide (-) cells in the large (MK)-sized cell population (day 8), as living mature MKs, were further cultured in the MKLI media in TWS119 (+/-) for 6 days. Platelet production from mature MKs in TWS119 (+) was approximately two-fold higher than that in TWS119 (-). The mature MKs were cultured in MKLI media in TWS119, in TPO (+/-), and platelet production was markedly decreased in TPO (-). This indicated that the GSK-3ß inhibition affects thrombopoiesis under these conditions with TPO. To identify the target(s) of GSK-3ß inhibition during differentiation into MK lineage cells, we performed a differential gene expression study and subsequent pathway analysis of the large (MK)-sized CD41 (+)/propidium iodide (-) cells cultured in TWS119 (+/-) for 3 days. The results of the analysis indicated that GSK-3ß inhibition during differentiation into MK lineage cells affected factors related to transcription, apoptosis, cell division, cell cycle, blood coagulation, lipid transport, keratin filament, metabolic processes, and the Wnt signaling and transforming growth factor-ß signaling pathways. These observations suggest that GSK-3ß inhibition and TPO treatment affect both megakaryopoiesis and thrombopoiesis in an in vitro differentiation system for primary human bone marrow cells.

Increased platelet GSK3B activity in patients with mild cognitive impairment and Alzheimer's disease.

The disruption of glycogen synthase kinase 3-beta (GSK3B) homeostasis has implications in the pathophysiology of neuropsychiatric disorders, namely Alzheimer's disease (AD). GSK3B activity is increased within the AD brain, favoring the hyperphosphorylation of microtubule-associated protein Tau and the formation of neurofibrillary tangles. Such abnormality has also been detected in leukocytes of patients with cognitive disorders. The aim of the present study was to determine the expression of total and phosphorylated GSK3B at protein level in platelets of older adults with varying degrees of cognitive impairment, and to compare GSK3B activity in patients with AD, mild cognitive impairment (MCI) and healthy controls. Sixty-nine older adults were included (24 patients with mild to moderate AD, 22 patients with amnestic MCI and 23 elderly controls). The expression of platelet GSK3B (total- and Ser-9 phosphorylated GSK3B) was determined by Western blot. GSK3B activity was indirectly assessed by means of the proportion between phospho-GSK3B to total GSK3B (GSK3B ratio), the former representing the inactive form of the enzyme. Ser-9 phosphorylated GSK3B was significantly reduced in patients with MCI and AD as compared to controls (p=0.04). Platelet GSK3B ratio was significantly decreased in patients with MCI and AD (p=0.04), and positively correlated with scores on memory tests (r=0.298, p=0.01). In conclusion, we corroborate previous evidence of increased GSK activity in peripheral tissues of patients with MCI and AD, and further propose that platelet GSK may be an alternative peripheral biomarker of this abnormality, provided samples are adequately handled in order to preclude platelet activation.

Antiplatelet activity of valproic acid contributes to decreased soluble CD40 ligand production in HIV type 1-infected individuals.

CD40L is a type II membrane glycoprotein of the TNF family that is found on activated T cells, B cells, and platelets. We previously reported that the soluble form of this inflammatory mediator (sCD40L) is elevated in the plasma and cerebrospinal fluid of HIV-1-infected, cognitively impaired individuals. In this study, we demonstrate that the mood-stabilizing drug valproic acid (VPA) reduces sCD40L levels in plasma samples of HIV-1-infected patients (n = 23) and in washed human platelets, which are the main source of circulating sCD40L. VPA also inhibited HIV-1 transactivator of transcription-induced release of sCD40L and platelet factor 4 in C57BL/6 mice. The mechanism by which VPA was able to do so was investigated, and we demonstrate that VPA, a known glycogen synthase kinase 3ß inhibitor, blocks platelet activating factor-induced activation of glycogen synthase kinase 3ß in platelets in a manner that alters sCD40L release from platelets. These data reveal that VPA has antiplatelet activity, and they convey important implications for the potential of VPA as an adjunct therapy not only for cognitively impaired patients with HIV-1 infection, but also numerous inflammatory diseases for which such antiplatelet therapies are currently lacking.

