American Society of Hematology 2019 guidelines for sickle cell disease: cardiopulmonary and kidney disease.

BACKGROUND: Prevention and management of end-organ disease represent major challenges facing providers of children and adults with sickle cell disease (SCD). Uncertainty and variability in the screening, diagnosis, and management of cardiopulmonary and renal complications in SCD lead to varying outcomes for affected individuals. OBJECTIVE: These evidence-based guidelines of the American Society of Hematology (ASH) are intended to support patients, clinicians, and other health care professionals in their decisions about screening, diagnosis, and management of cardiopulmonary and renal complications of SCD. METHODS: ASH formed a multidisciplinary guideline panel that included 2 patient representatives and was balanced to minimize potential bias from conflicts of interest. The Mayo Evidence-Based Practice Research Program supported the guideline development process, including performing systematic evidence reviews up to September 2017. The panel prioritized clinical questions and outcomes according to their importance for clinicians and patients. The panel used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach, including GRADE evidence-to-decision frameworks, to assess evidence and make recommendations, which were subject to public comment. RESULTS: The panel agreed on 10 recommendations for screening, diagnosis, and management of cardiopulmonary and renal complications of SCD. Recommendations related to anticoagulation duration for adults with SCD and venous thromboembolism were also developed. CONCLUSIONS: Most recommendations were conditional due to a paucity of direct, high-quality evidence for outcomes of interest. Future research was identified, including the need for prospective studies to better understand the natural history of cardiopulmonary and renal disease, their relationship to patient-important outcomes, and optimal management.

Hydroxyurea and Zileuton Differentially Modulate Cell Proliferation and Interleukin-2 Secretion by Murine Spleen Cells: Possible Implication on the Immune Function and Risk of Pain Crisis in Patients with Sickle Cell Disease.

BACKGROUND: Hydroxyurea (HU) reduces major complications associated with sickle cell disease in part because of the induction of fetal hemoglobin. However, because of its antiproliferative property, its long-term use may impair immunity. Zileuton, a derivative of HU, also induces fetal hemoglobin and has antiinflammatory properties, a feature that can reduce the risk of sickling. Our goal was to investigate the capacity of both drugs to modulate the secretion of interleukin-2 (IL-2), a regulatory cytokine for immune responses. METHODS: Spleen cells obtained from 11 4-month-old C57BL/6 female mice were incubated without and with 10 µg/mL HU or zileuton, 2.5 µg/mL concanavalin A (ConA), 20 µg/mL phytohemagglutinin (PHA), and 50 ng/mL anti-CD3 antibody for 12-48 h. IL-2 was measured in the supernatant by enzyme-linked immunosorbent assay and cell proliferation by (3)H-thymidine uptake. RESULTS: While HU reduced lymphocyte proliferation in response to mitogens (P<0.05), zileuton did not. Baseline IL-2 concentration and PHA-induced IL-2 were not significantly affected by either drug. Contrary to what we expected, while HU increased IL-2 supernatant levels 1.17-fold to 6.5-fold in anti-CD3 antibody-treated cells (P<0.05), zileuton decreased them 35%-65% (P<0.05). Zileuton likely reduced IL-2 levels by inhibiting 5-lipoxygenase, hence leukotriene B4 production, an IL-2 inducer. HU did not decrease IL-2 secretion likely because of its lack of effect on mRNA and protein synthesis. CONCLUSION: Modulation of IL-2 secretion by zileuton and/or reduced lymphocyte proliferation by HU may impair the immune response of patients with sickle cell disease but may also be beneficial by attenuating inflammation independently of fetal hemoglobin induction.

Iron overload in adults with sickle cell disease who have received intermittent red blood cell transfusions.

OBJECTIVE: To assess the prevalence of iron overload in adults with sickle cell disease (SCD) not on a chronic transfusion protocol. DESIGN: Retrospective chart review. DATA SOURCE: University of South Alabama Comprehensive Sickle Cell Center adult outpatient clinic. RESULTS: There was no significant difference in units transfused across the four genotypes (HbSS, HbSC, HbSß(0)-thalassemia, and HbSß(+)-thalassemia). Only individuals with HbSS (n = 63) met criteria for iron overload with ferritins of ≥1500 ng/mL. Forty-eight had ferritins <1500 ng/mL, eight (13%) had ferritins ≥3000 ng/mL, and seven (11%) had ferritins ≥1500 and <3000 ng/mL. The overall prevalence of iron overload was 9.74% in SCD cohort and 23.8% in the HbSS genotype. CONCLUSIONS: Our data support that patients with HbSS are at a particularly high risk for inadvertent iron overload as compared to HbSC, HbSß(0)-thalassemia, and HbSß(+)-thalassemia. IMPLICATIONS FOR PRACTICE: This study supports the need for healthcare providers to closely monitor the number of red blood cell (RBC) transfusions, RBC units transfused, and serial baseline, steady-state ferritin levels. With closer monitoring, the clinical significance of iron overload in SCD can be established and guide the healthcare provider's management in the prevention of iron overload.

