Jason M. Meunier

Ultrasound-enhanced thrombolysis with tPA-loaded echogenic liposomes

BACKGROUND AND PURPOSE Currently, the only FDA-approved therapy for acute ischemic stroke is the administration of recombinant tissue plasminogen activator (tPA). Echogenic liposomes (ELIP), phospholipid vesicles filled with gas and fluid, can be manufactured to incorporate tPA. Also, transcranial ultrasound-enhanced thrombolysis can increase the recanalization rate in stroke patients. However, there is little data on lytic efficacy of combining ultrasound, echogenic liposomes, and tPA treatment. In this study, we measure the effects of pulsed 120-kHz ultrasound on the lytic efficacy of tPA and tPA-incorporating ELIP (t-ELIP) in an in-vitro human clot model. It is hypothesized that t-ELIP exhibits similar lytic efficacy to that of rt-PA. METHODS Blood was drawn from 22 subjects after IRB approval. Clots were made in 20-microL pipettes, and placed in a water tank for microscopic visualization during ultrasound and drug treatment. Clots were exposed to combinations of [tPA]=3.15 microg/ml, [t-ELIP]=3.15 microg/ml, and 120-kHz ultrasound for 30 minutes at 37 degrees C in human plasma. At least 12 clots were used for each treatment. Clot lysis over time was imaged and clot diameter was measured over time, using previously developed imaging analysis algorithms. The fractional clot loss (FCL), which is the decrease in mean clot width at the end of lytic treatment, was used as a measure of lytic efficacy for the various treatment regimens. RESULTS The fractional clot loss FCL was 31% (95% CI: 26-37%) and 71% (56-86%) for clots exposed to tPA alone or tPA with 120 kHz ultrasound. Similarly, FCL was 48% (31-64%) and 89% (76-100%) for clots exposed to t-ELIP without or with ultrasound. CONCLUSIONS The lytic efficacy of tPA containing echogenic liposomes is comparable to that of tPA alone. The addition of 120 kHz ultrasound significantly enhanced lytic treatment efficacy for both tPA and t-ELIP. Liposomes loaded with tPA may be a useful adjunct in lytic treatment with tPA.

Tissue Plasminogen Activator Concentration Dependence of 120 kHz Ultrasound-Enhanced Thrombolysis

It has been known for some time that the application of ultrasound can enhance the efficacy of thrombolytic medications such as recombinant tissue plasminogen activator (rt-PA). Potential clinical applications of this ultrasound-enhanced thrombolysis (UET) include the treatment of myocardial infarction, acute ischemic stroke, deep venous thrombosis and other thrombotic disorders. It may be possible to reduce the dose of rt-PA while maintaining lytic efficacy; however there is little data on the rt-PA concentration dependence of UET. In this work, the rt-PA concentration dependence of clot lysis resulting from 120 kHz UET exposure was measured in an in vitro human clot model. Clots were exposed to rt-PA for 30 min, with (UET treated) or without 120 kHz ultrasound (rt-PA treated) at 37 degrees C, and the clot width measured as a function of time. The rt-PA concentration ranged from 0-10 microg/mL. The initial lytic rate for the UET-treated group was greater than that of the rt-PA group at almost all rt-PA concentrations, and exhibited a maximum over concentration values of 1-3 microg/mL.

Effect of low frequency ultrasound on combined rt-PA and eptifibatide thrombolysis in human clots

INTRODUCTION Fibrinolytics such as recombinant tissue plasminogen activator (rt-PA) are used to treat thrombotic disease such as acute myocardial infarction (AMI) and ischemic stroke. Interest in increasing efficacy and reducing side effects has led to the study of adjuncts such as GP IIb-IIIa inhibitors and ultrasound (US) enhanced thrombolysis. Currently, GP IIb-IIIa inhibitor and fibrinolytic treatment are often used in AMI, and are under investigation for stroke treatment. However, little is known of the efficacy of combined GP IIb-IIIa inhibitor, fibrinolytic and ultrasound treatment. We measure the lytic efficacy of rt-PA, eptifibatide (Epf) and 120 kHz ultrasound treatment in an in-vitro human clot model. MATERIALS AND METHODS Blood was drawn from 15 subjects after IRB approval. Clots were made in 20 microL pipettes, and placed in a water tank for microscopic visualization during lytic treatment. Clots were exposed to control, rt-PA (rt-PA), eptifibatide (Epf), or rt-PA+eptifibatide (rt-PA + Epf), with (+US) or without (-US) ultrasound for 30 minutes at 37 degrees C in human plasma. Clot lysis was measured over time, using a microscopic imaging technique. The fractional clot loss (FCL) and initial lytic rate (LR) were used to quantify lytic efficacy. RESULTS AND CONCLUSIONS LR values for (- US) treated clots were 0.8+/-0.1(control), 1.8+/-0.3 (Epf), 1.5+/-0.2 (rt-PA), and 1.3+/-0.4 (rt-PA + Epf) (% clot width/minute) respectively. In comparison, the (+ US) group exhibited LR values of 1.6+/-0.2 (control), 4.3+/-0.4 (Epf), 6.3+/-0.4 (rt-PA), and 4.6+/-0.6 (rt-PA + Epf). For (- US) treated clots, FCL was 6.0+/-0.8 (control), 9.2+/-2.5 (Epf), 15.6+/-1.7 (rt-PA), and 28.0+/-2.2% (rt-PA + Epf) respectively. FCL for (+ US) clots was 13.5+/-2.4 (control), 20.7+/-6.4 (Epf), 44.4+/-3.6 (rt-PA) and 30.3+/-3.6% (rt-PA + Epf) respectively. Although the addition of eptifibatide enhances the in-vitro lytic efficacy of rt-PA in the absence of ultrasound, the efficacy of ultrasound and rt-PA is greater than that of combined ultrasound, rt-PA and eptifibatide exposure.

