Off-Target drug effects resulting in altered gene expression events with epigenetic and "Quasi-Epigenetic" origins.

This review synthesizes examples of pharmacological agents who have off-target effects of an epigenetic nature. We expand upon the paradigm of epigenetics to include "quasi-epigenetic" mechanisms. Quasi-epigenetics includes mechanisms of drugs acting upstream of epigenetic machinery or may themselves impact transcription factor regulation on a more global scale. We explore these avenues with four examples of conventional pharmaceuticals and their unintended, but not necessarily adverse, biological effects. The quasi-epigenetic drugs identified in this review include the use of beta-lactam antibiotics to alter glutamate receptor activity and the action of cyclosporine on multiple transcription factors. In addition, we report on more canonical epigenome changes associated with pharmacological agents such as lithium impacting autophagy of aberrant proteins, and opioid drugs whose chronic use increases the expression of genes associated with addictive phenotypes. By expanding our appreciation of transcriptomic regulation and the effects these drugs have on the epigenome, it is possible to enhance therapeutic applications by exploiting off-target effects and even repurposing established pharmaceuticals. That is, exploration of "pharmacoepigenetic" mechanisms can expand the breadth of the useful activity of a drug beyond the traditional drug targets such as receptors and enzymes.

A multicenter phase II study incorporating high-dose rituximab and liposomal doxorubicin into the CODOX-M/IVAC regimen for untreated Burkitt's lymphoma.

BACKGROUND: Despite improvement with intensive multi-agent chemotherapy, 2-year progression-free survival (PFS) rates for adults with high-risk Burkitt's lymphoma (BL) remains <55%. PATIENTS AND METHODS: We conducted a phase II trial for newly diagnosed classic BL utilizing liposomal doxorubicin (Adriamycin) in lieu of doxorubicin and incorporating intravenous rituximab (at 500 mg/m(2) twice/cycle) into the CODOX-M/IVAC regimen. Correlative analyses included paired serum and cerebrospinal fluid (CSF) rituximab levels and close examination of cardiac function. RESULTS: Among 25 BL patients, the median age was 44 years (23-70) and 4 patients were HIV positive. There were 20 high-risk and 5 low-risk patients. At baseline, 40% of high-risk patients had bone marrow involvement, 35% had bulky disease and 15% had central nervous system involvement. The overall response rate was 100% (complete remission 92%). At 34-month median follow-up, the 2-year PFS and overall survival (OS) rates for all patients were 80% and 84%, respectively (low-risk: both 100%; high-risk: 76% and 81%, respectively). Furthermore, the 2-year PFS, OS, and disease-specific survival (DSS) rates for high-risk, HIV-negative patients were 84%, 89% and 100%, respectively. Adverse events (AEs) appeared to be consistent with prior CODOX-M/IVAC data, although there were several grade 3 cardiac events noted (all declined ejection fraction without clinical symptoms). The mean serum rituximab levels at 24 h after cycles 1 and 3 for patients without relapse were 258 and 306 µg/ml, respectively, versus 131 and 193 µg/ml, respectively, for patients with early progression (P = 0.002 and 0.002, respectively). The mean CSF rituximab levels for all patients were 0.11 and 0.24 µg/ml, respectively, at cycle 1 (24/72 h), which equated to serum:CSF ratios of 0.05% and 0.20%, respectively. CONCLUSIONS: The integration of rituximab into CODOX-M/IVAC for adult BL was feasible and tolerable, while changes in cardiac function warrant continued examination. This regimen was associated with excellent survival rates for HIV-negative BL. Further investigation of the predictive value of serum rituximab is needed. Clinicaltrials.gov NCT00392990.

Spatial and temporal patterns of neutral and adaptive genetic variation in the endangered African wild dog (Lycaon pictus).