A double-blind, placebo-controlled phase II study of the efficacy and safety of 2,2-dimethylbutyrate (HQK-1001), an oral fetal globin inducer, in sickle cell disease.

This placebo-controlled phase II study evaluated the pharmacodynamics, efficacy and safety of 2,2-dimethylbutyrate (HQK-1001), a fetal globin gene-inducing short-chain fatty acid derivative, administered orally at 15 mg/kg twice daily for 48 weeks in 76 subjects with sickle cell disease (SCD). The median age was 26 years (range: 12-55 years) and 37 subjects (49%) were treated previously with hydroxycarbamide. Sixty subjects (79%) had Hb SS and 16 (21%) had S/ß(0) thalassemia. The study was terminated after a planned interim analysis showed no significant increase in fetal hemoglobin (Hb F) and a trend for more pain crises in the HQK-1001 group. For 54 subjects with Week 24 data, the mean absolute increase in Hb F was 0.9% (95% confidence interval (CI): 0.1-1.6%) with HQK-1001 and 0.2% (95% CI: -0.7-1.1%) with placebo. Absolute increases in Hb F greater than 3% were noted in 9 of 38 subjects (24%) administered HQK-1001 and 1 of 38 subjects (3%) administered placebo. The mean changes in hemoglobin at Week 24 were comparable between the two groups. The mean annualized rate of pain crises was 3.5 with HQK-1001 and 1.7 with placebo. The most common adverse events in the HQK-1001 group, usually graded as mild or moderate, consisted of nausea, headache, vomiting, abdominal pain, and fatigue. Additional studies of HQK-1001 at this dose and schedule are not recommended in SCD. Intermittent HQK-1001 administration, rather than a daily regimen, may be better tolerated and more effective, as shown previously with arginine butyrate, and warrants further evaluation.

Pharmacological characterization of an imidazolopyrazole as novel selective androgen receptor modulator.

Selective androgen receptor modulators (SARMs) are androgens with tissue-selective activity. SARMs that have anabolic activity on muscle while having minimal stimulatory activity on prostate are classified as SARM agonists. They can be used to prevent the loss of lean body mass that is associated with cancer, immunodeficiency, renal disease and aging. They may also have anabolic activity on bone; thus, unlike estrogens, they may reverse the loss of bone strength associated with aging or hypogonadism. Our in-house effort on SARM program discovers a nonsteroidal androgen receptor ligand with a unique imidazolopyrazole moiety in its structure. In vitro, this compound is a weak androgen receptor binder and a weak androgen agonist. Despite this, in orchidectomized mature rats it is an effective SARM agonist, with an ED(50) on levator ani muscle of 3.3mg/kg and an ED(50) on ventral prostate of >30mg/kg. It has its maximal effect on muscle at the dose of 10mg/kg. In addition, this compound has mixed agonistic and antagonistic activities on prostate, reducing the weight of that tissue in intact rats by 22% at 10mg/kg. The compound does not have significant effect on gonadotropin levels or testosterone levels in both orchidectomized and intact male rats. It does not have notable progestin, estrogen or glucocorticoid agonistic or antagonistic activity in rats. In a female sexual behavior model, it improves the sexual desire of ovariectomized female rats for sexually mature intact males over nonsexually ovariectomized females. Overall, the imidazolopyrazole is a potent prostate-sparing candidate for development as a SARM agonist with an appropriate pharmacological profile for clinical benefit in muscle-wasting conditions and female sexual function disorders.

Misclassification of pulmonary hypertension in adults with sickle hemoglobinopathies using Doppler echocardiography.

