Myocardial Recovery in Recent Onset Dilated Cardiomyopathy: Role of CDCP1 and Cardiac Fibrosis.

BACKGROUND: Dilated cardiomyopathy (DCM) is a major cause of heart failure and carries a high mortality rate. Myocardial recovery in DCM-related heart failure patients is highly variable, with some patients having little or no response to standard drug therapy. A genome-wide association study may agnostically identify biomarkers and provide novel insight into the biology of myocardial recovery in DCM. METHODS: A genome-wide association study for change in left ventricular ejection fraction was performed in 686 White subjects with recent-onset DCM who received standard pharmacotherapy. Genome-wide association study signals were subsequently functionally validated and studied in relevant cellular models to understand molecular mechanisms that may have contributed to the change in left ventricular ejection fraction. RESULTS: The genome-wide association study identified a highly suggestive locus that mapped to the 5'-flanking region of the CDCP1 (CUB [complement C1r/C1s, Uegf, and Bmp1] domain containing protein 1) gene (rs6773435; P=7.12×10-7). The variant allele was associated with improved cardiac function and decreased CDCP1 transcription. CDCP1 expression was significantly upregulated in human cardiac fibroblasts (HCFs) in response to the PDGF (platelet-derived growth factor) signaling, and knockdown of CDCP1 significantly repressed HCF proliferation and decreased AKT (protein kinase B) phosphorylation. Transcriptomic profiling after CDCP1 knockdown in HCFs supported the conclusion that CDCP1 regulates HCF proliferation and mitosis. In addition, CDCP1 knockdown in HCFs resulted in significantly decreased expression of soluble ST2 (suppression of tumorigenicity-2), a prognostic biomarker for heart failure and inductor of cardiac fibrosis. CONCLUSIONS: CDCP1 may play an important role in myocardial recovery in recent-onset DCM and mediates its effect primarily by attenuating cardiac fibrosis.

Digital Tool-Assisted Hospitalization Detection in the Tailored Antiplatelet Initiation to Lessen Outcomes due to Decreased Clopidogrel Response After Percutaneous Coronary Intervention Study Compared to Traditional Site-Coordinator Ascertainment: Intervention Study.

BACKGROUND: Accurate, timely ascertainment of clinical end points, particularly hospitalizations, is crucial for clinical trials. The Tailored Antiplatelet Initiation to Lessen Outcomes Due to Decreased Clopidogrel Response after Percutaneous Coronary Intervention (TAILOR-PCI) Digital Study extended the main TAILOR-PCI trial's follow-up to 2 years, using a smartphone-based research app featuring geofencing-triggered surveys and routine monthly mobile phone surveys to detect cardiovascular (CV) hospitalizations. This pilot study compared these digital tools to conventional site-coordinator ascertainment of CV hospitalizations. OBJECTIVE: The objectives were to evaluate geofencing-triggered notifications and routine monthly mobile phone surveys' performance in detecting CV hospitalizations compared to telephone visits and health record reviews by study coordinators at each site. METHODS: US and Canadian participants from the TAILOR-PCI Digital Follow-Up Study were invited to download the Eureka Research Platform mobile app, opting in for location tracking using geofencing, triggering a smartphone-based survey if near a hospital for ≥4 hours. Participants were sent monthly notifications for CV hospitalization surveys. RESULTS: From 85 participants who consented to the Digital Study, downloaded the mobile app, and had not previously completed their final follow-up visit, 73 (85.8%) initially opted in and consented to geofencing. There were 9 CV hospitalizations ascertained by study coordinators among 5 patients, whereas 8 out of 9 (88.9%) were detected by routine monthly hospitalization surveys. One CV hospitalization went undetected by the survey as it occurred within two weeks of the previous event, and the survey only allowed reporting of a single hospitalization. Among these, 3 were also detected by the geofencing algorithm, but 6 out of 9 (66.7%) were missed by geofencing: 1 occurred in a participant who never consented to geofencing, while 5 hospitalizations occurred among participants who had subsequently turned off geofencing prior to their hospitalization. Geofencing-detected hospitalizations were ascertained within a median of 2 (IQR 1-3) days, monthly surveys within 11 (IQR 6.5-25) days, and site coordinator methods within 38 (IQR 9-105) days. The geofencing algorithm triggered 245 notifications among 39 participants, with 128 (52.2%) from true hospital presence and 117 (47.8%) from nonhospital health care facility visits. Additional geofencing iterative improvements to reduce hospital misidentification were made to the algorithm at months 7 and 12, elevating the rate of true alerts from 35.4% (55 true alerts/155 total alerts before month 7) to 78.7% (59 true alerts/75 total alerts in months 7-12) and ultimately to 93.3% (14 true alerts/5 total alerts in months 13-21), respectively. CONCLUSIONS: The monthly digital survey detected most CV hospitalizations, while the geofencing survey enabled earlier detection but did not offer incremental value beyond traditional tools. Digital tools could potentially reduce the burden on study coordinators in ascertaining CV hospitalizations. The advantages of timely reporting via geofencing should be weighed against the issue of false notifications, which can be mitigated through algorithmic refinements.

