Elucidating host cell response pathways and repurposing therapeutics for SARS-CoV-2 and other coronaviruses.

COVID-19, first reported in late 2019, is an ongoing pandemic that has been causing devastation across the globe. Although there are multiple vaccines that can prevent severe symptoms, effective COVID-19 therapeutics are still of importance. Using our proprietary in silico engine, we screened more than 22,000 unique compounds represented by over half a million gene expression profiles to uncover compounds that can be repurposed for SARS-CoV-2 and other coronaviruses in a timely and cost-efficient manner. We then tested 13 compounds in vitro and found three with potency against SARS-CoV-2 with reasonable cytotoxicity. Bortezomib and homoharringtonine are some of the most promising hits with IC50 of 1.39 µM and 0.16 µM, respectively for SARS-CoV-2. Tanespimycin and homoharringtonine were effective against the common cold coronaviruses. In-depth analysis highlighted proteasome, ribosome, and heat shock pathways as key targets in modulating host responses during viral infection. Further studies of these pathways and compounds have provided novel and impactful insights into SARS-CoV-2 biology and host responses that could be further leveraged for COVID-19 therapeutics development.

Restricted Delivery of Talazoparib Across the Blood-Brain Barrier Limits the Sensitizing Effects of PARP Inhibition on Temozolomide Therapy in Glioblastoma.

Poly ADP-ribose polymerase (PARP) inhibitors, including talazoparib, potentiate temozolomide efficacy in multiple tumor types; however, talazoparib-mediated sensitization has not been evaluated in orthotopic glioblastoma (GBM) models. This study evaluates talazoparib ± temozolomide in clinically relevant GBM models. Talazoparib at 1-3 nmol/L sensitized T98G, U251, and GBM12 cells to temozolomide, and enhanced DNA damage signaling and G2-M arrest in vitroIn vivo cyclical therapy with talazoparib (0.15 mg/kg twice daily) combined with low-dose temozolomide (5 mg/kg daily) was well tolerated. This talazoparib/temozolomide regimen prolonged tumor stasis more than temozolomide alone in heterotopic GBM12 xenografts [median time to endpoint: 76 days versus 50 days temozolomide (P = 0.005), 11 days placebo (P < 0.001)]. However, talazoparib/temozolomide did not accentuate survival beyond that of temozolomide alone in corresponding orthotopic xenografts [median survival 37 vs. 30 days with temozolomide (P = 0.93), 14 days with placebo, P < 0.001]. Average brain and plasma talazoparib concentrations at 2 hours after a single dose (0.15 mg/kg) were 0.49 ± 0.07 ng/g and 25.5±4.1 ng/mL, respectively. The brain/plasma distribution of talazoparib in Bcrp-/- versus wild-type (WT) mice did not differ, whereas the brain/plasma ratio in Mdr1a/b-/- mice was higher than WT mice (0.23 vs. 0.02, P < 0.001). Consistent with the in vivo brain distribution, overexpression of MDR1 decreased talazoparib accumulation in MDCKII cells. These results indicate that talazoparib has significant MDR1 efflux liability that may restrict delivery across the blood-brain barrier, and this may explain the loss of talazoparib-mediated temozolomide sensitization in orthotopic versus heterotopic GBM xenografts. Mol Cancer Ther; 16(12); 2735-46. ©2017 AACR.

Discovery of pyrazolopyrimidine derivatives as novel inhibitors of ataxia telangiectasia and rad3 related protein (ATR).

The ATR pathway is a critical mediator of the replication stress response in cells. In aberrantly proliferating cancer cells, this pathway can help maintain sufficient genomic integrity for cancer cell progression. Herein we describe the discovery of 19, a pyrazolopyrimidine-containing inhibitor of ATR via a strategic repurposing of compounds targeting PI3K.

Temozolomide analogs with improved brain/plasma ratios - Exploring the possibility of enhancing the therapeutic index of temozolomide.

