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BACKGROUND: New treatment alternatives have revolutionized the management of nAMD. However, there is limited evidence on the clinical and economic burden of nAMD in commercially insured US patients...

Introduction Admissions for acute bacterial skin and skin structure infections (ABSSSI) are often prolonged because of intravenous (IV) antibiotics. Use of a long-acting IV antibiotic may reduce length of stay (LOS) on a hospitalist service. The ENHANCE ABSSSI trial sought to determine the impact on LOS and work productivity in patients treated with a long-acting IV antibiotic, dalbavancin, vs. usual care at an urban tertiary-care center. Methods A single-center, pre- vs. post-period pragmatic trial at Weill-Cornell Medical Center assessed usual care for consecutively enrolled admitted ABSSSI patients during an observational period (pre-period). Identification and treatment of eligible admitted ABSSSI patients with dalbavancin were implemented in the post-period. Those with life-threatening infections, requiring multiple antibiotics/intensive care, or with unstable comorbidities were excluded. Outcomes were assessed over a 44-day follow-up period. Results Of 48 and 43 patients enrolled, respectively, in the pre- and post-periods, mean infection-related LOS was reduced in the post-period (3.2 days vs. 4.8 days; P = 0.003). Similar results were found in an adjusted LOS analysis. Work productivity and activity impairment outcomes significantly improved in the post-period (P ≤ 0.01). Complete response rates were similar: 50% (pre-period) and 57% (post-period). Among AEs identified, 17% (n = 7) were found to have possible causal relation to dalbavancin in the post-period. Few AEs were serious (n = 3; 7% post-period versus n = 1; 2% pre-period). Conclusion After implementing the ENHANCE ABSSSI pathway, LOS was significantly reduced by almost 2 days, with potential improvements in work productivity and ability to complete daily activities. Trial Registration ClinicalTrials.gov identifier, NCT03233438. Funding Allergan plc. Electronic supplementary material The online version of this article (10.1007/s40121-019-00275-4) contains supplementary material, which is available to authorized users.

Objectives Treatment of acute bacterial skin and skin structure infections (ABSSSIs) in the outpatient setting has potential advantages. We performed a subanalysis of outcomes for patients treated as outpatients versus inpatients with dalbavancin, a long-acting lipoglycopeptide, in a phase 3 clinical trial of ABSSSI. Methods The study was a double-blind trial of patients with ABSSSI randomised to receive dalbavancin 1500 mg intravenously as a single dose or two doses (1000 mg followed by 500 mg a week later). The primary endpoint was ≥20% reduction in erythema at 48–72 h after the start of therapy. Patient satisfaction and preference for antibiotic treatment and care setting were measured using the 10-item Skin and Soft Tissue Infection (SSTI) questionnaire at Day 14. Results A total of 698 patients were randomised (386 treated as outpatients and 312 as inpatients). Outpatients were more likely to be younger and to have major abscess or traumatic wound infection; inpatients were more likely to have cellulitis as the type of ABSSSI, to meet SIRS criteria and to have elevated plasma lactate at baseline. Efficacy and safety outcomes at 48–72 h, Days 14 and 28 were similar between patients treated in the outpatient and inpatient setting with either the single-dose or two-dose regimen. Outpatients reported significantly greater convenience and satisfaction with antibiotic treatment and care setting compared with inpatients (P Conclusion Single-dose dalbavancin is an effective treatment option for outpatients with ABSSSI and is associated with a high degree of patient treatment satisfaction and convenience.

Background Limited research has assessed patient preferences for treatment disposition and antibiotic therapy of acute bacterial skin and skin structure infection (ABSSSI) in the emergency department (ED). Understanding patient preference for the treatment of ABSSSI may influence treatment selection and improve satisfaction. Methods A survey was conducted across 6 US hospital EDs. Patients with ABSSSI completed a baseline survey assessing preferences for antibiotic therapy (intravenous versus oral) and treatment location. A follow-up survey was conducted within 30–40 days after ED discharge to reassess preferences and determine satisfaction with care. Results A total of 94 patients completed both baseline and follow-up surveys. Sixty (63.8%) participants had a history of ABSSSI, and 69 (73.4%) were admitted to the hospital. Treatment at home was the most common preference reported on baseline and follow-up surveys. Patients with higher education were 82.2% less likely to prefer treatment in the hospital. Single dose intravenous therapy was the most commonly preferred antibiotic regimen on baseline and follow-up surveys (39.8 and 19.1%, respectively). Median satisfaction scores for care in the ED, hospital, home, and with overall antibiotic therapy were all 8 out of a maximum of 10. Conclusions In these patients, the most common preference was for outpatient care and single dose intravenous antibiotics. Patient characteristics including higher education, younger age, and current employment were associated with these preferences. Opportunities exist for improving ABSSSI care and satisfaction rates by engaging patients and offering multiple treatment choices. Electronic supplementary material The online version of this article (10.1186/s12913-018-3751-0) contains supplementary material, which is available to authorized users.

