The F0F1-ATP Synthase Complex Contains Novel Subunits and Is Essential for Procyclic Trypanosoma brucei

The mitochondrial F0F1 ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F0F1 ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F1 subunits, three to F0 subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F1 α subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage) cells and are important for the structural integrity of the F0F1-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought

Mode of injection and treatment adherence: results of a survey characterizing the perspectives of health care providers and US women 18–45 years old

David L Gandell,1 E Jay Bienen,2 Jennifer Gudeman3 1Department of Obstetrics and Gynecology, University of Rochester, Rochester, NY, USA; 2The Curry Rockefeller Group, Tarrytown, NY, USA; 3Medical Affairs, AMAG Pharmaceuticals Inc., Waltham, MA, USA Purpose: A market research study was conducted to characterize perceptions of intramuscular (IM) and subcutaneous (SC) routes of injection, including use of autoinjectors, and how these perceptions affect adherence to injection regimens. The perspectives were from women of childbearing age (18–45 years old; consumers) and health care providers (HCPs) involved in women’s health care. Methods: Two telephone surveys, one of HCPs and the other of consumers, were conducted by KRC Research (New York, NY, USA) between May and July 2017. HCPs were recruited across the US; the consumer survey was administered to a nationally representative sample. Survey questions identified potential challenges of IM and SC administration, their impact on treatment adherence, and perceptions of autoinjectors. Results are reported using descriptive statistics and reflect an unweighted sample; margin of error is ±3% for the consumer survey. Results: HCP respondents included 100 generalist OB/GYNs, 101 maternal-fetal medicine specialists, and 519 nurses; there were 1,012 female consumer respondents. Nurses reported more experience than physicians in administering injections, including with autoinjectors. Consumers reported having received treatments via both IM and SC injections; 26% had received treatment with injections at regular intervals. Most HCPs (58%) said they preferred to administer SC injections, which was also the preference for receiving injections among consumers, who reported needle size as a concern regardless of administration mode. Other major concerns were perceptions of pain and fear/anxiety, and seeing the needle, all of which were greater for IM than for SC injections. HCPs and consumers both reported greater likelihood of adherence to therapy administered SC using an autoinjector because they believe that this method provides substantial HCP and patient benefits. Conclusion: HCPs and consumers identified similar challenges with adherence to injections. However, there was consistently higher preference for SC relative to IM. HCPs and consumers believe that autoinjectors may increase adherence. Keywords: women’s health care, intramuscular, subcutaneous, injection, patient preference, adherenc

Characterizing individual painDETECT symptoms by average pain severity

Alesia Sadosky,1 Vijaya Koduru,2 E Jay Bienen,3 Joseph C Cappelleri4 1Pfizer Inc, New York, NY, 2Eliassen Group, New London, CT, 3Outcomes Research Consultant, New York, NY, 4Pfizer Inc, Groton, CT, USA Background: painDETECT is a screening measure for neuropathic pain. The nine-item version consists of seven sensory items (burning, tingling/prickling, light touching, sudden pain attacks/electric shock-type pain, cold/heat, numbness, and slight pressure), a pain course pattern item, and a pain radiation item. The seven-item version consists only of the sensory items. Total scores of both versions discriminate average pain-severity levels (mild, moderate, and severe), but their ability to discriminate individual item severity has not been evaluated.Methods: Data were from a cross-sectional, observational study of six neuropathic pain conditions (N=624). Average pain severity was evaluated using the Brief Pain Inventory-Short Form, with severity levels defined using established cut points for distinguishing mild, moderate, and severe pain. The Wilcoxon rank sum test was followed by ridit analysis to represent the probability that a randomly selected subject from one average pain-severity level had a more favorable outcome on the specific painDETECT item relative to a randomly selected subject from a comparator severity level.Results: A probability >50% for a better outcome (less severe pain) was significantly observed for each pain symptom item. The lowest probability was 56.3% (on numbness for mild vs moderate pain) and highest probability was 76.4% (on cold/heat for mild vs severe pain). The pain radiation item was significant (P<0.05) and consistent with pain symptoms, as well as with total scores for both painDETECT versions; only the pain course item did not differ.Conclusion: painDETECT differentiates severity such that the ability to discriminate average pain also distinguishes individual pain item severity in an interpretable manner. Pain-severity levels can serve as proxies to determine treatment effects, thus indicating probabilities for more favorable outcomes on pain symptoms. Keywords: neuropathic pain, painDETECT, pain severity, psychometric propertie