Deciphering patterns of genetic variation within a species is essential for understanding population structure, local adaptation and differences in diversity between populations. Whilst neutrally evolving genetic markers can be used to elucidate demographic processes and genetic structure, they are not subject to selection and therefore are not informative about patterns of adaptive variation. As such, assessments of pertinent adaptive loci, such as the immunity genes of the major histocompatibility complex (MHC), are increasingly being incorporated into genetic studies. In this study, we combined neutral (microsatellite, mtDNA) and adaptive (MHC class II DLA-DRB1 locus) markers to elucidate the factors influencing patterns of genetic variation in the African wild dog (Lycaon pictus); an endangered canid that has suffered extensive declines in distribution and abundance. Our genetic analyses found all extant wild dog populations to be relatively small (N(e) < 30). Furthermore, through coalescent modelling, we detected a genetic signature of a recent and substantial demographic decline, which correlates with human expansion, but contrasts with findings in some other African mammals. We found strong structuring of wild dog populations, indicating the negative influence of extensive habitat fragmentation and loss of gene flow between habitat patches. Across populations, we found that the spatial and temporal structure of microsatellite diversity and MHC diversity were correlated and strongly influenced by demographic stability and population size, indicating the effects of genetic drift in these small populations. Despite this correlation, we detected signatures of selection at the MHC, implying that selection has not been completely overwhelmed by genetic drift.

Selective neutralization of prostaglandin E2 blocks inflammation, hyperalgesia, and interleukin 6 production in vivo.

The role of prostaglandin E2 (PGE2) in the development of inflammatory symptoms and cytokine production was evaluated in vivo using a neutralizing anti-PGE2 monoclonal antibody 2B5. In carrageenan-induced paw inflammation, pretreatment of rats with 2B5 substantially prevented the development of tissue edema and hyperalgesia in affected paws. The antibody was shown to bind the majority of PGE2 produced at the inflammatory site. In adjuvant-induced arthritis, the therapeutic administration of 2B5 to arthritic rats substantially reversed edema in affected paws. Anti-PGE2 treatment also reduced paw levels of IL-6 RNA and serum IL-6 protein without modifying tumor necrosis factor RNA levels in the same tissue. In each model, the antiinflammatory efficacy of 2B5 was indistinguishable from that of the nonsteroidal antiinflammatory drug indomethacin, which blocked the production of all PGs. These results indicate that PGE2 plays a major role in tissue edema, hyperalgesia, and IL-6 production at sites of inflammation, and they suggest that selective pharmacologic modulation of PGE2 synthesis or activity may provide a useful means of mitigating the symptoms of inflammatory disease.

Achilles' heel of sociality revealed by energetic poverty trap in cursorial hunters.

This study empirically tests two foundation ecological theories: (1) pack hunting is a driver for the evolution of sociality; and (2) species have a finite energy potential, whereby increased maintenance costs result in decreased reproductive effort. Using activity and prey data from 22 packs of African wild dogs (Lycaon pictus), we parameterized a model detailing the energetic cost/benefit of cooperative hunting. Larger pack size increased foraging time, prey size, and capture probability while reducing chase distance, resulting in a rapidly increasing net rate of energy intake up to a pack size of five, which peaked at 10 individuals and then declined. With a streamlined body plan necessary for hypercursoriality limiting stomach capacity in smaller packs, it was demonstrated that the group hunting benefit will rather accrue to widely foraging predators than to "sit-and-wait" ones. Reproductive effort, measured by the number of pups born, revealed smaller litters with decreasing pack size, validated finite energy theory, and highlighted a "poverty trap" where smaller groups have lower foraging gains, smaller litters, and increased vulnerability to extirpation. Consequently, these results demonstrated a mechanistic example of pervasive selection for maximal body size (Cope's rule), leading to a macroevolutionary ratchet, where sociality linked to hypercursoriality is betrayed by an Achilles' heel.

Tissue factor induction in human monocytes. Two distinct mechanisms displayed by different alloantigen-responsive T cell clones.