The extent to which pharmacogenomic-guided medication use has been adopted in various health systems is unclear. To assess the uptake of pharmacogenomic-guided medication use, we determined its frequency across our health system, which does not have a structured testing program. Using a multisite clinical data repository, we identified adult patients' first prescribed medications between January 2011 and December 2013 and investigated the frequency of germline and somatic pharmacogenomic testing, by the Pharmacogenomics Knowledgebase level of the US Food and Drug Administration label information. There were 268,262 medication orders for drugs with germline pharmacogenomic testing information in their drug labels. Pharmacogenomic testing was detected for 1.5% (129/8,718) of medication orders with recommended or required testing. Of the 3,817 medication orders associated with somatic pharmacogenomic testing information in their drug labels, 20% (372/1,819) of required tests were detected. The low rates of detectable pharmacogenomic testing suggest that structured testing programs are required to achieve the success of precision medicine.

Adherence and persistence to therapy, or how well a patient follows provider directions on frequency and time to discontinuation of prescribed medications, is associated with positive health outcomes, including decreased healthcare costs and patient mortality. A clear literature gap exists assessing adherence and persistence to antidepressants (ADs) in the major depressive disorder (MDD) population at clinically relevant time points and at the therapeutic class level. This study assessed adherence and persistence to specific ADs, therapeutic classes, and AD therapy overall at multiple time points among US individuals from commercial, Medicare supplemental, and Medicaid insurance plans. Patients with MDD without AD or MDD claims in the prior 6 months who initiated therapy in 2003–2014 with a selective serotonin reuptake inhibitor (SSRI), serotonin and norepinephrine reuptake inhibitor (SNRI), tricyclic AD (TCA), monoamine oxidase inhibitor (MAOI), or other AD were identified using MarketScan® databases. These databases contain information on diagnoses, billing codes, and dates of service. Adherence (proportion of days covered) and persistence (days until a 30-day gap in therapy) were calculated to AD medication, AD therapeutic class, and AD therapy overall over the first 3, 6, 9, and 12 months from the index prescription date. Multivariable logistic regression estimated the adjusted odds ratios (ORs) of adherence to initial AD medication comparing AD therapeutic classes. For 527,907 patients, adherence to initial AD medication decreased over 3, 6, 9, and 12 months (41, 31, 24, and 21%, respectively). Similar patterns were observed for adherence to initial AD therapeutic class, AD therapy overall, and all three persistence calculations. The odds of adherence to SNRIs versus SSRIs were 20–27% greater at 3, 6, 9, and 12 months (ORs 1.20, 1.23, 1.25, 1.27, respectively; p-values all 0.05)]. We found low adherence and persistence to ADs in the MDD population. Within the limitations of the insurance claims data we analysed, our results suggest that adherence may differ based on therapeutic class, as patients initiating SNRI therapy appeared to have a higher likelihood of adherence versus SSRIs over the year assessed, while the odds of adherence appeared similar or lower for other classes versus SSRIs. Further prospective research is needed to confirm these findings and determine additional drivers of these apparent differences by AD therapeutic class.

Babesiosis is a global zoonotic disease acquired by the bite of a Babesia-infected Ixodes tick or through blood transfusion with clinical relevance affecting humans and animals. In this study, we evaluated a series of small molecule compounds that have previously been shown to target specific apicomplexan enzymes in Plasmodium, Toxoplasma and Cryptosporidium. The compounds, bumped kinase inhibitors (BKIs), have strong therapeutic potential targeting apicomplexa-specific calcium dependent protein kinases (CDPKs). We investigated if BKIs also show inhibitory activities against piroplasms such as Babesia. Using a subset of BKIs that have promising inhibitory activities to Plasmodium and Toxoplasma, we determined that their actions ranged from 100% and no inhibition against Babesia bovis blood stages. One specific BKI, RM-1-152, showed complete inhibition against B. bovis within 48h and was the only BKI that showed noticeable phenotypic changes to the parasites. Focusing our study on this BKI, we further demonstrated that RM-1-152 has Babesia-static activity and involves the prohibition of merozoite egress while replication and re-invasion of host cells are unaffected. The distinct, abnormal phenotype induced by RM-1-152 suggests that this BKI can be used to investigate less studied cellular processes such as egression in piroplasm.