Effects of the cAMP-elevating agents cilostamide, cilostazol and forskolin on the phosphorylation of Akt and GSK-3beta in platelets.

Elevating intracellular cAMP has been shown to inhibit platelet function. cAMP interferes with platelet-activating signals which lead to aggregation inhibition, but the precise mechanism is unclear. The present study examined if cAMP-elevating agents inhibited phosphatidylinositol 3-kinase (PI3-kinase) signaling in rat platelets by immunoblotting. Akt is one of the key molecules downstream of PI3K, and is phosphorylated by collagen stimulation. The phosphodiesterase-3 (PDE3) inhibitors cilostamide and cilostazol, and the adenylate cyclase activator forskolin, inhibited collagen-induced Akt phosphorylation at Ser473. The inhibitory effects of these cAMP-elevating agents on Akt phosphorylation were unchanged in the presence of the PKA (cyclic AMP-dependent protein kinase) inhibitor H-89. These effects were consistent with inhibition of platelet aggregation. It is known that inhibition of Akt phosphorylation leads to inhibition of phosphorylation of glycogen synthase kinase 3-beta (GSK-3beta), which is an effector of Akt, but cAMP-elevating agents stimulated GSK-3beta phosphorylation at Ser9. The PKA inhibitor H-89 attenuated GSK-3beta phosphorylation. The cAMP-elevating agents cilostamide, cilostazol and forskolin did not directly affect the enzyme activity of PI3-kinase. These results suggested that cAMP-elevating agents have two effects on PI3K signalling: inhibition of Akt phosphorylation independent of PKA; and stimulation of GSK-3beta phosphorylation dependent on PKA. Our results provide new insights into the inhibitory effect of cAMP-elevating agents on platelet function.

A phase I trial of enzastaurin in patients with recurrent gliomas.

PURPOSE: Enzastaurin is a selective inhibitor of protein kinase C beta. Prior phase I studies did not show increased drug exposures with escalating once daily administration. Limits from gastrointestinal absorption may be overcome by twice daily dosing, potentially improving antitumor effects. EXPERIMENTAL DESIGN: We conducted a phase I dose escalation study in 26 patients with recurrent malignant glioma, stratified by use of enzyme-inducing antiepileptic drugs, to investigate whether divided twice daily dosing results in higher exposures compared with once daily dosing. Phosphorylated glycogen synthase 3 beta was analyzed as a potential biomarker of enzastaurin activity. RESULTS: Enzastaurin was poorly tolerated at all dose levels evaluated (500, 800, and 1,000 mg total daily), with thrombocytopenia and prolonged QTc as dose-limiting toxicities. The average drug concentration of enzastaurin under steady-state conditions was doubled by twice daily dosing compared with daily dosing [1.990; 90% confidence interval (CI), 1.450-2.730]. Additionally, geometric mean ratios doubled with 800 versus 500 mg dosing for both daily (2.687; 90% CI, 1.232-5.860) and twice daily regimens (1.852; 90% CI, 0.799-4.292). Two patients achieved long-term benefit (over 150 weeks progression free). CONCLUSIONS: Higher and more frequent dosing of enzastaurin resulted in improved drug exposure but with unacceptable toxicity at the doses tested. Phosphorylated glycogen synthase 3 beta may be a useful biomarker of the biological activity of enzastaurin. Enzastaurin has activity in a subset of malignant glioma patients and warrants continued study in combination with other agents using a maximal once daily dose of 500 mg.

Glycogen-synthase kinase-3beta is decreased in peripheral blood mononuclear cells of patients with mild cognitive impairment.