One component of the cellular immune response to antigens is the expression of procoagulant activity (PCA) by monocytes and macrophages. Induction of human monocyte PCA in response to alloantigenic stimulation requires the collaboration of HLA-DR-responsive T cells. In mixed lymphocyte cultures (MLCs), the induction of monocyte tissue factor appears to be mediated exclusively by a T cell-derived lymphokine. We have used a soft agar cloning method to generate alloantigen-responsive T cell clones from MLCs between irradiated Daudi lymphoblastoid cells and human peripheral blood mononuclear cells. Developing clones were screened for the ability to induce PCA in fresh autologous monocytes in response to Daudi stimulator cells. PCA induction was observed with some, but not all, proliferating T cell clones and two modes of induction were apparent. Some T cell clones mediated PCA induction exclusively by lymphokine production, whereas other clones delivered induction signals by direct cellular collaboration with the monocyte effector cells. These two inductive pathways were represented in distinct, non-inclusive functional subsets of T cell clones. Constitutive production of soluble inducer signals was not observed in T inducer clones. The magnitude of the monocyte PCA response increased in response to an increase in the allogeneic stimulator/T clone responder ratio, and third-party allogeneic cells were unable to elicit the PCA-inducing lymphokine signals from T inducer clones. Both modes of induction were shown to generate tissue factor protein activity in monocytes. Collectively, these results suggest that PCA induction can be initiated in response to alloantigens through collaboration with certain OKT3+, OKT4+, OKT8-, OKM1- T inducer clones, and that induction can be mediated by at least two different functional subsets of human T cells. Stimulation with the appropriate alloantigen may elicit both lymphokine and T cell-contact pathways of induction of tissue factor in human monocytes.

Selective inhibition of cyclooxygenase (COX)-2 reverses inflammation and expression of COX-2 and interleukin 6 in rat adjuvant arthritis.

Prostaglandins formed by the cyclooxygenase (COX) enzymes are important mediators of inflammation in arthritis. The contribution of the inducible COX-2 enzyme to inflammation in rat adjuvant arthritis was evaluated by characterization of COX-2 expression in normal and arthritic paws and by pharmacological inhibition of COX-2 activity. The injection of adjuvant induced a marked edema of the hind footpads with coincident local production of PGE2. PG production was associated with upregulation of COX-2 mRNA and protein in the affected paws. In contrast, the level of COX-1 mRNA was unaffected by adjuvant injection. TNF-alpha and IL-6 mRNAs were also increased in the inflamed paws as was IL-6 protein in the serum. Therapeutic administration of a selective COX-2 inhibitor, SC-58125, rapidly reversed paw edema and reduced the level of PGE2 in paw tissue to baseline. Interestingly, treatment with the COX-2 inhibitor also reduced the expression of COX-2 mRNA and protein in the paw. Serum IL-6 and paw IL-6 mRNA levels were also reduced to near normal levels by SC-58125. Furthermore, inhibition of COX-2 resulted in a reduction of the inflammatory cell infiltrate and decreased inflammation of the synovium. Notably, the antiinflammatory effects of SC-58125 were indistinguishable from the effects observed for indomethacin. These results suggest that COX-2 plays a prominent role in the inflammation associated with adjuvant arthritis and that COX-2 derived PGs upregulate COX-2 and IL-6 expression at inflammatory sites.

Regulation of tissue factor gene expression in the monocyte procoagulant response to endotoxin.

Tissue factor is the cellular receptor and cofactor for plasma factor VIIa which initiates the coagulation protease cascade on cell surfaces. Although normally absent from all intravascular cell types, tissue factor can be induced to appear on circulating monocytes and vascular endothelial cells by specific inflammatory or immunological mediators. In this study, we have examined the regulation of endotoxin-induced tissue factor gene expression in peripheral blood monocytes.

The evolving role of alemtuzumab in management of patients with CLL.

New insights into prognostic markers and the pathophysiology of chronic lymphocytic leukemia (CLL) are beginning to change the concept of CLL treatment. Alemtuzumab has evolved as a potent and effective therapeutic option for patients with CLL. Specifically, alemtuzumab has demonstrated substantial efficacy in fludarabine-refractory patients and has shown impressive responses when administered subcutaneously in first-line therapy. A group of experts gathered to discuss new data related to the use of alemtuzumab in CLL and to assess its place in the rapidly changing approach to treating patients with this disease. The main goals of this program were to update the management guidelines that were previously developed for alemtuzumab-treated patients and to provide community oncologists with guidance on the most effective way to integrate alemtuzumab into a CLL treatment plan.

Analysis of traditional versus three-dimensional augmented curriculum on anatomical learning outcome measures.