Point of care CYP2C19 genotyping after percutaneous coronary intervention.

Loss-of-function CYP2C19 variants are associated with increased cumulative ischemic outcomes warranting CYP2C19 genotyping prior to clopidogrel administration. TAILOR-PCI was an international, multicenter (40 sites), prospective, randomized trial comparing rapid point of care (POC) genotype-guided vs. conventional anti-platelet therapy. The performance of buccal-based rapid CYP2C19 genotyping performed by non-laboratory-trained staff in TAILOR-PCI was assessed. Pre-trial training and evaluation involved rapid genotyping of 373 oral samples, with 99.5% (371/373) concordance with Sanger sequencing. During TAILOR-PCI, 5302 patients undergoing PCI were randomized to POC rapid CYP2C19 *2, *3, and *17 genotyping versus no genotyping. At 12 months post-PCI, TaqMan genotyping determined 99.1% (2,364/2,385) concordance with the POC results, with 90.7-98.8% sensitivity and 99.2-99.6% specificity. In conclusion, non-laboratory personnel can be successfully trained for on-site instrument operation and POC rapid genotyping with analytical accuracy and precision across multiple international centers, thereby supporting POC genotyping in patient-care settings, such as the cardiac catheterization laboratory.Clinical Trial Registration: https://www.clinicalTrials.gov (Identifier: NCT01742117).

Safety and Efficacy of CYP2C19 Genotype-Guided Escalation of P2Y12 Inhibitor Therapy After Percutaneous Coronary Intervention in Chronic Kidney Disease: a Post Hoc Analysis of the TAILOR-PCI Study.

PURPOSE: Chronic kidney disease (CKD) is a risk factor for ischemic and bleeding events with dual antiplatelet therapy after percutaneous coronary intervention (PCI). Whether the presence of CYP2C19 loss of function (LOF) alleles modifies this risk, and whether a genotype-guided (GG) escalation of P2Y12 inhibitor therapy post PCI is safe in this population is unclear. METHODS: This was a post hoc analysis of randomized patients in TAILOR PCI. Patients were divided into two groups based on estimated glomerular filtration rate (eGFR) threshold of < 60 ml/min/1.73 m2 for CKD (n = 539) and non-CKD (n = 4276). The aggregate of cardiovascular death, stroke, myocardial infarction, stent thrombosis, and severe recurrent coronary ischemia at 12-months post-PCI was assessed as the primary endpoint. Secondary endpoint was major or minor bleeding. RESULTS: Mean (standard deviation) eGFR among patients with CKD was 49.5 (8.4) ml/min/1.72 m2. Among all patients, there was no significant interaction between randomized strategy and CKD status for any endpoint. Among LOF carriers, the interaction between randomized strategy and CKD status on composite ischemic outcome was not significant (p = 0.2). GG strategy was not associated with an increased risk of bleeding in either CKD group. CONCLUSIONS: In this exploratory analysis, escalation of P2Y12 inhibitor therapy following a GG strategy did not reduce the primary outcome in CKD. However, P2Y12 inhibitor escalation following a GG strategy was not associated with increased bleeding risk in CKD. Larger studies in CKD are needed. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT01742117?term=TAILOR-PCI&draw=2&rank=1 . NCT01742117.

Rationale and design of the TAILOR-PCI digital study: Transitioning a randomized controlled trial to a digital registry.