Background With the rise of antibiotic resistance, polymyxin use has re-emerged but with a concern of renal toxicity. This study aims to assess mortality, length of stay, and total hospitalization cost associated with acute kidney injury (AKI) among recipients of intravenous (IV) sodium colistimethate (CMS) or IV polymyxin B (PMB). Methods We conducted a retrospective database analysis using the Premier database from January 1, 2012, through September 30, 2015. Adults ≥18 years of age who were admitted for inpatient treatment with ≥3 consecutive days of CMS or PMB were included. Generalized linear models compared patients who developed AKI with those who did not. Models were adjusted for patient and clinical characteristics. Results A total of 4886 patients were included; 4103 patients received CMS, and 783 received PMB. In the multivariable analyses, the presence of AKI was associated with higher in-hospital mortality in both the CMS cohort (adjusted odds ratio [aOR], 2.3; 95% confidence interval [CI], 1.9–2.7; P < .001) and the PMB cohort (aOR, 2.7; 95% CI, 1.8–4.2; P < .001). In both cohorts, patients who developed AKI experienced longer hospital stays (9.7 days and 11.6 days in the CMS and PMB cohorts, respectively; P < .001). The mean total hospitalization costs for patients who developed AKI were $47 820 higher (95% CI, $34 918–$60 722) in the CMS cohort and $35 244 higher (95% CI, $17 561–$52 928) in the PMB cohort. Conclusions The clinical and economic burden of AKI in the context of polymyxin use is substantial. The use of effective antibiotics with limited toxicity should remain a priority.

Background The objective of this study was to characterize treatment of patients with acute bacterial skin and skin structure infections (ABSSSIs) and describe the association between hospital admission and emergency department (ED) visits or readmissions within 30 days after initial episode of care (IEC). Methods This was a retrospective, observational, cohort study of adults with ABSSSI who presented to an ED between July 1, 2012, and June 30, 2013. Patient, health care facility, and treatment characteristics, including unplanned ED visits or readmissions, were obtained through manual chart review and abstraction. Adjusted logistic regression analysis examined likelihood of all-cause unplanned ED visits or readmissions between admitted and nonadmitted patients. Results Records from 1527 ED visits for ABSSSI from 40 centers were reviewed (admitted, n = 578 [38%]; nonadmitted, n = 949 [62%]). Admitted patients were typically older (mean age, 52.2 years vs 43.0 years), more likely to be morbidly obese (body mass index > 40 kg/m2; 17.3% vs 9.1%), and had more comorbidities (Charlson Comorbidity Index ≥ 4; 24.4% vs 6.8%) compared with those not admitted. In the primary analysis, adjusted logistic regression, controlling for comorbidities and severity of illness, demonstrated that there was a similar likelihood of all-cause unplanned ED visits or readmissions between admitted and nonadmitted patients (odds ratio, 1.03; 95% confidence interval, 0.74–1.43; P = .87). Conclusions ABSSSI treatment pathways leveraging outpatient treatment vs hospital admission support similar likelihood of unplanned 30-day ED visits or readmissions, an important clinical outcome and quality metric at US hospitals. Further research regarding the decision criteria around hospital admission to avoid potentially unnecessary hospitalizations is warranted.