OBJECTIVE: To compare the diagnostic utility of Doppler echocardiography-derived tricuspid regurgitant jet velocity (TRV) ≥ 2.5 m/s to right heart catheterization (RHC) in defining pulmonary hypertension (PH) in adult patients with sickle cell disease (SCD). METHODS: This is a retrospective chart review of adults with SCD who had a TRV ≥ 2.5 m/s and RHC. A TRV ≥ 2.5 m/s is suggestive of PH. Pulmonary arterial hypertension (PAH) was defined as a mean pulmonary artery pressure (mPAP) ≥ 25 mm Hg and pulmonary capillary wedge pressure ≤ 15 mm Hg. Pulmonary venous hypertension was defined as an mPAP ≥ 25 mm Hg and pulmonary capillary wedge pressure >15 mm Hg. RESULTS: Twenty-five patients with SCD met the inclusion criteria. Nine of the 25 (36%) patients had an mPAP ≥ 25 mm Hg. Of these 9, 3 (33%) had PAH and 6 (66%) had pulmonary venous hypertension. Patients with PH did not have a higher TRV (3.1 ± 0.68 vs 2.70 ± 0.16 m/s; P = 0.12), but they did have higher cardiac outputs (10.4 ± 2.7 vs 7.81 ± 1.85 L/min; P = 0.012. The specificity of TRV equal to 2.51 m/s in diagnosing PH was 18.8%. At a TRV of 2.88 m/s, the specificity increased to 81%. CONCLUSIONS: In adults with SCD, a TRV of 2.5 m/s lacks specificity for use as a screening tool in the diagnosis of PH. Using a TRV of ≥ 2.88 m/s allows the TRV to be used as a screening tool and reduces the false-positive rate and need for unnecessary RHC.

A primary care provider's guide to preventive and acute care management of adults and children with sickle cell disease.

PURPOSE: To familiarize primary care providers (PCPs) with the pathophysiological processes, diagnostic evaluation, and medical management of sickle hemoglobinopathies and their complications. Current standards of care, clinical research advances, and new treatment options will also be addressed to assist PCPs in the management of sickle cell disease (SCD). DATA SOURCES: A selective search and review of the current literature on SCD and the authors' experience. CONCLUSIONS: Management of individuals with SCD is very complex, requiring a multidisciplinary approach that includes the patient or parent, PCP, specialist, nurse, and social worker. More patients living with SCD are relying on PCPs in nonspecialty practices for comprehensive disease management. IMPLICATIONS FOR PRACTICE: Newborn screening detects new cases of SCD annually. The median life expectancy has more than doubled for individuals with sickle cell anemia. Healthcare providers are now in an era of increased routine screening, assessment, and management of chronic complications from this illness not previously seen in the care of adults with SCD.

A selective androgen receptor modulator with minimal prostate hypertrophic activity restores lean body mass in aged orchidectomized male rats.

Androgens are required for the maintenance of normal sexual activity in adulthood and for enhancing muscle growth and lean body mass in adolescents and adults. Androgen receptor (AR) ligands with tissue selectivity (selective androgen receptor modulators, or SARMs) have potential for treating muscle wasting, hypogonadism of aging, osteoporosis, female sexual dysfunction, and other indications. JNJ-37654032 is a nonsteroidal AR ligand with mixed agonist and antagonist activity in androgen-responsive cell-based assays. It is an orally active SARM with muscle selectivity in orchidectomized rat models. It stimulated growth of the levator ani muscle with ED(50) 0.8 mg/kg, stimulating maximal growth at a dose of 3mg/kg. In contrast, it stimulated ventral prostate growth to 21% of its full size at 3mg/kg. At the same time, JNJ-37654032 reduced prostate weight in intact rats by 47% at 3mg/kg, while having no inhibitory effect on muscle. Using magnetic resonance imaging to monitor body composition, JNJ-37654032 restored about 20% of the lean body mass lost following orchidectomy in aged rats. JNJ-37654032 reduced follicle-stimulating hormone levels in orchidectomized rats and reduced testis size in intact rats. JNJ-37654032 is a potent prostate-sparing SARM with the potential for clinical benefit in muscle-wasting diseases.

Hydroxyurea attenuates activated neutrophil-mediated sickle erythrocyte membrane phosphatidylserine exposure and adhesion to pulmonary vascular endothelium.