Characterizing neuropathic pain profiles: enriching interpretation of painDETECT

Joseph C Cappelleri,1 Vijaya Koduru,2 E Jay Bienen,3 Alesia Sadosky4 1Pfizer Inc, Groton, CT, USA; 2Eliassen Group, New London, CT, USA; 3Outcomes Research Consultant, New York, NY, USA; 4Pfizer Inc, New York, NY, USA Purpose: To psychometrically evaluate painDETECT, a patient-reported screening questionnaire for neuropathic pain (NeP), for discriminating among sensory pain symptoms (burning, tingling/prickling, light touching, sudden pain attacks/electric shock-type pain, cold/heat, numbness, and slight pressure). Methods: The seven-item version of painDETECT provides an overall score that targets only sensory symptoms, while the nine-item version adds responses on two items to the overall score, covering pain course pattern and pain radiation. Both versions have relevance in terms of characterizing broad NeP. The nine- and seven-item versions of painDETECT were administered to subjects with confirmed NeP across six conditions identified during office visits to US community-based physicians. Responses on the sensory symptom items were dichotomized into “at least moderate” (ie, moderate, strongly, very strongly) relative to the combined other responses (never, hardly noticed, slightly). Logistic regression of dichotomized variables on the total painDETECT score provided probabilities of experiencing each symptom across the range of painDETECT scores. Results: Both painDETECT versions discriminated among the symptoms with similar probabilities across the score ranges. Using these data, the probability of moderately experiencing each pain sensory item was estimated for a particular score, providing a pain profile. Additionally, the likelihood of experiencing each sensation was determined for a discrete increase in score, ie, the odds of at least a moderate sensation of burning (versus less than a moderate sensation) was 1.29 for a 1-point increase, 3.52 for a 5-point increase, and 12.42 for every 10-point increase in the nine-item painDETECT score. Conclusion: painDETECT differentiates pain profiles across the range of scores such that, for a particular score, the probability of experiencing at least a moderate sensation of each symptom was determined and compared. These results can help characterize NeP symptomatology, enrich interpretation of painDETECT scores, and provide a basis for individualizing NeP management. Keywords: neuropathic pain, painDETECT, sensory symptoms, pain profile, interpretation, patient-reported outcome

A cross-sectional study examining the psychometric properties of the painDETECT measure in neuropathic pain

Joseph C Cappelleri,1 Vijaya Koduru,2 E Jay Bienen,3 Alesia Sadosky41Pfizer Inc, Groton, CT, USA; 2Eliassen Group, New London, CT, USA; 3Outcomes Research Consultant, New York, NY, USA; 4Pfizer Inc, New York, NY, USABackground: Similarities and differences on the nine-item and seven-item versions of painDETECT, a patient-reported screener to identify neuropathic pain (NeP), have not been psychometrically explored across NeP conditions.Methods: Scores on the nine-item painDETECT (seven pain symptom items, one pain course pattern item, one pain radiation item) range from -1 to 38; scores ≥19 indicate NeP is likely (>90% probability). The seven-item version (only pain symptoms) score range is 0 to 35. painDETECT was administered to subjects with confirmed diagnoses of human immunodeficiency virus-related peripheral NeP (HIVP) (n=103), spinal cord injury-related NeP (SCI) (n=103), small fiber neuropathy (n=100), painful diabetic peripheral neuropathy (n=112), posttrauma/postsurgical NeP (n=100), and NeP in chronic low back pain (n=106) identified during office visits to US community-based physicians. Analysis of covariance compared mean scores (adjusted for age, sex, race, ethnicity, time since NeP diagnosis, and number of comorbidities) on the nine-item and seven-item versions of painDETECT. Cronbach's alpha assessed internal consistency reliability, and corrected item-to-total correlations assessed item-level discrimination.Results: The adjusted mean nine-item scores ranged from 21.0 (SCI) to 24.3 (small fiber neuropathy). Differences between conditions were either trivial or small-to-medium in magnitude. Cronbach's alpha gave overall internal consistency reliability of 0.76, with a range of 0.63 (SCI) to 0.82 (HIVP). Mean scores and Cronbach's alphas for the seven-item version were generally similar to the nine-item version. Corrected item-to-total correlations adequately discriminated all pain symptom items on both painDETECT versions for each condition (0.3–0.7), but the two nonsensory items on the nine-item version showed lackluster discrimination (<0.3).Conclusion: painDETECT scores were within the range indicating high probability of NeP. Differences between conditions were generally modest or not large. Both versions showed evidence of internal consistency reliability and item-level discrimination, suggesting that painDETECT is a useful screening measure for identifying NeP across NeP conditions.Keywords: painful diabetic neuropathy, spinal cord injury, back pain, small fiber neuropathy, HIV-related peripheral neuropathy, posttrauma neuropath