Glycogen-synthase kinase-3 (GSK-3) plays a central role in Alzheimer's disease (AD). It is involved in the hyper-phosphorylation of Tau and the increased production of beta-amyloid. Despite its eminent role, only one study has been published so far in AD blood samples, reporting an increase of GSK-3alpha and -3beta levels in white blood cells. In this study, we measured GSK-3alpha and -3beta by quantitative ELISA in peripheral blood mononuclear cells of patients with mild cognitive impairment (MCI), AD and depression in comparison to healthy subjects. In contrast to the previous study, we observed a significant reduction of GSK-3beta levels in MCI patients and less pronounced in AD but not in depression. The data indicate that high GSK-3 brain activity is not reflected in peripheral blood mononuclear cells. Therefore, we conclude that more longitudinal studies have to be performed to clarify whether GSK-3 blood levels may qualify as disease specific biological markers.

A dose-ranging study of the pharmacokinetics and pharmacodynamics of the selective apoptotic antineoplastic drug (SAAND), OSI-461, in patients with advanced cancer, in the fasted and fed state.

PURPOSE: To evaluate the safety, pharmacokinetics and determine the recommended dose of the selective apoptotic antineoplastic drug, OSI-461 administered on a twice-daily regimen to patients with advanced solid malignancies. METHODS: In this phase I trial, 33 patients were treated with OSI-461 doses ranging from 400 to 1,200 mg given twice daily in 4-week cycles. Pharmacokinetic studies were performed to characterize the plasma disposition of OSI-461 and the effect of food intake on OSI-461 absorption. Secondary biomarker studies were performed to assess the biologic activity of OSI-461 including the measurement of pGSK-3beta, a PKG substrate, and pharmacogenetic studies to identify polymorphisms of CYP3A that influence drug metabolism and of ABCG2, involved in drug resistance. RESULTS: Thirty-three patients were treated with 86 courses of OSI-461. The dose-limiting toxicities were grade 3 abdominal pain, found in one patient at the 1,000 mg BID fed dose level and all patients at the 1,200 mg BID fed dose level. There was also one episode each of grade 3 fatigue and grade 3 constipation at the 1,000 and 1,200 mg BID fed dose levels, respectively. Other common toxicities included mild to moderate fatigue, nausea, anorexia and mild elevation in bilirubin. Pharmacokinetic studies of OSI-461 revealed approximately a twofold increase in AUC(0-24) when OSI-461 was administered with food. An increase in pGSK-3beta post-dose was seen in the majority of patients and was greater at higher dose levels. No patients exhibited CYP3A4 polymorphisms, while 100% of patients were found to have the CYP3A5*3/CYP3A5*3 polymorphism. Two known polymorphisms of the ABCG2 gene, G34 --> A34 and C421 --> A421, occurred at frequencies of 11.76 and 29%, respectively. CONCLUSIONS: Toxicity and pharmacodynamic data show that the recommended oral dose of OSI-461 is 800 mg twice daily administered with food. The drug appears to be well-tolerated, and overall bioavailability appears to be markedly increased when the drug is administered with food. These results support further disease-directed evaluations of OSI-461 at a dose of 800 mg BID in combination with other chemotherapeutic agents.

Use of a variably sensitive selected reaction monitoring method to extend the linear dynamic range for quantitative high-performance liquid chromatography/tandem mass spectrometry.

A method has been devised with the capacity to extend the linear dynamic range of a triple quadrupole mass spectrometer operated in the selected reaction monitoring (SRM) mode of analysis. This extended range experiment can be realized by simultaneously acquiring variably sensitive data, via collision energy adjustment, for the same precursor-to-product ion transition within a single SRM method. While this method can be applied universally to many different study types without any detrimental effect to the analysis or throughput, it was applied herein to acquire and quantify, within a single analysis, the concentrations of GSK-A in a multiple-dose rodent study, that previously required a dilution scheme. Using this methodology, the linear dynamic range of GSK-A was increased over traditional methods by nearly two orders of magnitude, from 2.00-10,000 ng/mL to 0.500-100,000 ng/mL.