Temozolomide is a chemotherapeutic agent that is used in the treatment of glioblastoma and other malignant gliomas. It acts through DNA alkylation, but treatment is limited by its systemic toxicity and neutralization of DNA alkylation by upregulation of the O6-methylguanine-DNA methyltransferase gene. Both of these limiting factors can be addressed by achieving higher concentrations of TMZ in the brain. Our research has led to the discovery of new analogs of temozolomide with improved brain:plasma ratios when dosed in vivo in rats. These compounds are imidazotetrazine analogs, expected to act through the same mechanism as temozolomide. With reduced systemic exposure, these new agents have the potential to improve efficacy and therapeutic index in the treatment of glioblastoma.

Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide.

INTRODUCTION: The androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer. METHODS: We examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR. RESULTS: In a cohort of 192 women with ER + breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HR = 4.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HR = 4.04, 95% CI: 1.68, 9.69; p = 0.002) and disease specific survival (HR = 2.75, 95% CI: 1.11, 6.86; p = 0.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/AR + breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis. CONCLUSIONS: AR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR+ tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth.

Enzalutamide, an androgen receptor signaling inhibitor, induces tumor regression in a mouse model of castration-resistant prostate cancer.

BACKGROUND: Enzalutamide (formerly MDV3100 and available commercially as Xtandi), a novel androgen receptor (AR) signaling inhibitor, blocks the growth of castration-resistant prostate cancer (CRPC) in cellular model systems and was shown in a clinical study to increase survival in patients with metastatic CRPC. Enzalutamide inhibits multiple steps of AR signaling: binding of androgens to AR, AR nuclear translocation, and association of AR with DNA. Here, we investigate the effects of enzalutamide on AR signaling, AR-dependent gene expression and cell apoptosis. METHODS: The expression of AR target gene prostate-specific antigen (PSA) was measured in LnCaP and C4-2 cells. AR nuclear translocation was assessed in HEK-293 cells stably transfected with AR-yellow fluorescent protein. The in vivo effects of enzalutamide were determined in a mouse xenograft model of CRPC. Differential gene expression in LNCaP cells was measured using Affymetrix human genome microarray technology. RESULTS: We found that unlike bicalutamide, enzalutamide lacked AR agonistic activity at effective doses and did not induce PSA expression or AR nuclear translocation. Additionally, it is more effective than bicalutamide at inhibiting agonist-induced AR nuclear translocation. Enzalutamide induced the regression of tumor volume in a CRPC xenograft model and apoptosis in AR-over-expressing prostate cancer cells. Finally, gene expression profiling in LNCaP cells indicated that enzalutamide opposes agonist-induced changes in genes involved in processes such as cell adhesion, angiogenesis, and apoptosis. CONCLUSIONS: These results indicate that enzalutamide efficiently inhibits AR signaling, and we suggest that its lack of AR agonist activity may be important for these effects.

Novel p38α mitogen-activated protein kinase inhibitor shows analgesic efficacy in acute postsurgical dental pain.

SCIO-469 is a selective p38α mitogen-activated protein kinase (MAPK) inhibitor for preclinical models of acute pain. This prospective, double-blind, randomized clinical study compared efficacy and safety of oral SCIO-469, ibuprofen, and placebo in postsurgical dental pain. Subjects (n = 263) undergoing extraction of 1 or more impacted mandibular third molars received preoperative treatment with SCIO-469 (150, 210, or 300 mg), ibuprofen (400 mg), or placebo; the 210-mg group received 90 mg postoperatively. A 4-point categorical scale and a 100-mm visual analogue scale were used to measure pain intensity. The primary end point was median time from first incision to first rescue medication using the Kaplan-Meier product limit estimator. All SCIO-469 groups had significantly longer times to rescue medication compared with placebo; preoperative and postoperative treatment with 210 + 90 mg SCIO-469 resulted in 8.1 hours versus 4.1 hours to rescue for placebo (P = .003). Ibuprofen also increased time to rescue medication (6.6 hours) versus placebo (P = .04). Dizziness, headache, and nausea were the most frequently reported adverse events. This is the first clinical demonstration of antinociceptive effects in acute pain with preoperative administration of a p38α MAPK inhibitor.