Activated neutrophils increase erythrocyte phosphatidylserine (PS) exposure. PS-exposed sickle red blood cells (SSRBCs) are more adhesive to vascular endothelium than non-PS-exposed cells. An increase in SSRBC fetal hemoglobin (HbF) concentration has been associated with improved rheology and decreased numbers of vasoocclusive episodes. This study examined the effects of HbF, PS-exposed SSRBCs, and chronic hydroxyurea (HU) treatment on activated neutrophil-mediated SSRBC retention/adherence in isolated-perfused rat lungs. Lungs were perfused with erythrocyte suspensions from 1) individuals homozygous for hemoglobin S with 0-7% HbF (SS), 2) with > or =8% HbF (SS + F), and 3) individuals homozygous for hemoglobin S treated with HU therapy for > or =1 yr (SS + HU). Retention of SSRBCs from the SS + HU group was significantly less than that seen in both the SS and SS + F groups. No difference was observed between the SS and SS + F groups. The percentage of HbF and F-cells did not differ between the SS + F and SS + HU groups. At baseline, the proportion of PS-exposed SSRBCs was not different between the SS and SS + HU groups. However, SSRBC treatment with activated neutrophil supernatant caused a twofold increase in PS-exposed SSRBCs in the SS control and no change in the SS + HU group. We conclude that 1) HU attenuates SSRBC retention/adherence in the pulmonary circulation seen in response neutrophil activation, 2) HU stabilizes SSRBC membrane PS, and 3) HU attenuation SSRBC retention/adherence in the pulmonary circulation occurs through a mechanism(s) independent of HbF.

A selective androgen receptor modulator with minimal prostate hypertrophic activity enhances lean body mass in male rats and stimulates sexual behavior in female rats.

Androgen receptor (AR) ligands with tissue selectivity (selective androgen receptor modulators, or SARMs) have potential for treating muscle wasting, hypogonadism of aging, osteoporosis, female sexual dysfunction, and other indications. JNJ-28330835 is a nonsteroidal AR ligand with mixed agonist and antagonist activity in androgen-responsive cell-based assays. It is an orally active SARM with muscle selectivity in orchidectomized rat models. It stimulated growth of the levator ani muscle, stimulating maximal growth at a dose of 10 mg/kg. At the same time, JNJ-28330835 reduced prostate weight in intact rats by a mean of 30% at 10 mg/kg, while having no inhibitory effect on muscle. Using magnetic resonance imaging (MRI) to monitor body composition, it prevented half of the loss of lean body mass associated with orchidectomy, and restored about 30% of lost lean mass to aged orchidectomized rats. It had agonist effects on markers of both osteoclast and osteoblast activity, suggesting that it reduces bone turnover. In a model of sexual behavior, JNJ-28330835 enhanced the preference of ovariectomized female rats for sexually intact male rats over nonsexual orchidectomized males. JNJ-28330835 is a prostate-sparing SARM with the potential for clinically beneficial effects in muscle-wasting diseases and sexual function disorders.

A selective androgen receptor modulator that reduces prostate tumor size and prevents orchidectomy-induced bone loss in rats.

The pharmacological activity of JNJ-26146900 is described. JNJ-26146900 is a nonsteroidal androgen receptor (AR) ligand with tissue-selective activity in rats. The compound was evaluated in in vitro and in vivo models of AR activity. It binds to the rat AR with a K(i) of 400nM and acts as a pure androgen antagonist in an in vitro cell-based assay. Its in vitro profile is similar to the androgen antagonist bicalutamide (Casodex). In intact rats, JNJ-26146900 reduces ventral prostate weight with an oral potency (ED(50)) of 20-30mg/kg, again comparable to that of bicalutamide. JNJ-26146900 prevented prostate tumor growth in the Dunning rat model, maximally inhibiting growth at a dose of 10mg/kg. It slowed tumor growth significantly in a CWR22-LD1 mouse xenograft model of human prostate cancer. It was tested in aged male rats for its ability to prevent bone loss and loss of lean body mass following orchidectomy. After 6 weeks of dosing, bone volume decreased by 33% in orchidectomized versus intact vehicle-treated rats with a probability (P) of less than 0.05, as measured by micro-computerized tomography analysis. At a dose of 30mg/kg, JNJ-26146900 significantly reduced castration-induced tibial bone loss as indicated by the following parameters: bone volume, trabecular connectivity, trabecular number and spacing between trabeculae. Bone mineral density decreased from 229+/-34mg/cm(3) of hydroxyapatite to 166+/-26mg/cm(3) following orchidectomy, and was maintained at 194+/-20mg/cm(3) with JNJ-26146900 treatment (P<0.05 relative to orchidectomy alone). Using magnetic resonance imaging, the compound was found to partially prevent orchidectomy-induced loss of lean body mass. Our data show that selective androgen receptor modulators (SARMs) have the potential for anabolic effects on bone and muscle while maintaining therapeutic efficacy in prostate cancer.