Background Acute bacterial skin and skin structure infections (ABSSSIs) are a frequent cause of emergency department (ED) visits. Providers in the ED have many decisions to make during the initial treatment of ABSSSI. There are limited data on the patient factors that influence these provider decisions. Methods An anonymous survey was administered to providers at 6 EDs across the United States. The survey presented patient cases with ABSSSIs ≥75 cm2 and escalating clinical scenarios including relapse, controlled diabetes, and sepsis. For each case, participants were queried on their decision for admission vs discharge and antibiotic therapy (intravenous, oral, or both) and to rank the factors that influenced their antibiotic decision. Results The survey was completed by 130 providers. For simple ABSSSI, the majority of providers chose an oral antibiotic and discharged patients home. The presence of recurrence or controlled diabetes resulted in more variation in responses. Thirty-four (40%) and 51 (60%) providers chose intravenous followed by oral antibiotics and discharged the recurrence and diabetes cases, respectively. Presentation with sepsis resulted in initiation with intravenous antibiotics (122, 95.3%) and admission (125, 96.1%) in most responses. Conclusions Variability in responses to certain patient scenarios suggests opportunities for education of providers in the ED and the development of an ABSSSI clinical pathway to help guide treatment.

Objectives: We estimated the total US hospital costs associated with acute bacterial skin and skin structure infection (ABSSSI) admissions as well as the admissions that may have been potential candidates for outpatient parenteral antimicrobial therapy (OPAT). Methods: We assessed inpatient admissions for ABSSSI from the Premier database (2011–2014), focusing on all admissions of adults with length of stay (LOS) ≥ 1 days and a primary diagnosis of erysipelas, cellulitis/abscess, or wound infection. We performed a detailed analysis of 2014 admissions for patient, treatment, hospital, and economic characteristic variables. Using published selection criteria, we identified a subset of patients admitted in 2014 who may have been potential candidates for OPAT. Results: We analyzed 277,971 admissions. In 2014, most admissions were for cellulitis without major complications or comorbidities; mean ± SD LOS was 4.0 ± 3.0 days, and total hospital cost per admission was $6400 ± $6874, 54% of which was attributable to room costs. Among 2014 admissions, 14% involved patients with clinical characteristics suggesting that they were consistent with guideline recommendations for exclusive treatment with OPAT. Compared with all admissions in the year, these admissions were of younger patients (aged 50 vs. 55 years), admitted more frequently for cellulitis (90% vs. 70%), with shorter LOS (2.8 ± 1.8 days), and lower mean total hospital cost per admission ($4080 ± $3066). Conclusions: Admissions for ABSSSI impose a substantial cost to US hospitals, with half of costs attributable to room costs. When extrapolated to all US patients admitted to the hospital for ABSSSI during 2014, had OPAT guidelines been universally followed, admissions may have been reduced by 14%, thereby saving US hospitals $161 million.

Background Prolonged admissions for acute bacterial skin and skin structure infections (ABSSSI) present an opportunity to improve efficiency and quality of care. A primary reason for admission for ABSSSI is to receive intravenous (IV) antibiotics, where multiple guidelines support shifting care to outpatient settings for appropriate patients. A hospital pathway for ABSSSI that leverages long-acting IV antibiotic therapy, such as dalbavancin, may reduce the length of stay (LOS). The ENHANCE ABSSSI trial (NCT03233438) sought to quantify LOS vs. that of usual care after implementing a new ABSSSI pathway. Methods A single-center, pre- vs. post-period pragmatic trial at Weill-Cornell Medical Center assessed usual care for consecutively enrolled ABSSSI patients during an observational period (pre-period). A new ABSSSI pathway was implemented in the post-period, which included (1) identification of eligible admitted ABSSSI patients and (2) treatment with dalbavancin. Those with life-threatening infections, requiring multiple antibiotics/intensive care, or with unstable comorbidities were excluded. Outcomes were assessed over a 44-day follow-up period. Results Of 48 and 43 patients enrolled in pre- and post-periods (Figure 1), mean infection-related LOS was reduced in the post-period (3.2 days vs. 4.8 days; P = 0.003; Figure 2 and 3). Similar results were found in an adjusted LOS analysis. Work productivity and activity impairment outcomes significantly improved in the post-period, apart from absenteeism, while quality of life was similar between periods (Figure 4). Complete response to treatment was similar between periods: 50% (pre-period) and 57% (post-period). A greater proportion of total adverse events (AEs) occurred in the post-period (n = 20; 48%) vs. pre-period (n = 3; 6%) with most AEs being mild in severity and not related to antibiotic use; few AEs were serious (7% [n = 3] post-period vs. 2% [n = 1] pre-period). The most common AEs were unrelated infection in the pre-period and fever in the post-period. Conclusion After implementing the ENHANCE ABSSSI pathway among eligible patients, LOS was significantly reduced by almost 2 days, with potential improvements in work productivity and the ability to complete daily activities. Disclosures All authors: No reported disclosures.