BACKGROUND: Tailored Antiplatelet Initiation to Lessen Outcomes Due to Decreased Clopidogrel Response after Percutaneous Coronary Intervention (TAILOR-PCI) is the largest cardiovascular genotype-based randomized pragmatic trial (NCT#01742117) to evaluate the role of genotype-guided selection of oral P2Y12 inhibitor therapy in improving ischemic outcomes after PCI. The trial has been extended from the original 12- to 24-month follow-up, using study coordinator-initiated telephone visits. TAILOR-PCI Digital Study tests the feasibility of extending the trial follow-up in a subset of patients for up to 24 months using state-of-the-art digital solutions. The rationale, design, and approach of extended digital study of patients recruited into a large, international, multi-center clinical trial has not been previously described. METHODS: A total of 930 patients from U.S. and Canadian sites previously enrolled in the 5,302 patient TAILOR-PCI trial within 23 months of randomization are invited by mail to the Digital Study website (http://tailorpci.eurekaplatform.org) and by up to 2 recruiting telephone calls. Eureka, a direct-to-participant digital research platform, is used to consent and collect prospective data on patients for the digital study. Patients are asked to answer health-related surveys at fixed intervals using the Eureka mobile app and or desktop platform. The likelihood of patients enrolled in a randomized clinical trial transitioning to a registry using digital technology, the reasons for nonparticipation and engagement rates are evaluated. To capture hospitalizations, patients may optionally enable geofencing, a process that allows background location tracking and triggering of surveys if a hospital visit greater than 4 hours is detected. In addition, patients answer digital hospitalization surveys every month. Hospitalization data received from the Digital Study will be compared to data collected from study coordinator telephone visits during the same time frame. CONCLUSIONS: The TAILOR-PCI Digital Study evaluates the feasibility of transitioning a large multicenter randomized clinical trial to a digital registry. The study could provide evidence for the ability of digital technology to follow clinical trial patients and to ascertain trial-related events thus also building the foundation for conducting digital clinical trials. Such a digital approach may be especially pertinent in the era of COVID-19.

Cocaine Hydrolase Gene Transfer Demonstrates Cardiac Safety and Efficacy against Cocaine-Induced QT Prolongation in Mice.

Cocaine addiction is associated with devastating medical consequences, including cardiotoxicity and risk-conferring prolongation of the QT interval. Viral gene transfer of cocaine hydrolase engineered from butyrylcholinesterase offers therapeutic promise for treatment-seeking drug users. Although previous preclinical studies have demonstrated benefits of this strategy without signs of toxicity, the specific cardiac safety and efficacy of engineered butyrylcholinesterase viral delivery remains unknown. Here, telemetric recording of electrocardiograms from awake, unrestrained mice receiving a course of moderately large cocaine doses (30 mg/kg, twice daily for 3 weeks) revealed protection against a 2-fold prolongation of the QT interval conferred by pretreatment with cocaine hydrolase vector. By itself, this prophylactic treatment did not affect QT interval duration or cardiac structure, demonstrating that viral delivery of cocaine hydrolase has no intrinsic cardiac toxicity and, on the contrary, actively protects against cocaine-induced QT prolongation.

Reward and Toxicity of Cocaine Metabolites Generated by Cocaine Hydrolase.

Butyrylcholinesterase (BChE) gene therapy is emerging as a promising concept for treatment of cocaine addiction. BChE levels after gene transfer can rise 1000-fold above those in untreated mice, making this enzyme the second most abundant plasma protein. For months or years, gene transfer of a BChE mutated into a cocaine hydrolase (CocH) can maintain enzyme levels that destroy cocaine within seconds after appearance in the blood stream, allowing little to reach the brain. Rapid enzyme action causes a sharp rise in plasma levels of two cocaine metabolites, benzoic acid (BA) and ecgonine methyl ester (EME), a smooth muscle relaxant that is mildly hypotensive and, at best, only weakly rewarding. The present study, utilizing Balb/c mice, tested reward effects and cardiovascular effects of administering EME and BA together at molar levels equivalent to those generated by a given dose of cocaine. Reward was evaluated by conditioned place preference. In this paradigm, cocaine (20 mg/kg) induced a robust positive response but the equivalent combined dose of EME + BA failed to induce either place preference or aversion. Likewise, mice that had undergone gene transfer with mouse CocH (mCocH) showed no place preference or aversion after repeated treatments with a near-lethal 80 mg/kg cocaine dose. Furthermore, a single administration of that same high cocaine dose failed to affect blood pressure as measured using the noninvasive tail-cuff method. These observations confirm that the drug metabolites generated after CocH gene transfer therapy are safe even after a dose of cocaine that would ordinarily be lethal.