Cognition-enhancing properties of Dimebon in a rat novel object recognition task are unlikely to be associated with acetylcholinesterase inhibition or N-methyl-D-aspartate receptor antagonism.

Dimebon (dimebolin) treatment enhances cognition in patients with Alzheimer's disease (AD) or Huntington's disease. Although Dimebon was originally thought to improve cognition and memory through inhibition of acetylcholinesterase (AChE) and the N-methyl-d-aspartate (NMDA) receptor, the low in vitro affinity for these targets suggests that these mechanisms may not contribute to its clinical effects. To test this hypothesis, we assessed whether Dimebon enhances cognition in rats and if such an action is related to either mechanism or additional candidate mechanisms. Acute oral administration of Dimebon to rats (0.05, 0.5, and 5 mg/kg) enhanced cognition in a novel object recognition task and produced Dimebon brain concentrations of 1.7 +/- 0.43, 14 +/- 5.1, and 172 +/- 94 nM, respectively. At these concentrations, Dimebon did not alter the activity of recombinant human or rat brain AChE. Unlike the AChE inhibitors donepezil and galantamine, Dimebon did not change acetylcholine levels in the hippocampus or prefrontal cortex of freely moving rats. Dimebon displays affinity for the NMDA receptor (K(i) = 105 +/- 18 microM) that is considerably higher than brain concentrations associated with cognition enhancement in the novel object recognition task and 200-fold weaker than that of memantine (K(i) = 0.54 +/- 0.05 microM). Dimebon did not block NMDA-induced calcium influx in primary neuronal cells (IC(50) > 50 microM), consistent with a lack of significant effect on this pathway. The cognition-enhancing effects of Dimebon are unlikely to be mediated by AChE inhibition or NMDA receptor antagonism, and its mechanism of action appears to be distinct from currently approved medications for AD.

Topical alpha-selective p38 MAP kinase inhibition reduces acute skin inflammation in guinea pig.

Certain skin pathologies, including psoriasis, are thought to be immune-mediated inflammatory diseases. Available literature clearly indicates the involvement of inflammatory cells (neutrophils, T cells, and macrophages), their cytokines, and the p38 mitogen-activated protein kinase (MAPK) signaling pathway in the pathophysiology of psoriasis. Neutrophils play an important role in the formation of acute inflammatory changes in psoriasis. Acute inflammation or acute flares in psoriasis remain poorly addressed in clinical medicine. In this communication, we first establish a simple and reproducible model for studying neutrophil-mediated acute skin inflammation. Using the hairless guinea pig, due to the similarity of skin architecture to that of human, acute inflammation was induced with an intradermal injection of 50 µg/mL lipopolysaccharide (LPS) in 50 µL solution. Myeloperoxidase (MPO) activity was measured by MPO-positive neutrophils and shown to increase for 24-hours post-injection. Simultaneously, the level of phosphorylated p38 MAPK was documented for 48-hours post-LPS injection in the skin. Next, we used this model to examine the therapeutic potential of an α-selective p38 MAPK inhibitor, SCIO-469. A comparison of topical application of SCIO-469 at 5 mg/mL or 15 mg/mL to vehicle revealed that SCIO-469 dose-dependently reduces acute skin inflammation and that this effect is statistically significant at the higher dose. Further examination of tissues that received this dose also revealed statistically significant reduction of MPO activity, phosphorylated p38 MAPK, interleukin-6, and cyclooxygenase-2. These data suggest that the α-selective p38 MAPK inhibitor, SCIO-469, acts as a topical anti-inflammatory agent via the p38 MAPK pathway to reduce neutrophil induced acute inflammation in the skin. These observations suggest that α-selective p38 MAPK inhibition may be an effective therapeutic strategy to manage acute skin inflammation.

Long-term effects of B-type natriuretic peptide infusion after acute myocardial infarction in a rat model.