Malaria remains a major health concern for a large percentage of the world's population. While great strides have been made in reducing mortality due to malaria, new strategies and therapies are still needed. Therapies that are capable of blocking the transmission of Plasmodium parasites are particularly attractive, but only primaquine accomplishes this, and toxicity issues hamper its widespread use. In this study, we describe a series of pyrazolopyrimidine- and imidazopyrazine-based compounds that are potent inhibitors of PfCDPK4, which is a calcium-activated Plasmodium protein kinase that is essential for exflagellation of male gametocytes. Thus, PfCDPK4 is essential for the sexual development of Plasmodium parasites and their ability to infect mosquitoes. We demonstrate that two structural features in the ATP-binding site of PfCDPK4 can be exploited in order to obtain potent and selective inhibitors of this enzyme. Furthermore, we demonstrate that pyrazolopyrimidine-based inhibitors that are potent inhibitors of the in vitro activity of PfCDPK4 are also able to block Plasmodium falciparum exflagellation with no observable toxicity to human cells. This medicinal chemistry effort serves as a valuable starting point in the development of safe, transmission-blocking agents for the control of malaria.

Background In an effort to lower costs and improve quality of care, there is potential to change the treatment landscape for low-risk (i.e., less severe) ABSSSI patients who historically required inpatient management, a costly option. Outpatient IV treatment pathways have been shown to be a cost-saving option for hospitals and insurers. The objective was to quantify the potential opportunity for reducing cost of ABSSSI treatment in an insured Commercial and Medicare Aetna population. Methods Adult patients between January 2013 and July 2016 were identified with a primary ABSSSI claim (Table 1) in the Aetna fully-insured Commercial and Medicare insurance claims database. ABSSSI encounters were identified with insurance eligibility for the 7 months prior to and no evidence of ABSSSI in the 30 days prior to the ABSSSI claim. Demographic and clinical data were described, including length of stay (LOS) and allowed cost for inpatient encounters with data. Inpatient encounters without evidence of severity (e.g., codes for major complications or comorbidities) were considered potential candidates for an outpatient LAA pathway. A sensitivity analysis for LOS and cost was run including all ABSSSI patients with LAA dispenses through 2016 (i.e., inclusion/exclusion criteria did not need to be met). Results 194,023 ABSSSI encounters were identified, most receiving non-IV treatment (90%). 18,603 received IV treatment, where 83% initially presented to the emergency room and the majority were admitted (97%). Of the 28 encounters with LAA use, 7 were inpatient. Of all current inpatient encounters (N = 9,019 after Jan 1, 2015), the majority (N = 7,005; 78%) where considered potential LAA pathway candidates. Comparing inpatient encounters with vs. without LAA use, mean LOS and cost differed (Table 2: 4.1 days and $14,295 vs. 9.0 days and $23,194, respectively). A sensitivity analysis supported similar mean LOS and cost for all inpatient LAA dispenses. Conclusion Current use of LAA in an inpatient population is limited but resulted in potential cost-savings. Most of the inpatient population was identified as potential candidates for an outpatient LAA pathway. Research on utilization and quality of care for outpatient IV treatment pathways with LAA is warranted. Disclosures K. R. Keyloun, Allergan: Employee, Salary N. Bonine, Allergan: Employee, Salary