5-Aminosalicylic acid attenuates allergen-induced airway inflammation and oxidative stress in asthma.

Pro-inflammatory cytokines regulate the magnitude of allergic reactions during asthma. Tumor necrosis factor--alpha (TNF-α), interleukin-6 (IL-6) and interleukin-13 (IL-13) play a crucial role in aggravating the inflammatory conditions during allergic asthma. In addition, oxidative stress contributes to the pathogenesis of asthma by altering the physiological condition resulting in the development of status asthmaticus. Anti-inflammatory corticosteroids are being widely used for treating allergic asthma. In the present study 5-aminosalicylic acid (5-ASA), a salicylic acid derivative, was evaluated, in vivo for its potential to suppress TNF-α, IL-6 and IL-13 using ovalbumin (OVA) induced allergic asthma in Balb/C mice. Oral administration of 65, 130 and 195 mg/kg 5-ASA significantly reduced the OVA induced total and differential leucocyte count, TNF-α, IL-6, IL-13, nitrite, nitrate, MDA, MPO and TPL levels in the lung lavage samples. Collectively, these findings suggest that 5-ASA is a potent immunomodulator and suppresses key Th2 cytokines production and oxidative stress in OVA-induced asthma.

Diagnostic Utility of a New Assay for Thyroid Stimulating Immunoglobulins in Graves' Disease and Thyroid Eye Disease.

Background: The syndrome of thyrotoxicosis typically relies on radioactive iodine scans for establishing its etiology. Alternatively, the determination of thyrotropin (TSH) receptor antibodies (TRAbs) helps to distinguish Graves' disease (GD) from thyrotoxic thyroiditis. Current assays are impacted by limitations in sensitivity and/or turnaround time. Therefore, we decided to test a new assay for the detection of TSH receptor stimulating antibodies (TSAbs) and compare it with the clinically available assays. Methods: We enrolled 110 individuals in 5 cohorts: patients with incident or recurrent GD (cohort 1); patients with thyroiditis, painless or subacute (cohort 2); patients with Graves' orbitopathy/thyroid eye disease (TED) in cohort 3; patients with Hashimoto's thyroiditis (cohort 4), and a control group of normal volunteers (cohort 5). The patients were tested with the two clinically available assays: Roche Elecsys anti-TSHR assay (ROC-TBII) from Roche Diagnostics and Thyretain™ TSI Reporter BioAssay Kit (QUI-TSI) from Quidel. In addition, the samples were tested with the aequorin TSAb assay (OTS-TSI) provided by Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan). The data were collected in a cross-sectional manner before initiation of therapy. Results: We had 36 cases of GD, 17 cases of thyroiditis, 27 cases of TED, 10 cases of Hashimoto's thyroiditis, and 20 normal volunteers. OTS-TSI had 100% sensitivity and specificity in distinguishing GD from thyroiditis, identical with QUI-TSI but superior to ROC-TBII (sensitivity 86% and specificity 94.1%). OTS-TSI had 93% sensitivity and 100% specificity for the diagnosis of TED, compared with normal controls. QUI-TSI and ROC-TBII performed similarly in this analysis, demonstrating 82% sensitivity and 100% specificity. The range of detectable values for OTS-TSI was 20-29,000 mIU/L and the turnaround time was ≤6 hours, without the need for cell culture equipment. Conclusions: OTS-TSI performed excellently, though similarly to QUI-TSI, for the differential diagnosis of GD vs. thyroiditis, while being superior in that respect to ROC-TBII. Furthermore, OTS-TSI has superior sensitivity to QUI-TSI and ROC-TBII for TED diagnosis, while retaining high specificity. It has a short turnaround time and avoids the need for cell culture and sterility. Larger studies in U.S. populations are needed for its validation.

Sex-Specific Differences in Clinical Outcomes After Percutaneous Coronary Intervention: Insights from the TAILOR-PCI Trial.