This study examined whether student learning outcome measures are influenced by the addition of three-dimensional and digital teaching tools to a traditional dissection and lecture learning format curricula. The study was performed in a semester long graduate level course that incorporated both gross anatomy and neuroanatomy curricula. Methods compared student examination performance on material taught using lecture and cadaveric dissection teaching tools alone or lecture and cadaveric dissection augmented with computerized three-dimensional teaching tools. Additional analyses were performed to examine potential correlations between question difficulty and format, previous student performance (i.e., undergraduate grade point average), and a student perception survey. The results indicated that students performed better on material in which three-dimensional (3D) technologies are utilized in conjunction with lecture and dissection methodologies. The improvement in performance was observed across the student population primarily on laboratory examinations. Although, student performance was increased, students did not perceive that the use of the additional 3D technology significantly influenced their learning. The results indicate that the addition of 3D learning tools can influence long-term retention of gross anatomy material and should be considered as a beneficial supplement for anatomy courses. Anat Sci Educ 9: 529-536. © 2016 American Association of Anatomists.

Lenalidomide treatment and prognostic markers in relapsed or refractory chronic lymphocytic leukemia: data from the prospective, multicenter phase-II CLL-009 trial.

Efficacy of lenalidomide was investigated in 103 patients with relapsed/refractory chronic lymphocytic leukemia (CLL) treated on the prospective, multicenter randomized phase-II CLL-009 trial. Interphase cytogenetic and mutational analyses identified TP53 mutations, unmutated IGHV, or del(17p) in 36/96 (37.5%), 68/88 (77.3%) or 22/92 (23.9%) patients. The overall response rate (ORR) was 40.4% (42/104). ORRs were similar irrespective of TP53 mutation (36.1% (13/36) vs 43.3% (26/60) for patients with vs without mutation) or IGHV mutation status (45.0% (9/20) vs 39.1% (27/68)); however, patients with del(17p) had lower ORRs than those without del(17p) (21.7% (5/22) vs 47.1% (33/70); P=0.049). No significant differences in progression-free survival and overall survival (OS) were observed when comparing subgroups defined by the presence or absence of high-risk genetic characteristics. In multivariate analyses, only multiple prior therapies (⩾3 lines) significantly impacted outcomes (median OS: 21.2 months vs not reached; P=0.019). This analysis indicates that lenalidomide is active in patients with relapsed/refractory CLL with unfavorable genetic profiles, including TP53 inactivation or unmutated IGHV. (ClinicalTrials.gov identifier: NCT00963105).

Suppression of telomerase activity and cytokine messenger RNA levels in acute myelogenous leukemia cells in vivo in patients by amifostine and interleukin 4.

High levels of telomerase activity and high rates of cell proliferation are associated with a poor prognosis in acute myelogenous leukemia. Furthermore, cytokine production by leukemia cells is believed to play an important role in determining the proliferative characteristics of leukemia. The in vivo effects of two noncytotoxic agents on these parameters were determined in 33 acute myelogenous leukemia patients. Three daily doses of interleukin (IL) 4 or a single dose of amifostine reduced telomerase activity in the leukemia marrow cells in 7 of 9 and 11 of 13 patients, respectively. The administration of a single dose of amifostine resulted in a reduction in tumor necrosis factor alpha and IL-6 transcript levels in the marrow cells of 10 of 13 and 12 of 13 patients in which these transcripts were present. The administration of only three doses of IL-4 or a single dose of amifostine has a significant effect on leukemia cell parameters, which are believed to have a significant impact on the in vivo biology of the disease and on its response to remission induction therapy.

Cell cycle kinetic studies in 68 patients with myelodysplastic syndromes following intravenous iodo- and/or bromodeoxyuridine.

Sixty-eight patients with myelodysplastic syndromes (MDS) received sequential infusions of iodo- and/or bromodeoxyuridine for cell kinetic analysis. Bone marrow biopsy sections were treated by appropriate antibodies and a labeling index (LI), duration of S-phase (Ts), and total cell cycle time (Tc) of myeloid cells were determined. The mean LI was 28.4%, Ts was 11.8 hours and Tc was 40.7 hours. The %LI decreased as the disease evolved from refractory anemia toward transformation to acute leukemia (p = 0.04). Double-labeling of biopsy sections for apoptosis and proliferation showed that 30-90% of S-phase cells in MDS patients were simultaneously apoptotic or "antonymous." We conclude that MDS are highly proliferative disorders in which the ineffective hematopoiesis is probably the result of excessive apoptosis rather than slow proliferation.