Sickle acute lung injury: role of prevention and early aggressive intervention strategies on outcome.

Acute chest syndrome in sickle cell disease is a form of acute lung injury that may progress to acute respiratory distress syndrome and death. Despite recent advances in diagnosis and treatment that have resulted in improved survival in sickle cell disease, acute chest syndrome remains the most common cause of death in this population. The current standards of treatment for acute chest syndrome have been reviewed. Biomedical re-search forms the basis for sound clinical decision making and implementation of interventions that target prevention, diagnosis, and effective treatment options. Although current clinical trials are ongoing to address several new potential therapeutic options,more research using preventative and interventional strategies in sickle acute lung injury is warranted.

Activated neutrophil-mediated sickle red blood cell adhesion to lung vascular endothelium: role of phosphatidylserine-exposed sickle red blood cells.

Activated neutrophils (ANs) increase sickle red blood cell (SRBC) retention/adhesion in the pulmonary circulation. This study investigates the role of neutrophil activation and SRBC retention/adhesion in the pulmonary circulation through a mechanism that involves increasing phosphatidylserine (PS) exposure on the external membrane surface of the SRBCs (PS-exposed). With the use of flow cytometry, double-labeling studies were performed with a calcium-dependent phospholipid-binding protein, annexin V-fluorescein isothiocyanate fluorescence, and the erythroid-specific marker glycophorin A to assess for the percentage of PS-exposed normal and SRBCs at baseline and after coincubation with ANs. Additional studies were performed that assessed retention/adhesion of SRBCs in the isolated rat lung using (51)Cr-labeled SRBC alone, SRBC + AN, SRBC + AN + zileuton, and SRBC + AN + annexin V. Specific activities of lung and perfusate were measured, and the number of retained SRBCs per gram lung was calculated. Flow cytometry demonstrated that ANs increased the percentage of PS-exposed normal and SRBCs. The 5-lipoxygenase inhibitor zileuton attenuated AN-mediated increases in PS-exposed SRBCs and decreased SRBC retention/adherence in the lung on histological sections. Similarly, in the isolated perfused lung and in histological lung sections, retention/adherence of SRBCs cloaked with annexin V was attenuated in the presence of ANs. We conclude that ANs enhance the adhesion of SRBCs to vascular endothelium by increasing red blood cell membrane externalization of PS. Zileuton attenuation of AN-mediated SRBC PS externalization suggests that a 5-lipoxygenase product(s), secreted by the AN, plays a vital role in altering the adhesive properties of PS-exposed SRBCs to vascular endothelium.

JNJ-10280205 and JNJ-10287069: Novel PDE5 inhibitors as clinical candidates for erectile dysfunction.

Phosphodiesterase 5 (PDE5) inhibitors are efficacious in treating patients with erectile dysfunction. New PDE5 inhibitors with different selectivity and pharmacokinetic profiles have been vigorously pursued. Here we report two novel, potent, and selective PDE5 inhibitors, JNJ-10280205 and JNJ-10287069, with Ki values of 0.05 and 0.12 nM, respectively. Both compounds displayed superior selectivity against PDE1-4 and -6 when compared to sildenafil. In the anesthetized dogs, JNJ-10280205 and JNJ-10287069 exhibited similar efficacy as sildenafil in enhancing erectile functions, with no significant effect on cardiovascular parameters. Pharmacokinetic studies showed that JNJ-10287069 had better oral bioavailability than JNJ-10280205 in several animal species. In vitro study suggested that cytochrome P450 (CYP) 3A4 played a major role in the metabolism of both compounds. The compounds inhibited some of the CYP450 enzymes and the human ether-a-go-go (HERG) channel at much higher concentrations than that required to inhibit PDE5, thus, no cross inhibition would be expected at therapeutic doses. Both compounds are suitable clinical candidates.

Expression of PDE11A in normal and malignant human tissues.