Background TAILOR-PCI (Tailored Antiplatelet Initiation to Lessen Outcomes due to decreased Clopidogrel Response After Percutaneous Coronary Intervention) studied genotype-guided selection of antiplatelet therapy after percutaneous coronary intervention versus conventional therapy with clopidogrel. The presence of CYP2C19 loss-of-function alleles in patients treated with clopidogrel may be associated with increased risk for ischemic events. We report a prespecified sex-specific analysis of genotyping and associated cardiovascular outcomes from this study. Methods and Results Associations between sex and major adverse cardiac events (MACE: cardiovascular death, myocardial infarction, stroke, stent thrombosis, and severe recurrent ischemia) and Bleeding Academic Research Consortium (BARC) bleeding at 12 months were analyzed using Cox proportional-hazards models. Among 5276 randomized patients, loss-of-function carriers were observed in ≈36% of both sexes, and >80% of carriers were heterozygotes. At 12 months, after adjustment for baseline differences, risks of MACE (HR , 1.28 [0.97 to 1.68]; P=0.088) and BARC bleeding (hazard ratio [HR], 1.36 [0.91 to 2.05]; P=0.14) were comparable among women and men. There were no significant interactions between sex and treatment strategy for MACE interaction P value (Pint=0.59) or BARC bleeding (Pint=0.47) nor for sex and genotype (MACE Pint=0.15, and BARC bleeding Pint=0.60). Conclusions CYP2C19 loss-of-function alleles were present in ≈1 in 3 women and men. Women had similar adjusted risks of MACE and bleeding as men following percutaneous coronary intervention. Genotype-guided therapy did not significantly reduce the risk of MACE or bleeding relative to conventional therapy for both sexes. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT01742117.

Patient Onboarding and Engagement to Build a Digital Study After Enrollment in a Clinical Trial (TAILOR-PCI Digital Study): Intervention Study.

BACKGROUND: The Tailored Antiplatelet Initiation to Lessen Outcomes Due to Decreased Clopidogrel Response After Percutaneous Coronary Intervention (TAILOR-PCI) Digital Study is a novel proof-of-concept study that evaluated the feasibility of extending the TAILOR-PCI randomized controlled trial (RCT) follow-up period by using a remote digital platform. OBJECTIVE: The aim of this study is to describe patients' onboarding, engagement, and results in a digital study after enrollment in an RCT. METHODS: In this intervention study, previously enrolled TAILOR-PCI patients in the United States and Canada within 24 months of randomization were invited by letter to download the study app. Those who did not respond to the letter were contacted by phone to survey the reasons for nonparticipation. A direct-to-patient digital research platform (the Eureka Research Platform) was used to onboard patients, obtain consent, and administer activities in the digital study. The patients were asked to complete health-related surveys and digitally provide follow-up data. Our primary end points were the consent rate, the duration of participation, and the monthly activity completion rate in the digital study. The hypothesis being tested was formulated before data collection began. RESULTS: After the parent trial was completed, letters were mailed to 907 eligible patients (representing 18.8% [907/4837] of total enrolled in the RCT) within 15.6 (SD 5.2) months of randomization across 24 sites. Among the 907 patients invited, 290 (32%) visited the study website and 110 (12.1%) consented-40.9% (45/110) after the letter, 33.6% (37/110) after the first phone call, and 25.5% (28/110) after the second call. Among the 47.4% (409/862) of patients who responded, 41.8% (171/409) declined to participate because of a lack of time, 31.2% (128/409) declined because of the lack of a smartphone, and 11.5% (47/409) declined because of difficulty understanding what was expected of them in the study. Patients who consented were older (aged 65.3 vs 62.5 years; P=.006) and had a lower prevalence of diabetes (19% vs 30%; P=.02) or tobacco use (6.4% vs 24.8%; P<.001). A greater proportion had bachelor's degrees (47.2% vs 25.7%; P<.001) and were more computer literate (90.5% vs 62.3% of daily internet use; P<.001) than those who did not consent. The average completion rate of the 920 available monthly electronic visits was 64.9% (SD 7.6%); there was no decrease in this rate throughout the study duration. CONCLUSIONS: Extended follow-up after enrollment in an RCT by using a digital study was technically feasible but was limited because of the inability to contact most eligible patients or a lack of time or access to a smartphone. Among the enrolled patients, most completed the required electronic visits. Enhanced recruitment methods, such as the introduction of a digital study at the time of RCT consent, smartphone provision, and robust study support for onboarding, should be explored further. TRIAL REGISTRATION: Clinicaltrails.gov NCT01742117; https://clinicaltrials.gov/ct2/show/NCT01742117.

Missed critical value callbacks due to middleware flaw.