Genetic diseases conferring resistance to infectious diseases.

This review considers available evidence for mechanisms of conferred adaptive advantages in the face of specific infectious diseases. In short, we explore a number of genetic conditions, which carry some benefits in adverse circumstances including exposure to infectious agents. The examples discussed are conditions known to result in resistance to a specific infectious disease, or have been proposed as being associated with resistance to various infectious diseases. These infectious disease-genetic disorder pairings include malaria and hemoglobinopathies, cholera and cystic fibrosis, tuberculosis and Tay-Sachs disease, mycotic abortions and phenylketonuria, infection by enveloped viruses and disorders of glycosylation, infection by filoviruses and Niemann-Pick C1 disease, as well as rabies and myasthenia gravis. We also discuss two genetic conditions that lead to infectious disease hypersusceptibility, although we did not cover the large number of immunologic defects leading to infectious disease hypersusceptibilities. Four of the resistance-associated pairings (malaria/hemogloginopathies, cholera/cystic fibrosis, tuberculosis/Tay-Sachs, and mycotic abortions/phenylketonuria) appear to be a result of selection pressures in geographic regions in which the specific infectious agent is endemic. The other pairings do not appear to be based on selection pressure and instead may be serendipitous. Nonetheless, research investigating these relationships may lead to treatment options for the aforementioned diseases by exploiting established mechanisms between genetically affected cells and infectious organisms. This may prove invaluable as a starting point for research in the case of diseases that currently have no reliably curative treatments, e.g., HIV, rabies, and Ebola.

1,2-Diarylcyclopentenes as selective cyclooxygenase-2 inhibitors and orally active anti-inflammatory agents.

A series of 1,2-diarylcyclopentene methyl sulfones and sulfonamides have been shown to be remarkably potent and selective cyclooxygenase-2 (COX-2) inhibitors. The methyl sulfone analogs 7 showed excellent COX-2 activity, with IC50s ranging from 0.003 (7f,n) to 0.87 (7o) microM. In addition, most analogs of 7 showed no activity (IC50 > 100 microM) against the COX-1 enzyme. Replacement of the methyl sulfone moiety with a sulfonamide group gave a slightly more potent (typically 2-5-fold) but less selective COX-2 inhibitor, mainly due to an increase (20- > 100-fold) in COX-1 activity. However, in vitro COX-1/COX-2 selectivity for the sulfonamides 8 could be increased in many cases by simply incorporating a substituent at the 3-position of the phenyl group. Furthermore, in vitro selectivity increased with the size and number of substituents, as demonstrated in the selectivity trend of 8k (8000) > 8j (1900) > 8i (500) > 8h (100). More importantly, the sulfonamide COX-2 inhibitors showed greatly enhanced oral activity in the rat model of established adjuvant-induced arthritis, with inhibition values of 79.0% (8a), 81.5% (8c), and 83.0% (8g) at 1 mg/kg. On the basis of its overall biological profile, sulfonamide 8c was evaluated as a potential clinical candidate, displaying an ED50 of 22 mpk in the rat carrageenan-induced paw edema model and an ED50 of 0.16 mpk in the rat established adjuvant-induced arthritis model with no indication of gastrointestinal toxicity in rats and mice at 200 mpk. In addition, a preparative-scale synthetic route to sulfonamide 8c has been developed.

Novel terphenyls as selective cyclooxygenase-2 inhibitors and orally active anti-inflammatory agents.