INTRODUCTION: The effects of exogenous B-type natriuretic peptide (BNP) on postmyocardial infarction (MI) are not known. We tested the hypothesis that in vivo infusion of BNP would improve cardiac function and affect left ventricular (LV) remodeling in an experimental model of MI. METHODS: MI was induced by coronary ligation in rats and confirmed by echocardiography. 19 rats were randomized to 1 of 3 groups: sham (n = 7), MI + saline (n = 5), MI + BNP (400 ng.kg(-1).minute(-1)) (n = 7). Infusions were delivered for 7 days via venous catheters tunneled to an infusion pump. Rats were followed for 8 weeks. Echocardiography, hemodynamics, histology, and in vivo and ex vivo pressure-volume relationships were examined. RESULTS: LV systolic pressure, LV dP/dtmax, and infarct size improved with BNP treatment versus control MI group (132 +/- 4 vs.110 +/- 2 mm Hg, 8097 +/- 317 vs. 5816 +/- 378 mm Hg/s, 19.3% +/- 1.6% vs. 23.3% +/- 1.9%, respectively; all P < 0.05). Ex vivo end-diastolic pressure-volume relationship demonstrated reduced diastolic dysfunction after BNP therapy (P < 0.05 vs. control MI). Serum BNP levels confirmed delivery of BNP. CONCLUSIONS: We demonstrate beneficial effects on LV function and decreased LV remodeling with BNP infusion in an experimental model of acute MI.

Clinical implications of defective B-type natriuretic peptide.

Our understanding of the natriuretic peptide system continues to evolve rapidly. B-type natriuretic peptide (BNP), originally thought to be a simple volume-regulating hormone that is produced in response to cardiac stretch, has been shown to also play important roles in modulating bronchodilation, endothelial function, and cardiac remodeling. Recent data demonstrate that elevated levels of BNP in patients with heart failure do not represent a simple ratcheting up of normal production in response to increased stimulus. Instead, we now know that chronic stimulation of BNP synthesis induces a reversion to fetal gene expression, resulting in production of high molecular weight forms of BNP that are functionally deficient. Standard point-of-care BNP assays are immunoassays that will detect any molecule containing the target epitopes. Consequently, these assays cannot distinguish between defective, high molecular weight forms of BNP and normal, physiologically active BNP. In 2 separate evaluations, mass spectroscopy detected little, if any, normal BNP in patients with heart failure, despite the appearance of high circulating levels of immunoreactive BNP (iBNP) using commercial assays. Therefore, these commercial assays should be considered to be only an indication of myocardial stress. They do not measure physiologic BNP activity. This accounts for the "BNP paradox," namely, that administration of exogenous recombinant human BNP (rhBNP, nesiritide) has substantial clinical and hemodynamic impact in the presence of high levels of circulating iBNP using commercial assays. In addition to its short-term hemodynamic impact, rhBNP may have other important effects in this setting, and further investigation is warranted.

Prolonged effects of B-type natriuretic peptide infusion on cardiac remodeling after sustained myocardial injury.

B-type natriuretic peptide (BNP) is an established first-line therapy for acute decompensated heart failure (HF), but its efficacy in preventing left ventricular (LV) remodeling after myocardial injury is unknown. The goal of this study was to evaluate the effects of BNP therapy on remodeling after ischemic injury in an awake canine model. Dogs were chronically instrumented for hemodynamics. Ischemia was created by daily coronary embolization (Embo; 3.1 x 10(4) beads/day) for 3 wk; 60 min after the first embolization, BNP (100 ng x kg(-1) x min(-1); n = 6) or saline (control; n = 6) was continuously infused via a left atrial catheter for 3 wk. Hemodynamics and echocardiography were performed in an awake state at baseline, 3 wk after Embo + BNP infusion, and 4 wk after stopping Embo + BNP infusion. End-systolic elastance (E(es)) and LV change in pressure over time (dP/dt) were preserved throughout Embo + BNP therapy versus control therapy (E(es): 3.76 +/- 1.01 vs. 1.41 +/- 0.16 mmHg/ml; LV dP/dt: 2,417 +/- 96 vs. 2,068 +/- 95 mmHg/s; both P < 0.05 vs. control). LV end-diastolic dimension was significantly smaller in BNP-treated dogs compared with control dogs (4.29 +/- 0.10 vs. 4.77 +/- 0.17 cm), and ejection fraction was maintained in treated dogs vs. control dogs (53 +/- 1% vs. 46 +/- 2%) (both P < 0.05 vs. control). Cyclooxygenase (COX)-2 expression in terminal LV tissue was significantly reduced after BNP therapy. Treatment with continuous infusion of BNP preserved LV geometry, improved systolic function, and prevented the progression of systolic HF after persistent ischemic injury.

p38 MAPK inhibition reduces diabetes-induced impairment of wound healing.