OBJECTIVES: In 2018, our clinical laboratory was alerted to back-to-back plasma sodium critical value callback failures on the same patient, occurring on different shifts and involving different technologists. Therefore, we set forth to investigate the root cause for the critical value callback failures. DESIGN AND METHODS: We conducted a thorough investigation focused on the processes associated with critical value identification and notification for plasma sodium measurement performed on the Siemens Vista. RESULTS: Our investigation uncovered a flaw in the Siemens CentraLink middleware software. A default dark blue bar in the top row of the results review display was determined to obscure the red color which highlights critical values for lab staff identification. Sodium was disproportionately impacted by this flaw, as it is commonly ordered as part of metabolic panels, and is listed first among the panel analytes in the top row of the CentraLink display. Retrospective data review comparing critical callback failure rates for sodium to potassium and hemoglobin confirmed that sodium had significantly higher critical callback failure rates than these other analytes. After alerting the product manufacturer, Siemens programmed the CentraLink display so that the top row was blank and devoid of patient results, so that the blue color in the top row would no longer obscure the red visual cue of a patient's critical result. Sodium critical value callback failures were reduced to 0% after this middleware display correction. CONCLUSIONS: Middleware design flaws can have unexpected consequences on clinical laboratory operations. We encourage clinical laboratories to closely examine user interfaces utilized by laboratory staff, and be wary of potential impacts that the display format may have on results reporting.

Transient hyperphosphatasemia following pediatric liver transplantation in a patient with hepatic and skeletal abnormalities.

BACKGROUND: We report a unique case of transient hyperphosphatasemia in a pediatric patient with a history of hepatic and skeletal abnormalities. PATIENT AND METHODS: A 2-month old male was diagnosed with progressive familial intrahepatic cholestasis type-2 and osteoporosis after marked increases in liver function tests were noted at 1 month of age. He underwent a second liver transplantation at 1 y. The increased liver function test trend resolved a few weeks post-transplantation. Four months after successful liver transplantation, unexplained significant increases in alkaline phosphatase (ALP) were observed, and they persisted for almost 9 months. Among the etiologies under consideration for the isolated increased ALP activity were viral infections and macro-ALP. RESULTS: A persistent trend in abnormally increased ALP for 9 months was investigated leading to a confirmed diagnosis of transient hyperphosphatasemia (TH). CONCLUSION: Pediatric post-liver transplant patients with skeletal and hepatic abnormalities including isolated markedly increased ALP activities represent a previously undescribed TH patient population. The 4.3% prevalence of TH in pediatric liver transplant recipients within our healthcare system is considerably higher than the previously reported prevalence of 2.1% for patients within the United States.

Clinical and Biochemical Phenotypes in a Family With ENPP1 Mutations.

Inactivating mutations of the ENPP1 gene are associated with generalized arterial calcification of infancy (GACI) and less often autosomal-recessive hypophosphatemic rickets type 2 (ARHR2). We aimed to investigate the spectrum of phenotypes in a family with monoallelic and biallelic mutations of ENPP1 after identification through whole exome sequencing of a 54-year-old female with biallelic mutation of ENPP1, c.323G > T; p.Cys108Phe and c.1441C > T; p.Arg481Trp. Including the proband, 2 subjects had biallelic mutations, 5 had monoallelic mutations, and 2 had no mutation of ENPP1. The maternal mutation, a known pathogenic variant associated with GACI, was found in 3 subjects with monoallelic mutations, while the paternal mutation, which was not previously reported, was present in 2 subjects with monoallelic mutations. Both subjects with biallelic mutations had bowing of bilateral femurs, periarticular mineral deposition, normocalcemic primary hyperparathyroidism with multigland parathyroidectomy, increased carotid intima-media thickness, and enthesopathy was also noted in one subject. Intact FGF23 was elevated in both subjects with biallelic mutations, while C-terminal FGF23 was only elevated in one and PPi was reduced in one. Subjects with monoallelic mutations did not have periarticular calcifications or bone deformities. To conclude, patients with biallelic GACI causing mutations can survive well into adulthood, and despite the same biallelic ENPP1 pathogenic variants, clinical and biochemical manifestations can significantly differ, and include enthesopathy and primary hyperparathyroidism, which have not been previously described. Although carriers of monoallelic ENPP1 variants appear unaffected by classic disease manifestations, there may be subtle biochemical and clinical findings that warrant further investigation. © 2019 American Society for Bone and Mineral Research.

