Pharmacodynamics and proteomic analysis of acalabrutinib therapy: similarity of on-target effects to ibrutinib and rationale for combination therapy.

Acalabrutinib, a highly selective Bruton's tyrosine kinase inhibitor, is associated with high overall response rates and durable remission in previously treated chronic lymphocytic leukemia (CLL); however, complete remissions were limited. To elucidate on-target and pharmacodynamic effects of acalabrutinib, we evaluated several laboratory endpoints, including proteomic changes, chemokine modulation and impact on cell migration. Pharmacological profiling of samples from acalabrutinib-treated CLL patients was used to identify strategies for achieving deeper responses, and to identify additive/synergistic combination regimens. Peripheral blood samples from 21 patients with relapsed/refractory CLL in acalabrutinib phase I (100-400 mg/day) and II (100 mg BID) clinical trials were collected prior to and on days 8 and 28 after treatment initiation and evaluated for plasma chemokines, reverse phase protein array, immunoblotting and pseudoemperipolesis. The on-target pharmacodynamic profile of acalabrutinib in CLL lymphocytes was comparable to ibrutinib in measures of acalabrutinib-mediated changes in CCL3/CCL4 chemokine production, migration assays and changes in B-cell receptor signaling pathway proteins and other downstream survival proteins. Among several CLL-targeted agents, venetoclax, when combined with acalabrutinib, showed optimal complementary activity in vitro, ex vivo and in vivo in TCL-1 adoptive transfer mouse model system of CLL. These findings support selective targeting and combinatorial potential of acalabrutinib.

Development of Multiwell-Plate Methods Using Pure Cultures of Methanogens To Identify New Inhibitors for Suppressing Ruminant Methane Emissions.

Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H2 and CO2 that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen Methanococcus maripaludis strain S2 and the rumen methanogen Methanobrevibacter species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against M. maripaludis as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against M. maripaludis and M. AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions.IMPORTANCE Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions, and new technologies are required to control emissions in the agriculture technology (agritech) sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimized, and validated a high-throughput 96-well microtiter plate assay for growing environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions.

Sci-Thurs PM: Delivery-12: Correction and calibration of megavoltage cone-beam CT images for the calculation of the dose of the day.

PURPOSE: To show that accurate dose calculations can be achieved with megavoltage cone-beam CT (MVCBCT) images of head-and-neck (H&N) and prostate sites, allowing the verification of the daily dose distribution received by these patients. METHOD AND MATERIALS: Corrections for the cupping and missing data artifacts seen on MVCBCT images were developed for both H&N and pelvic imaging. MVCBCT images of six H&N and two prostate patients were acquired weekly during the course of their treatment. Several regions of interest were contoured including: the prostate and rectum and the spinal cord and parotids. Dose calculation was performed with the MVCBCT images using the plan beams. Variations from treatment plan dosimetric endpoints were analyzed. RESULTS: Dose calculations with kVCT and corrected MVCBCT images of the H&N (pelvic) regions show standard deviations of 1.9% (0.6%). The mean dose to the right parotid of H&N patients had an average increase of 18% during treatment. The maximum dose to 1% of the spinal cord went up by 2% on average. For prostate patients on one fraction the dose received by 95% of the prostate diminished by 3%. One patient had an average increase of 3.6% of the maximum dose received by 1% of the rectum. CONCLUSION: MVCBCT can be used to verify daily dose distributions for H&N and prostate patients. An increase in the mean dose to normal tissues was observed during H&N treatment. Underdosage of the prostate and the dosimetric consequences of volume changes in rectum and bladder were observed. Research supported by Siemens.

Mechanisms of the vasorelaxant effect of Danshen (Salvia miltiorrhiza) in rat knee joints.