In healthy tissue, a wound initiates an inflammatory response characterized by the presence of a hematoma, infiltration of inflammatory cells into the wound and, eventually, wound healing. In pathological conditions like diabetes mellitus, wound healing is impaired by the presence of chronic nonresolving inflammation. p38 mitogen-activated protein kinase (MAPK) inhibitors have demonstrated anti-inflammatory effects, primarily by inhibiting the expression of inflammatory cytokines and regulating cellular traffic into wounds. The db/db mouse model of type 2 diabetes was used to characterize the time course of expression of activated p38 during impaired wound healing. The p38α-selective inhibitor, SCIO-469, was applied topically and effects on p38 activation and on wound healing were evaluated. A topical dressing used clinically, Promogran™, was used as a comparator. In this study, we established that p38 is phosphorylated on Days 1 to 7 post-wounding in db/db mice. Further, we demonstrated that SCIO-469, at a dose of 10 µg/wound, had a positive effect on wound contraction, granulation tissue formation, and re-epithelialization, and also increased wound maturity during healing. These effects were similar to or greater than those observed with Promogran™. These results suggest a novel approach to prophylactic and therapeutic management of chronic wounds associated with diabetes or other conditions in which healing is impaired.

Transforming growth factor-beta blockade down-regulates the renin-angiotensin system and modifies cardiac remodeling after myocardial infarction.

After myocardial infarction (MI), the heart may undergo progressive ventricular remodeling, resulting in a deterioration of cardiac function. TGF-beta is a key cytokine that both initiates and terminates tissue repair, and its sustained production underlies the development of tissue fibrosis, particularly after MI. We investigated the effects of a novel orally active specific inhibitor of the TGF-beta receptor 1 (SD-208) in an experimental model of MI. Mice underwent ligation of the left coronary artery to induce MI and were subsequently treated for 30 d after infarction with either SD-208 or a vehicle control. Blockade of TGF-beta signaling reduced mean arterial pressure in all groups. SD-208 treatment after MI resulted in a trend for reduced ventricular and renal gene expression of TGF-beta-activated kinase-1 (a downstream modulator of TGF-beta signaling) and a significant decrease in collagen 1, in association with a marked decrease in cardiac mass. Post-MI SD-208 treatment significantly reduced circulating levels of plasma renin activity as well as down-regulating the components of the cardiac and renal renin-angiotensin system (angiotensinogen, angiotensin converting enzyme, and angiotensin II type I receptor). Our findings indicate that blockade of the TGF-beta signaling pathway results in significant amelioration of deleterious cardiac remodeling after infarction.

Role of p38 mitogen activated protein kinase in a model of osteosarcoma-induced pain.

The focus of this work was to examine the potential role of p38 mitogen activated protein kinase (p38) in a mouse model of bone cancer (osteosarcoma) pain. To generate osteosarcoma and sham animals, osteosarcoma cells or medium were injected into the medullary canal of the femur. Initially, ipsilateral tactile allodynia was observed in both groups, but by 12 days post-surgery, thresholds in the sham group returned towards baseline while hypersensitivity in the osteosarcoma group lasted throughout the study. An increase in phosphorylated p38 was detected by western blotting in dorsal root ganglia (DRG) and spinal cord day 14 after surgery. Immunohistochemistry showed that p38 was phosphorylated in DRG and spinal dorsal horn neurons at this time point. Two doses of a selective p38 inhibitor, SCIO-469, were administered in the chow starting 5 days post-surgery and continued throughout the study. Treatment with SCIO-469 led to a decrease in osteosarcoma-induced clinical score but had no effect on the allodynia. Bone erosion and tumor growth were also examined but no significant reduction of bone erosion or tumor growth was observed in the SCIO-469 treated mice. These data suggest that the p38 signaling pathway does not play a major role in bone cancer-mediated pain.