A cross-sectional study examining the psychometric properties of the painDETECT measure in neuropathic pain

Joseph C Cappelleri,1 Vijaya Koduru,2 E Jay Bienen,3 Alesia Sadosky41Pfizer Inc, Groton, CT, USA; 2Eliassen Group, New London, CT, USA; 3Outcomes Research Consultant, New York, NY, USA; 4Pfizer Inc, New York, NY, USABackground: Similarities and differences on the nine-item and seven-item versions of painDETECT, a patient-reported screener to identify neuropathic pain (NeP), have not been psychometrically explored across NeP conditions.Methods: Scores on the nine-item painDETECT (seven pain symptom items, one pain course pattern item, one pain radiation item) range from -1 to 38; scores ≥19 indicate NeP is likely (>90% probability). The seven-item version (only pain symptoms) score range is 0 to 35. painDETECT was administered to subjects with confirmed diagnoses of human immunodeficiency virus-related peripheral NeP (HIVP) (n=103), spinal cord injury-related NeP (SCI) (n=103), small fiber neuropathy (n=100), painful diabetic peripheral neuropathy (n=112), posttrauma/postsurgical NeP (n=100), and NeP in chronic low back pain (n=106) identified during office visits to US community-based physicians. Analysis of covariance compared mean scores (adjusted for age, sex, race, ethnicity, time since NeP diagnosis, and number of comorbidities) on the nine-item and seven-item versions of painDETECT. Cronbach's alpha assessed internal consistency reliability, and corrected item-to-total correlations assessed item-level discrimination.Results: The adjusted mean nine-item scores ranged from 21.0 (SCI) to 24.3 (small fiber neuropathy). Differences between conditions were either trivial or small-to-medium in magnitude. Cronbach's alpha gave overall internal consistency reliability of 0.76, with a range of 0.63 (SCI) to 0.82 (HIVP). Mean scores and Cronbach's alphas for the seven-item version were generally similar to the nine-item version. Corrected item-to-total correlations adequately discriminated all pain symptom items on both painDETECT versions for each condition (0.3–0.7), but the two nonsensory items on the nine-item version showed lackluster discrimination (<0.3).Conclusion: painDETECT scores were within the range indicating high probability of NeP. Differences between conditions were generally modest or not large. Both versions showed evidence of internal consistency reliability and item-level discrimination, suggesting that painDETECT is a useful screening measure for identifying NeP across NeP conditions.Keywords: painful diabetic neuropathy, spinal cord injury, back pain, small fiber neuropathy, HIV-related peripheral neuropathy, posttrauma neuropath

Identification of compounds with anti-proliferative activity against Trypanosoma brucei brucei strain 427 by a whole cell viability based HTS campaign

Human African Trypanosomiasis (HAT) is caused by two trypanosome sub-species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Drugs available for the treatment of HAT have significant issues related to difficult administration regimes and limited efficacy across species and disease stages. Hence, there is considerable need to find new alternative and less toxic drugs. An approach to identify starting points for new drug candidates is high throughput screening (HTS) of large compound library collections. We describe the application of an Alamar Blue based, 384-well HTS assay to screen a library of 87,296 compounds against the related trypanosome subspecies, Trypanosoma brucei brucei bloodstream form lister 427. Primary hits identified against T.b. brucei were retested and the IC(50) value compounds were estimated for T.b. brucei and a mammalian cell line HEK293, to determine a selectivity index for each compound. The screening campaign identified 205 compounds with greater than 10 times selectivity against T.b. brucei. Cluster analysis of these compounds, taking into account chemical and structural properties required for drug-like compounds, afforded a panel of eight compounds for further biological analysis. These compounds had IC(50) values ranging from 0.22 µM to 4 µM with associated selectivity indices ranging from 19 to greater than 345. Further testing against T.b. rhodesiense led to the selection of 6 compounds from 5 new chemical classes with activity against the causative species of HAT, which can be considered potential candidates for HAT early drug discovery. Structure activity relationship (SAR) mining revealed components of those hit compound structures that may be important for biological activity. Four of these compounds have undergone further testing to 1) determine whether they are cidal or static in vitro at the minimum inhibitory concentration (MIC), and 2) estimate the time to kill