A novel series of terphenyl methyl sulfones and sulfonamides have been shown to be highly potent and selective cyclooxygenase-2 (COX-2) inhibitors. The sulfonamide analogs 17 and 21 were found to be much more potent COX-2 inhibitors and orally active anti-inflammatory agents than the corresponding methyl sulfone analogs 16 and 20, respectively, albeit with some decrease in COX-2 selectivity. Structure-activity relationship studies have determined that incorporation of two fluorine atoms in the central phenyl group, as in 20 and 21, is extremely advantageous for both in vitro COX-2 potency and selectivity as well as in vivo activity. Several noticeable examples in the 1,2-diaryl-4,5-difluorobenzenesulfonamide series are 21a-c,k,l,n (COX-2, IC50 = 0.002-0.004 microM), in which all have in vitro COX-1/COX-2 selectivity > 1000. In addition, sulfonamides 21a,b,d,g,j,m,n,q were shown to have greatly enhanced oral activity with more than 90% inhibition of prostaglandin E2 production in the air pouch model of inflammation. Furthermore, sulfonamide 21b was found to be very active in the rat adjuvant-induced arthritis model (ED50 = 0.05 mg/kg) and carrageenan-induced hyperalgesia assay (ED50 = 38.7 mg/kg) with no indication of gastrointestinal toxicity in rats at doses as high as 200 mg/kg.

1,2-Diarylimidazoles as potent, cyclooxygenase-2 selective, and orally active antiinflammatory agents.

Series of 1,2-diarylimidazoles has been synthesized and found to contain highly potent and selective inhibitors of the human COX-2 enzyme. The paper describes a short synthesis of the target 1,2-diarylimidazoles starting with aryl nitriles. Different portions of the diarylimidazole (I) were modified to establish SAR. Systematic variations of the substituents in the aryl ring B have yielded very potent (IC50 = 10-100 nm) and selective (1000-12500) inhibitors of the COX-2 enzyme. The study on the influence of substituents in the imidazole ring established that a CF3 group at position 4 gives the optimum oral activity. A number of the diarylimidazoles showed excellent inhibition in the adjuvant induced arthritis model (e.g., ED50 = 0.02 mpk for 22 and 34). The diarylimidazoles are also potent inhibitors of carrageenan-induced edema (ED50 = 9-30 mpk) and hyperalgesia (ED50 = 11-40 mpk). Several orally active diarylimidazoles show no GI toxicity in the rat and mouse up to 200 mpk.

Diarylspiro[2.4]heptenes as orally active, highly selective cyclooxygenase-2 inhibitors: synthesis and structure-activity relationships.

A novel series of 5,6-diarylspiro[2.4]hept-5-enes was shown to provide highly potent and selective cyclooxygenase-2 (COX-2) inhibitors. A study of structure-activity relationships in this series suggests that 3,4-disubstituted phenyl analogs are generally more selective than 4-substituted phenyl analogs and that replacement of the methyl sulfone group on the 6-phenyl ring with a sulfonamide moiety results in compounds with superior in vivo pharmacological properties, although with lower COX-2 selectivity. Several compounds have been shown to possess promising pharmacological properties in adjuvant-induced arthritis and edema analgesia models. The absence of gastrointestinal (GI) toxicity at 200 mpk of several selected compounds in rats and mice corresponds well with the weak potency for inhibition of COX-1 observed in the enzyme assay. Methyl sulfone 55 and sulfonamide 24 were shown to have superior in vivo pharmacological profiles, low GI toxicity, and good oral bioavailability and duration of action.

Synthesis and biological evaluation of the 1,5-diarylpyrazole class of cyclooxygenase-2 inhibitors: identification of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benze nesulfonamide (SC-58635, celecoxib).

A series of sulfonamide-containing 1,5-diarylpyrazole derivatives were prepared and evaluated for their ability to block cyclooxygenase-2 (COX-2) in vitro and in vivo. Extensive structure-activity relationship (SAR) work was carried out within this series, and a number of potent and selective inhibitors of COX-2 were identified. Since an early structural lead (1f, SC-236) exhibited an unacceptably long plasma half-life, a number of pyrazole analogs containing potential metabolic sites were evaluated further in vivo in an effort to identify compounds with acceptable pharmacokinetic profiles. This work led to the identification of 1i (4-[5-(4-methylphenyl)-3-(trifluoromethyl)- H-pyrazol-1-yl]benzenesulfonamide, SC-58635, celecoxib), which is currently in phase III clinical trials for the treatment of rheumatoid arthritis and osteoarthritis.