Role of the TGF-beta/Alk5 signaling pathway in monocrotaline-induced pulmonary hypertension.

RATIONALE: Pulmonary arterial hypertension is a progressive disease characterized by an elevation in the mean pulmonary artery pressure leading to right heart failure and a significant risk of death. Alterations in two transforming growth factor (TGF) signaling pathways, bone morphogenetic protein receptor II and the TGF-beta receptor I, Alk1, have been implicated in the pathogenesis of pulmonary hypertension (PH). However, the role of TGF-beta family signaling in PH and pulmonary vascular remodeling remains unclear. OBJECTIVES: To determine whether inhibition of TGF-beta signaling will attenuate and reverse monocrotaline-induced PH (MCT-PH). METHODS: We have used an orally active small-molecule TGF-beta receptor I inhibitor, SD-208, to determine the functional role of this pathway in MCT-PH. MEASUREMENTS AND MAIN RESULTS: The development of MCT-PH was associated with increased vascular cell apoptosis, which paralleled TGF-beta signaling as documented by psmad2 expression. Inhibition of TGF-beta signaling with SD-208 significantly attenuated the development of the PH and reduced pulmonary vascular remodeling. These effects were associated with decreased early vascular cell apoptosis, adventitial cell proliferation, and matrix metalloproteinase expression. Inhibition of TGF-beta signaling with SD-208 in established MCT-PH resulted in a small but significant improvement in hemodynamic parameters and medial remodeling. CONCLUSIONS: These findings provide evidence that increased TGF-beta signaling participates in the pathogenesis of experimental severe PH.

Pharmacological properties of SD-282 - an alpha-isoform selective inhibitor for p38 MAP kinase.

The effects of small-molecule p38 inhibitors in numerous models of different disease states have been published, including those of SD-282, an indole-5-carboxamide inhibitor. The aim of the present study was to evaluate the pharmacological activity of SD-282 on cytokine production in vitro as well as in 2 in vivo models of inflammation in order to illuminate the role of this particular inhibitor in diverse disease states. The results presented here provide further characterization of SD-282 and provide a context in which to interpret the activity of this p38 inhibitor in models of arthritis, pain, myocardial injury, sepsis and asthma; all of which have an inflammatory component. SD-282 represents a valuable tool to elucidate the role of p38 MAP kinase in multiple models of inflammation.

p38alpha-selective MAP kinase inhibitor reduces tumor growth in mouse xenograft models of multiple myeloma.

Multiple myeloma (MM) is a clonal plasma cell malignancy, which is currently incurable. Therefore, new mono- or combined therapy treatment regimens in the early and advanced phases of MM are urgently needed to combat this disease. Recently, p38 mitogen-activated protein kinase (MAPK) has been implicated as playing an important role in MM. Therefore, the effect of a p38alpha-selective MAPK inhibitor, SCIO-469 (indole-5-carboxamide, ATP-competitive inhibitor), or its structural analog, SD-282 (indole-5-carboxamide, ATP-competitive inhibitor) was examined in mouse xenograft models of MM using human RPMI-8226 or H-929 plasmacytoma inocula. Oral treatment with SCIO-469 (10, 30, 90 mg/kg) twice daily was initiated in mice with palpable tumors of RPMI-8226 origin, a condition that mimics early human myeloma disease. In mice with palpable tumors, 14 days of SCIO-469 treatment significantly reduced RPMI-8226 tumor growth in a dose-dependent manner. A significant dose-dependent reduction in RPMI-8226 tumor growth was also observed when SCIO-469 oral treatment at doses of 10, 30 and 90 mg/kg twice daily was initiated in mice with tumors of pronounced size, a condition that mimics advanced human myeloma disease. In a similar set of studies employing the SCIO-469 analogue SD-282 at 90 mg/kg/bid orally, histological assessment at the end of the study demonstrated a significant reduction in RPMI-8226 tumor growth and angiogenesis. SD-282 treatment was additionally shown to significantly reduced expression of heat-shock protein-27 (HSP-27) and phospho-p38 in the tumor cells. Furthermore, co-administration of SCIO-469 with dexamethasone elicited antitumor properties in dexamethasone-sensitive H-929 tumors at much lower than the typically effective doses of dexamethasone, suggesting its potential for combined therapy. In conclusion, p38 inhibitors reduced human myeloma cell growth in vivo both at early and advanced phases of the disease. The current study also provides evidence of potential for co-therapy with dexamethasone.