Background Reducing hospital admission and improving transitions of care can lessen the burden of ABSSSIs in EDs and hospitals. Limited research to date has assessed the patient’s preference for ABSSSI treatment. Understanding patient preferences may provide insights that encourage optimal treatment plans and improve satisfaction with their care. Methods A patient survey was conducted across 5 hospital EDs in the US. After providing informed consent, patients with ABSSSI completed a baseline survey assessing their ABSSSI history and preferences for antibiotic (ABX) therapy [intravenous (IV) vs. oral] and treatment location. Patient characteristics and ensuing treatment details were collected from the medical record after the ED or hospitalization was completed. Descriptive statistics were used for analyses. Results Seventy-one patients were enrolled and completed the baseline survey. The mean ± SD age was 50 ± 17 years, 22 (31%) had diabetes, and 47 (66.2%) had a previous ABSSSI. The median (25th-75th quartile) lesion size was 190 (53-613) cm2; 51 (71.8%) presented with cellulitis, an abscess, or both. Fifty-four (76.1%) were admitted to the hospital with a median (25th-75thquartile) length of stay of 4.5 (2-9) days. In the ED, vancomycin (39.4%) and β-lactams (36.6%) were the most common ABX; IV was prescribed in 58/63 (92.1%) patients. Once admitted, 37 (68.5%) and 34 (63%) patients received vancomycin and β-lactams, respectively. When surveyed, 26 (36.6%) patients preferred to receive ABX at home, while 22 (31.0%) chose hospital stay for one or more nights. The most common ABX preference was a single IV dose to complete treatment, selected by 29 (40.8%) patients. The most important ABX factors were efficacy and their doctor’s opinion, then convenience and route of administration; least important were treatment location, adverse events, and cost. Conclusion In these patients presenting to the ED with ABSSSI, the majority were admitted to the hospital and received IV ABX. Patient preferences for treatment location varied, but many valued therapies that could prevent admission. These data suggest opportunities for improving ABSSSI care by engaging the patient and offering treatment alternatives they may not be aware of. Disclosures K. R. Keyloun, Allergan: Employee, Salary. D. P. Nicolau, Allergan: Grant Investigator, Scientific Advisor and Speaker’s Bureau, Consulting fee, Research grant and Speaker honorarium. J. L. Kuti, Allergan: Grant Investigator, Investigator, Scientific Advisor and Speaker’s Bureau, Consulting fee, Research grant and Speaker honorarium

The protozoans Cryptosporidium parvum and Toxoplasma gondii are parasites of major health concern to humans. Both parasites contain a group of calcium-dependent protein kinases (CDPKs), which are found in plants and ciliates but not in humans or fungi. Here we describe a series of potent inhibitors that target CDPK1 in C. parvum (CpCDPK1) and T. gondii (TgCDPK1). These inhibitors are highly selective for CpCDPK1 and TgCDPK1 over the mammalian kinases SRC and ABL. Furthermore, they are able to block an early stage of C. parvum invasion of HCT-8 host cells, which is similar to their effects on T. gondii invasion of human fibroblasts.

Background: The availability of high fidelity electronic health record (EHR) data is a hallmark of the learning health care system. Washington State’s Surgical Care Outcomes and Assessment Program (SCOAP) is a network of hospitals participating in quality improvement (QI) registries wherein data are manually abstracted from EHRs. To create the Comparative Effectiveness Research and Translation Network (CERTAIN), we semi-automated SCOAP data abstraction using a centralized federated data model, created a central data repository (CDR), and assessed whether these data could be used as real world evidence for QI and research.Objectives: Describe the validation processes and complexities involved and lessons learned.Methods: Investigators installed a commercial CDR to retrieve and store data from disparate EHRs. Manual and automated abstraction systems were conducted in parallel (10/2012-7/2013) and validated in three phases using the EHR as the gold standard: 1) ingestion, 2) standardization, and 3) concordance of automated versus manually abstracted cases. Information retrieval statistics were calculated.Results: Four unaffiliated health systems provided data. Between 6 and 15 percent of data elements were abstracted: 51 to 86 percent from structured data; the remainder using natural language processing (NLP). In phase 1, data ingestion from 12 out of 20 feeds reached 95 percent accuracy. In phase 2, 55 percent of structured data elements performed with 96 to 100 percent accuracy; NLP with 89 to 91 percent accuracy. In phase 3, concordance ranged from 69 to 89 percent. Information retrieval statistics were consistently above 90 percent.Conclusions: Semi-automated data abstraction may be useful, although raw data collected as a byproduct of health care delivery is not immediately available for use as real world evidence. New approaches to gathering and analyzing extant data are required.

New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds designed to be inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site at residue 128. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii cells expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to this class of selective kinase inhibitors. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable.

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds' mechanisms of action--i.e., the specific molecular targets by which they kill the parasite--would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children's Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC50 < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible.