The aim of this study is to investigate the mechanisms of the vasorelaxant effect of the crude extract of Salvia miltiorrhiza (family: Labiatae), also known as "Danshen", in rat knee joints. Changes in blood flow of rat knee joints were measured in vivo by a laser Doppler perfusion imager. Topical administration of Danshen onto the exposed knee joint blood vessels produced dose-dependent increases in blood flow. Treatment of the rat knee joint with 2x 1 nmol of atropine, 2x 0.1 nmol of propranolol, or 2x 0.1 nmol of a mixture of pyrilamine plus cimitedine produced no change on the vasodilator response to Danshen. However, significant inhibition of the Danshen-induced vasodilator response was observed in knee joints treated with 2x 100 nmol of N(G)-nitro-L-arginine methyl ester (L-NAME), 2x 100 nmol of flurbiprofen, 2x 10 nmol of the calcitnonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37), and also in knee joints but had been denervated by capsaicin treatment or by surgery. Intravenous administration of low doses of Danshen (2.5 and 6 mg/kg) did not affect the systemic blood pressure but significantly increased knee joint blood flow, whereas, high doses of Danshen (167 and 381 mg/kg) produced hypotension with concurrent decreases in knee joint blood flow. These findings indicate that the knee joint blood vessels are more sensitive to the relaxant effect of Danshen compared to blood vessels in the general circulation. The vasorelaxant effect of Danshen was found to be partly mediated by CGRP released from sensory nerves, and nitric oxide and prostaglandins also played a part. However, there is no evidence to support a role for muscarinic receptors, adrenoceptors, or histamine receptors.

Anti-cancer and pro-apoptotic effects of an herbal medicine and Saccharomyces cerevisiae product (CKBM) on human hepatocellular carcinoma HepG2 cells in vitro and in vivo.

Hepatocellular carcinoma is a major health problem worldwide. Different treatment strategies have been developed to cope with this problem. Herbal medicine is now widely studied in both Eastern and Western countries. In this study, we used both in vitro and in vivo model to illustrate the anti-tumor effect of a product, CKBM, consisting of herbal medicine and specially processed Saccharomyces cerevisiae. Dose-dependent anti-proliferation effect was observed on in vitro growth of human hepatoma HepG2 cells after 48 hours incubation with CKBM. At the 50% inhibitory concentration (IC50) no significant toxic effect was observed on normal human fibroblasts Hs68 and human liver WRL-68 cells. The results of morphological changes, detection of DNA fragmentation, flow cytometric analysis and Western blot analysis indicated that this anti-tumor effect of CKBM was mediated via the process of apoptosis. In addition, HepG2 cells- bearing nude mice model was used for in vivo anti-tumor study. Our results showed that 14-day treatment with 0.8 ml daily dosage of CKBM could inhibit 54.1% of tumor growth. The plasma activities of enzymes specific for heart and liver, namely creatine kinase, lactate dehydrogenase, aspartate transaminase and alanine transaminase, remained at normal levels, indicated that CKBM did not produce toxicity to the host.

Isolation of various forms of sterol beta-D-glucoside from the seed of Cycas circinalis: neurotoxicity and implications for ALS-parkinsonism dementia complex.

The factors responsible for ALS-parkinsonism dementia complex (ALS-PDC), the unique neurological disorder of Guam, remain unresolved, but identification of causal factors could lead to clues for related neurodegenerative disorders elsewhere. Earlier studies focused on the consumption and toxicity of the seed of Cycas circinalis, a traditional staple of the indigenous diet, but found no convincing evidence for toxin-linked neurodegeneration. We have reassessed the issue in a series of in vitro bioassays designed to isolate non-water soluble compounds from washed cycad flour and have identified three sterol beta-d-glucosides as potential neurotoxins. These compounds give depolarizing field potentials in cortical slices, induce alterations in the activity of specific protein kinases, and cause release of glutamate. They are also highly toxic, leading to release of lactate dehydrogenase (LDH). Theaglycone form, however, is non-toxic. NMDA receptor antagonists block the actions of the sterol glucosides, but do not compete for binding to the NMDA receptor. The most probable mechanism leading to cell death may involve glutamate neuro/excitotoxicity. Mice fed cycad seed flour containing the isolated sterol glucosides show behavioral and neuropathological outcomes, including increased TdT-mediated biotin-dUTP nick-end labelling (TUNEL) positivity in various CNS regions. Astrocytes in culture showed increased caspase-3 labeling after exposure to sterol glucosides. The present results support the hypothesis that cycad consumption may be an important factor in the etiology of ALS-PDC and further suggest that some sterol glucosides may be involved in other neurodegenerative disorders.