In vivo characterization of a novel phenylisothiocyanate tropane analog at monoamine transporters in rat brain.

Previous studies have shown that the phenylisothiocyanate tropane analog 2-beta-propanoyl-3-beta-(2-naphthyl)-8-[4-isothiocyanato)benzyl]nortropane (HD-205) binds covalently to dopamine and serotonin transporters (DAT and SERT, respectively) in rat brain membranes (Biochem Pharmacol 74:336-344, 2007). The present study evaluated the irreversible effects of HD-205 in vivo in rats after intracranial injection. Rats were implanted with unilateral cannulae in rat striatum, and HD-205 (0.001-3 nmol) was administered by intrastriatal injection. In vitro autoradiography of DAT binding with [125I]2-carbomethoxy-3-(4-iodophenyl)tropane (RTI-55) on brain sections obtained 24 h after injection showed a highly localized blockade of binding in striatum, with maximal blockade of binding by 1 to 3 nmol HD-205. Similar blockade of SERT binding (using [3H]-citalopram) was observed in the same area. No blockade of DAT or SERT binding was observed after intrastriatal injections of the reversible analog 2-beta-propanoyl-3-beta-(2-naphthyl)-8-benzyl nortropane (HD-206), and HD-205 treatment had no effect on D(2)- and mu-opioid-stimulated guanosine 5'-O-(3-[35S]thio)-triphosphate binding in sections from the same animals. In a time course study, rats administered with 1 nmol HD-205 showed recovery of 50% DAT binding after 3 to 4 days postinjection, and full recovery after 6 weeks. Rats implanted with bilateral cannulae were tested for cocaine-induced locomotor activity. Two days after intrastriatal injection of 1 nmol of HD-205, systemic (20 mg/kg i.p.) cocaine-induced locomotor activity was not affected; however, locomotor activity induced by intrastriatal administration of cocaine (6 nmol) was eliminated. This strategy of site-specific chemical blockade of transporters could serve as a valuable tool to evaluate the neuroanatomical basis of DAT-mediated cocaine effects.

Irreversible binding of a novel phenylisothiocyanate tropane analog to monoamine transporters in rat brain.

Irreversible tropane analogs have been useful in identifying binding sites of cocaine on biogenic amine transporters, including transporters for dopamine (DAT), serotonin (SERT) and norepinephrine (NET). The present study characterizes the properties of the novel phenylisothiocyanate tropane HD-205, synthesized from the highly potent 2-napthyl tropane analog WF-23. In radioligand binding studies in brain membranes, direct IC(50) values of HD-205 were 4.1, 14 and 280nM at DAT, SERT and NET, respectively. Wash-resistant binding was characterized by preincubation of HD-205 with brain membranes, followed by extensive washing before performing transporter radioligand binding. Results for HD-205 showed wash-resistant IC(50) values of 191, 230 and 840nM at DAT, SERT and NET, respectively. Saturation binding studies with [(125)I]RTI-55 in membranes pretreated with 100nM HD-205 showed that HD-205 significantly decreased the B(max) but not K(D) of DAT and SERT binding. To further characterize its irreversible binding, an iodinated analog of HD-205, HD-244, was prepared from a trimethylsilyl precursor. The direct IC(50) of HD-244 at DAT was 20nM. [(125)I]HD-244 was synthesized with chloramine-T, purified on HPLC, reacted with rat striatal membranes, and proteins were separated by SDS-PAGE. Results showed several non-specific labeled bands, but only a single specific band of radioactivity co-migrating with an immunoreactive DAT band at approx. 80 kilodaltons was detected, suggesting that [(125)I]HD-244 covalently labeled DAT protein in striatal membranes. These results demonstrate that phenylisothiocyanate analogs of WF-23 can be used as potential ligands to map distinct binding sites of cocaine analogs at DAT.

Brain Targeting of 1,9-Pyrazoloanthrone an c-Jun-N-terminal Kinase Inhibitor Using Liposomes for Effective Management of Parkinson's Disease.