Detection of endogenous B-type natriuretic peptide at very low concentrations in patients with heart failure.

BACKGROUND: The myocardium secretes B-type natriuretic peptide (BNP) in response to stimuli associated with heart failure (HF). However, high immunoreactive-BNP levels in patients with HF are associated with a paradoxical lack of natriuretic response. We hypothesized that commercially available assays for immunoreactive BNP do not reflect the bioactivity of the natriuretic peptide system, because they measure both unprocessed inactive pro-BNP and mature BNP 1-32. We describe an assay for the detection of bioactive BNP 1-32 and confirm very low concentrations in plasma from HF patients. METHODS AND RESULTS: We developed a quantitative mass spectrometry immunoassay to capture endogenous BNP peptides using high affinity antibodies. Bound BNP and its truncated fragments were detected by matrix assisted laser desorption ionization-time of flight mass spectrometry based on their predicted masses. Mass spectrometry immunoassay revealed rapid in vitro degradation of BNP 1-32 in plasma, which requires plasma collection in the presence of high protease inhibitor concentrations. In 11 of 12 HF patients BNP 1-32 was detectable, ranging from 25 to 43 pg/mL. Several degraded forms of BNP were also detected at similarly low levels. In contrast, parallel measurements of immunoreactive BNP using the Biosite assay ranged from 900 to 5000 pg/mL. CONCLUSIONS: Detection of endogenous BNP 1-32 requires special preservation of plasma samples. Mass spectrometry immunoassay technology demonstrates that HF patients have low levels of BNP 1-32. Commercially available immunoreactive-BNP assays overrepresent biological activity of the natriuretic peptide system because they cannot distinguish between active and inactive forms. This observation may, in part, explain the "natriuretic paradox."

Selective p38α mitogen-activated protein kinase inhibitor attenuates lung inflammation and fibrosis in IL-13 transgenic mouse model of asthma.

p38 Mitogen-activated protein kinase (MAPK) plays a critical role in the activation of inflammatory cells. We investigated the anti-inflammatory effects of a p38α-selective MAPK inhibitor (SD-282) in a mouse transgenic (CC10:IL-13) asthma model. The CC-10-driven over-expression of IL-13 in the mouse lung/airway has been shown to result in a remarkable phenotype recatitulating many features of asthma and characterized by eosinophilic and mononuclear inflammation, with airway epithelial cell hypertrophy, mucus cell metaplasia, the hyperproduction of neutral and acidic mucus, the deposition of Charcot-Leyden-like crystal, and airway sub-epitheilial fibrosis. Here we show how activated p38 MAPK can be observed in the lungs at the onset of asthma ie, around 8 weeks of age in both female and male mice. We also show that administration of a p38α MAPK selective inhibitor, SD-282 at 30 or 90 mg/kg, twice a day for a period of four weeks beginning at the onset of asthma, significantly reduced the inflammation (p < 0.001); hyperplasia of airway epithelium (p < 0.05); goblet cell metaplasia and mucus hypersecretion (p < 0.001) and reduced lung remodeling and fibrosis (p < 0.01), alleviating the severity of lung damage as measured by a composite score (p < 0.05). Furthermore, SD-282 significantly reduced activated p38 MAPK in the lymphocytes and epithelial cells (p < 0.001). Simultaneously, identical studies were conducted with an anti-fibrotic TGFßR1 kinase inhibitor (SD-208) which demonstrated anti-fibrotic but not anti-inflammatory properties. These findings suggest that the p38α-selective MAPK inhibitor may have dual therapeutic potential in attenuating both the inflammatory component and the fibrotic component of asthma and other Th2-polarized inflammatory lung diseases.