Novel terpenoid-type quinones isolated from Pycnanthus angolensis of potential utility in the treatment of type 2 diabetes.

Using an ethnomedical-based drug discovery program, two previously unknown compounds (SP-18904 and SP-18905) from Pycnanthus angolensis were isolated that lower glucose concentrations in mouse models of type 2 diabetes. SP-18904 and SP-18905 are terpenoid-type quinones that significantly lowered plasma glucose concentration (p <.05) when given orally to either ob/ob or db/db mice, both of which are hyperglycemic and hyperinsulinemic. The antihyperglycemic actions of SP-18904 and SP-18905 were associated with significant decreases in plasma insulin concentrations (p <.05), suggesting that both compounds lowered glucose by enhancing insulin-mediated glucose uptake. This was supported by the insulin suppression test in ob/ob mice. Studies in hyperglycemic, insulin-deficient mice and in vitro experiments on 3T3-L1 adipocytes further supported this conclusion. As such, these two terpenoid-type quinones represent a new class of compounds of potential use in the treatment of type 2 diabetes.

Masoprocol (nordihydroguaiaretic acid): a new antihyperglycemic agent isolated from the creosote bush (Larrea tridentata).

An ethnomedically-driven approach was used to evaluate the ability of a pure compound isolated from the creosote bush (Larrea tridentata) to lower plasma glucose concentration in two mouse models of type 2 diabetes. The results indicated that plasma glucose concentration fell approximately 8 mmol/l in male C57BL/ks-db/db or C57BL/6J-ob/ob mice following the oral administration of masoprocol (nordihydroguaiaretic acid), a well known lipoxygenase inhibitor. The decline in plasma glucose concentration following masoprocol treatment in the mice was achieved without any change in plasma insulin concentration. In addition, oral glucose tolerance improved and the ability of insulin to lower plasma glucose concentrations was accentuated in masoprocol-treated db/db mice. These data raise the possibility that masoprocol, or other lipoxygenase inhibitors, represents a new approach to the pharmacological treatment of Type 2 diabetes.

Effect of electro-acupuncture on chronic painful conditions in general medical practice--a 4 years' study.

A total of 204 consecutive, recalcitrant painful conditions were treated by electroacupuncture and closely observed up to 4 years in a group medical practice in Australia. The majority (151 cases or 74%) had enjoyed significant pain reduction well beyond 3 months. The causes of failure in the remainders were analysed and discussed. The encouraging overall results led to a renewed call for this mode of therapy to be used more widely as an adjunct in family medicine.

Mechanism of protection by verapamil and nifedipine from anoxic injury in isolated cardiac myocytes.

This study was designed to determine whether slow Ca2+ channel blocking agents exert a direct protective effect on the anoxic myocardial cell and, if so, what the mechanism of protection is. Isolated Ca2+-tolerant rat cardiac myocytes were incubated under aerobic or anaerobic conditions, with or without verapamil or nifedipine, in the resting and contractile state. Protection against cell injury was assessed by preservation of rod-shaped morphology, cellular ATP levels, intracellular ionic composition, and lactate dehydrogenase release. Resting myocytes incubated anaerobically lost their rod-shaped appearance, accumulated Na+ and lost K+, and suffered a significant loss of cellular ATP. The release of lactate dehydrogenase into the medium was increased twofold, indicating significant membrane injury. Verapamil (1 microM) or nifedipine (1 microM) did not afford any protection against anoxic injury as measured by these parameters. Furthermore, on reoxygenation, anoxic verapamil- and nifedipine-treated myocytes had significantly higher cellular Ca2+ levels than control aerobic cells. When anoxic myocytes were paced at a rate of 300/min for 10 min, there were marked decreases in the number of rod-shaped cells and cellular ATP levels, whereas identically paced aerobic cells sustained no significant injury. Verapamil (1 microM) or nifedipine (1 microM) protected cells paced at 300/min from anoxic injury, but the cells were unable to sustain contraction rates at the frequency of the imposed pacing.(ABSTRACT TRUNCATED AT 250 WORDS)