The major challenge to treat Parkinson's disease (PD) is penetration of target molecule into the brain to improve the efficacy of drugs. To achieve better brain penetration and targeted delivery, 1,9-Pyrazoloanthrone (1,9-P) loaded liposomes were developed by solvent injection technique using ultrasonication and evaluated for particle size, morphology, entrapment efficiency, FT-IR, and in-vitro drug release studies. The potential of 1,9-Pyrazoloanthrone (1,9-P), a c-Jun-N-terminal Kinase (JNK-3) inhibitor which could stop or retard the rate of apoptosis of neuronal cells was also evaluated. In-vivo pharmacokinetic and brain uptake studies of liposomes were performed to investigate the bioavailability and brain distribution of 1,9-P. Cytotoxicity and neuroprotection were done on SH-SY5Y cell line using MTT and AO/EB apoptosis assay. The optimized batch of liposomes showed an average size of 112.33 ± 0.84 nm with a zeta potential value of -19.40 mV and 78.96 ± 0.28% drug entrapment efficiency. The in-vitro release studies demonstrated the sustained release profile of liposome up to 24 h. The pharmacokinetic data in Wistar rats over the period of 12 h demonstrated 4.82-folds greater AUC(0-12 h) for liposome in brain compared with 1,9-P suspension. Cytotoxicity assay showed no sign of toxicity, whereas apoptosis assay revealed a neuroprotective action of liposomes. The results demonstrated successful targeting of the 1,9-P, to brain as a novel strategy having significant therapeutic potential to treat PD.

Salicylic acid derivatives as potential anti asthmatic agents using disease responsive drug delivery system for prophylactic therapy of allergic asthma.

Asthma is a multi-factorial and complicated lung disorder of the immune system which has expanded to a wider ambit unveiling its etiology to be omnipresent at both ends of the spectrum involving basic pharmacology and in-depth immunology. As asthma occurs through triggered activation of various immune cells due to different stimuli, it poses a great challenge to uncover specific targets for therapeutic interventions. Recent pharmacotherapeutic approaches for asthma have been focused on molecular targeting of transcription factors and their signaling pathways; mainly nucleus factor kappa B (NFκB) and its associated pathways which orchestrate the synthesis of pro-inflammatory cytokines (IL-1ß, TNF-α, GM-CSF), chemokines (RANTES, MIP-1a, eotaxin), adhesion molecules (ICAM-1, VCAM-1) and inflammatory enzymes (cyclooxygenase-2 and iNOS). 5-aminosalicylic acid (5-ASA) and sodium salicylate are known to suppress NFκB activation by inhibiting inhibitor of kappa B kinase (IKκB). In order to target the transcription factor, a suitable carrier system for delivering the drug to the intracellular space is essential. 5-ASA and sodium salicylate loaded liposomes incorporated into PEG-4-acrylate and CCRGGC microgels (a polymer formed by crosslinking of trypsin sensitive peptide and PEG-4-acrylate) could probably suit the needs for developing a disease responsive drug delivery system which will serve as a prophylactic therapy for asthmatic patients.

Pharmacokinetic and tissue distribution studies of 1,9-pyrazoloanthrone, a c-Jun-N-terminal kinase inhibitor in Wistar rats by a simple and sensitive HPLC method.

JNK pathway activates c-Jun(s) which are responsible for cell apoptosis; as a result, inhibitors of JNK pathway have the potential to prevent dopaminergic neurons from death and decrease the loss of dopamine in substantia nigra pars compacta (SNpc). Recent in-vitro studies show that 1,9-pyrazoloanthrone (1,9-P) a potent JNK-3 inhibitor prevents the apoptosis of dopaminergic cells of brain. In the present study we formulated liposomes to increase the bioavailability of 1,9-P in the brain and developed a simple, sensitive and selective high performance liquid chromatographic method and validated for the estimation of 1,9-P in Wistar rat plasma and tissue samples. Plasma and tissue samples were extracted by protein precipitation technique using acetonitrile (ACN) and rasagiline as the internal standards. Chromatography was performed on Hibar C18 column with mobile phase of ammonium acetate (10mM, pH 8.0 adjusted with ammonia) and ACN at a flow rate of 1mL/min. The lower limit of quantification of the developed method was found to be 2.0ng/mL and 4.0ng/g in plasma and tissue samples respectively. The liposomes of 1,9-P administered to animals at the dose equivalent to 15mg/kg orally demonstrated remarkable absorption into the systemic circulation with maximum concentration (∼7500ng/mL) within 2.0h. The order of the area under curve was found to be kidney>liver>brain>lungs>spleen>heart. The liposomes of 1,9-P were rapidly taken up into brain and showed a good brain concentration after 2.0h; sustenance up to 4.0h was achieved which is better than 1,9-P solution.