Cyclic nucleotide phosphodiesterase 11A (PDE11A) is the newest member in the PDE family. Although the tissue distribution of PDE11A mRNA has been shown, its protein expression pattern has not been well studied. The goal of this report is to investigate the distribution of PDE11A proteins in a wide range of normal and malignant human tissues. We utilized a polyclonal antibody that recognized all four PDE11A isoforms. Its specificity was demonstrated by Western blot analysis on a recombinant human PDE11A protein and native PDE11A proteins in various human tissues. Immunohistochemistry showed that PDE11A is widely expressed. Various degrees of immunoreactivity were observed in the epithelial cells, endothelial cells, and smooth muscle cells of all tissues examined. The highest expression was in the epithelial, endothelial, and smooth muscle cells of the prostate, Leydig, and spermatogenic cells of the testis, the tubule epithelial cells in the kidney, the epithelial and endothelial cells in the adrenal, the epithelial cells and macrophages in the colon, and the epidermis in the skin. Furthermore, PDE11A expression was also detected in several human carcinomas. Our results suggest that PDE11A might be involved in multiple physiological processes in various organs via its ability to modulate intracellular cAMP and cGMP levels.

Differential effects of hydroxyurea and zileuton on interleukin-13 secretion by activated murine spleen cells: implication on the expression of vascular cell adhesion molecule-1 and vasoocclusion in sickle cell anemia.

BACKGROUND: Interleukin-13 (IL-13), a TH2 cytokine, upregulates the expression of vascular cell adhesion molecule-1 on endothelial cells, a factor involved in vasoocclusion in sickle cell disease (SCD). Hydroxyurea improves clinical status of SCD patients in part by induction of fetal hemoglobin. Its effect on IL-13 secretion has not been investigated. OBJECTIVE: To determine whether hydroxyurea and zileuton, a hydroxyurea derivative with antiinflammatory properties, affect IL-13 secretion. METHODS: We measured IL-13 in the supernatant of murine spleen cells incubated without and with hydroxyurea, zileuton (10 microg/ml), concanavalin A (2.5 microg/ml), and anti-CD3 (50 ng/ml) (n=8). RESULTS: Hydroxyurea and zileuton do not affect baseline IL-13 secretion. Unexpectedly, hydroxyurea increases IL-13 levels above baseline (120%, 216.5%, [p<0.05] after 24 h and 48 h, respectively) in lymphocytes activated by anti-CD3, while zileuton reduces them by 59%-78% (p<0.005). In lymphocytes activated by concanavalin A, hydroxyurea and zileuton reduce IL-13 secretion by 24-36% and 50-87%, respectively (p<0.05). Hydroxyurea, but not zileuton, significantly inhibits spleen cell proliferative responses to mitogens (p<0.005). CONCLUSION: Data suggest that hydroxyurea up-regulates IL-13 secretion in anti-CD3-activated lymphocytes through gene activation but not by altered cell proliferation. Increased IL-13 secretion may contribute to unresponsiveness of certain SCD patients to hydroxyurea. The potential benefit of zileuton in the management of vasoocclusion is discussed.

Pyrroloquinolone PDE5 inhibitors with improved pharmaceutical profiles for clinical studies on erectile dysfunction.

We previously reported a series of potent and selective pyrimidinyl pyrroloquinolone PDE5 inhibitors such as 2a for potential use in male erectile dysfunction (MED) (Sui, Z.; Guan, J.; Macielag, M. J.; Jiang, W.; Zhang, S.; Qiu, Y.; Kraft, P., Bhattacharjee, S.; John, T. M.; Craig, E.; Haynes-Johnson, D.; Clancy, J. J. Med. Chem. 2002, 45, 4094-4096). Unfortunately, the low aqueous solubility and poor oral bioavailability rendered them undesirable development candidates. To address this issue, we designed a series of analogues using two approaches: increasing the overall basicity and reducing molecular weight of the lead. Through earlier SAR studies, we discovered that the PDE5 potency of the pyrroloquinolones is insensitive to substitution on the pyrrole nitrogen. Basic functional groups such as pyridines and benzimidazoles were appended via the aromatic ring connected to the pyrrole nitrogen. Several truncated analogues were also designed and synthesized to improve oral absorption. These modifications allowed us to identify analogues with good oral bioavailability in rats, dogs, and monkeys while the high potency against PDE5 and desirable selectivity versus other PDE isozymes were maintained. Compounds R-11e and R-11l were selected as development candidates for MED and other indications.