Background ABSSSI are a frequent cause of emergency room visits. Physicians (PHY) and Advanced Practice Providers (APP) have many decisions to make during the initial treatment of ABSSSI, including intravenous (IV) vs. oral (PO) ABX, and hospital admission vs. discharge home. There are limited data, however, on factors that influence the provider’s decision for the treatment of ABSSSI. Methods An anonymous survey was offered to EM providers at 5 hospitals across the US. The survey presented patient cases with ABSSSI ≥ 75cm2 and escalating clinical scenarios including relapse, controlled diabetes (DM), and SIRS. For each case, participants were queried on their decision for admission vs. discharge, ABX therapy (IV, PO, or both), and to rank factors that influenced their ABX decision. Descriptive and inferential statistics were used for analyses. Results Across the 5 hospitals, 104 providers completed the survey; 59 (56.7%) were PHY and 45 (43.3%) were APP. Experience was evenly represented with 50 (48.1%) providers practicing for ≥5 years and 54 (51.9%) less than 5 years. For the first case with no relapse, DM, or SIRS, most providers selected PO ABX [95 (91.4%)] and discharge [100 (96.2%)]. In case 2, the presence of relapse resulted in PO ABX for 50 (48.1%) and discharge in 54 (51.9%) of responses. In case 3, the presence of controlled DM resulted in PO ABX for 55 (53.4%) and discharge home in 65 (63.1%) of responses. Four (3.8%) and 13 (12.5%) providers chose to give 1–2 IV doses followed by PO and discharge the relapsed and DM cases, respectively. Compared with APP, PHY more frequently prescribed PO ABX for the DM case (62.7% vs. 40.9%, P = 0.046) and incorporated PO ABX into the treatment of the SIRS case (48.3% vs. 22.7%, P = 0.015). The addition of SIRS criteria in case 4 resulted in initiation with IV ABX [97 (95.1%)] and admission [100 (96.2%)]. The highest ranked factors influencing ABX selection were infection severity and co-morbidities; the lowest were patient convenience, adverse events, and cost. Conclusion These data highlight factors influencing EM provider decisions in the treatment of ABSSSI. Variability in responses to patient cases suggests opportunities for education and the development of an ABSSSI clinical pathway to guide treatment. Disclosures K. R. Keyloun, Allergan: Employee, Salary. D. P. Nicolau, Allergan: Grant Investigator, Scientific Advisor and Speaker’s Bureau, Consulting fee, Research grant and Speaker honorarium. J. L. Kuti, Allergan: Grant Investigator, Investigator, Scientific Advisor and Speaker’s Bureau, Consulting fee, Research grant and Speaker honorarium

SUMMARYSpecific roles of individual CDPKs vary, but in general they mediate essential biological functions necessary for parasite survival. A comparative analysis of the structure-activity relationships (SAR) of Neospora caninum, Eimeria tenella and Babesia bovis calcium-dependent protein kinases (CDPKs) together with those of Plasmodium falciparum, Cryptosporidium parvum and Toxoplasma gondii was performed by screening against 333 bumped kinase inhibitors (BKIs). Structural modelling and experimental data revealed that residues other than the gatekeeper influence compound–protein interactions resulting in distinct sensitivity profiles. We subsequently defined potential amino-acid structural influences within the ATP-binding cavity for each orthologue necessary for consideration in the development of broad-spectrum apicomplexan CDPK inhibitors. Although the BKI library was developed for specific inhibition of glycine gatekeeper CDPKs combined with low inhibition of threonine gatekeeper human SRC kinase, some library compounds exhibit activity against serine- or threonine-containing CDPKs. Divergent BKI sensitivity of CDPK homologues could be explained on the basis of differences in the size and orientation of the hydrophobic pocket and specific variation at other amino-acid positions within the ATP-binding cavity. In particular, BbCDPK4 and PfCDPK1 are sensitive to a larger fraction of compounds than EtCDPK1 despite the presence of a threonine gatekeeper in all three CDPKs.

Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.

5-Aminopyrazole-4-carboxamide was used as an alternative scaffold to substitute for the pyrazolopyrimidine of a known "bumped kinase inhibitor" to create selective inhibitors of calcium-dependent protein kinase-1 from both Toxoplasma gondii and Cryptosporidium parvum. Compounds with low nanomolar inhibitory potencies against the target enzymes were obtained. The most selective inhibitors also exhibited submicromolar activities in T. gondii cell proliferation assays and were shown to be non-toxic to mammalian cells.

IMPORTANCE Genetic biomarkers that predict a drug’s efficacy or likelihood of toxicity are assuming increasingly important roles in the personalization of pharmacotherapy, but concern exists that evidence that links use of some biomarkers to clinical benefit is insufficient. Nevertheless, information about the use of biomarkers appears in the labels of many prescription drugs, whichmay add confusion to the clinical decision-making process.