The putative tumor suppressor deleted in malignant brain tumors 1 is an estrogen-regulated gene in rodent and primate endometrial epithelium.

Deleted in malignant brain tumors 1 (DMBT1) is a candidate suppressor of malignancies of the brain, lung, gut, and breast. We have been studying gene expression in the uterus in the presence of estrogens and their antagonists. Here, we show that DMBT1 RNA levels are robustly increased by estrogen treatment in the uteri of ovariectomized monkeys and rats. In monkeys, the progestin antagonist mifepristone inhibits estrogen-dependent uterine proliferation. As determined by a microarray experiment and quantitative analysis of RNA levels, mifepristone inhibited estrogenic induction of DMBT1. DMBT1 was not expressed in intact monkeys that were treated with a gonadotropin agonist to suppress steroidogenesis. An in vitro transfection study with human DMBT1 promoter constructs showed that an Alu site approximately 3000 nucleotides upstream of the gene mediates estrogenic regulation. Surprisingly, the estrogen antagonists tamoxifen, raloxifene, and ICI 182,780 also induced gene expression via this Alu site. Rodents represent a more convenient model system for studying uterine biology than monkeys. In rats, uterine DMBT1 RNA levels were dramatically up-regulated by estrogen. Consistent with the transfection study, tamoxifen and raloxifene increased DMBT1 RNA levels in vivo, but ICI 182,780 inhibited an estrogen-induced increase. Immunohistochemical studies showed that DMBT1 is specifically induced in glandular and luminal epithelia of the rat endometrium. Our experiments establish that DMBT1 is an estrogen-responsive gene with a possible role in endometrial proliferation or differentiation, and they have implications for the putative tumor suppressive and mucosal protective functions of DMBT1 in the uterus.

Physicians' attitude and practices in sickle cell disease pain management.

Many physicians believe that patients with sickle cell disease (SCD) are more likely to become addicted to pain medication than are other patient populations. This study hypothesizes that physicians' attitudes towards addiction in patients with SCD affects pain management practices. The Physician Attitudes Survey was sent to 286 physicians at seven National Institutes of Health-funded university-based comprehensive sickle cell centres. The survey assessed demographic information; and physician's attitudes toward and knowledge of pain, pain treatment, and drug addiction and abuse. Significant Pearson product-moment correlations were found between attitudes towards pain and beliefs regarding addiction to prescribed opioids. Physicians reported varied pain management strategies, however, many believe that attitudes toward addiction and to patients in pain crises may result in undertreatment of pain. These results indicate that physicians might benefit from additional education regarding sickle cell disease, addiction to pain medication, the pharmacology of opioids, and the assessment and treatment of pain.

Altered activities of cyclic nucleotide phosphodiesterases and soluble guanylyl cyclase in cultured RFL-6 cells.

We utilized rat fetal lung fibroblasts (RFL-6) to evaluate our PDE5 inhibitors at cellular level and observed a decrease in cGMP accumulation induced by sodium nitroprusside (SNP) and PDE5 inhibitors with passage. To further investigate this observation, we examined cGMP synthesis via soluble guanylyl cyclase (sGC) and degradation via phosphodiesterases (PDEs) at different passages. At passage (p)4, p9, p14, major cGMP and cAMP degradation activities were contributed by PDE5 and PDE4, respectively. The PDE5 activity decreased 50% from p4 to p14, while PDE4 activity doubled. The cGMP accumulation was evaluated in the presence of sodium nitroprusside (SNP) and/or PDE inhibitors in p4 and p14 cells. SNP together with sildenafil, a PDE5 inhibitor, induced dose-dependent increase in cGMP levels in cells at p4, but showed little effect on cells at p14. The possible down regulation of sGC at mRNA level was explored using real-time RT-PCR. The result showed the mRNA level of the alpha1 subunit of sGC decreased about 98% by p9, while the change on beta1 mRNA was minimal. Consistently, sGC activities in cell lysate decreased by 94% at p9. Forskolin stimulated a dramatic increase in cAMP levels in cells at all passages examined. Our results show that sGC activity decreased significantly and rapidly with passage due to a down regulation of the alpha1 subunit mRNA, yet the adenylyl cyclase activity was not compromised. This study further emphasized the importance of considering passage number when using cell culture as a model system to study NO/cGMP pathway.