Long-term stability of recombinant tissue plasminogen activator at -80 C

BackgroundRecombinant tissue plasminogen activator (tPA) is a thrombolytic widely used clinically in the treatment of acute thrombotic disease such as ischemic stroke, myocardial infarction, and deep venous thrombosis. This has led to much interest in tPA based lytic therapies leading to laboratory based in-vitro and in-vivo investigations using this drug. However, tPA reconstituted in solution exhibits full activity for only 6–8 hours, according to the manufacturer. Therefore, methods to store reconstituted tPA for long durations while maintaining activity would be of assistance to laboratories using this enzyme.FindingsIn this work, the enzymatic activity of tPA stored at -80 C over time was measured, using an ELISA technique that measured the amount of active tPA bound to plasminogen activator inhibitor 1 (PAI-1) in a given sample. Sample of tPA solution mixed to a concentration of 1 (mg/ml) were stored in cryogenic vials at -80 C for up to 7 years. For a given sample, aliquots were assayed for tPA activity, and compared with a tPA standard to determine relative enzymatic activity. Results are reported as means with standard errors, and 12 measurements were performed for each sample age.ConclusionThere was no decrease in tPA activity for samples stored up to 7 years. Such cryogenic storage is a viable method for the preservation of tPA solution for laboratory investigations of tPA-based lytic therapies.

Making the Right Choice: Optimizing rt-PA and eptifibatide lysis, an in vitro study

INTRODUCTION Recombinant tissue plasminogen activator (rt-PA) is the only FDA approved lytic therapy for acute ischemic stroke. However, there can be complications such as intra-cerebral hemorrhage. This has led to interest in adjuncts such as GP IIb-IIIa inhibitors. However, there is little data on combined therapies. Here, we measure clot lysis for rt-PA and eptifibatide in an in vitro human clot model, and determine the drug concentrations maximizing lysis. A pharmacokinetic model is used to compare drug concentrations expected in clinical trials with those used here. The hypothesis is that there is a range of rt-PA and eptifibatide concentrations that maximize in vitro clot lysis. MATERIALS AND METHODS Whole blood clots were made from blood obtained from 28 volunteers, after appropriate institutional approval. Sample clots were exposed to rt-PA and eptifibatide in human fresh-frozen plasma; rt-PA concentrations were 0, 0.5, 1, and 3.15 μg/ml, and eptifibatide concentrations were 0, 0.63, 1.05, 1.26 and 2.31 μg/ml. All exposures were for 30 minutes at 37 C. Clot width was measured using a microscopic imaging technique and mean fractional clot loss (FCL) at 30 minutes was used to determine lytic efficacy. On average, 28 clots (range: 6-148) from 6 subjects (3-24) were used in each group. RESULTS AND CONCLUSIONS FCL for control clots was 14% (95% Confidence Interval: 13-15%). FCL was 58% (55-61%) for clots exposed to both drugs at all concentrations, except those at an rt-PA concentration of 3.15 μg/ml, and eptifibatide concentrations of 1.26 μg/ml (Epf) or 2.31 μg/ml. Here, FCL was 43% (36-51) and 35% (32-38) respectively. FCL is maximized at moderate rt-PA and eptifibatide concentration; these values may approximate the average concentrations used in some rt-PA and eptifibatide treatments.

120 kHz pulsed ultrasound enhanced thrombolysis with tissue plasminogen activator‐loaded echogenic liposomes

Echogenic liposomes (ELIP), phospholipid vesicles filled with gas and fluid, can be manufactured to incorporate the thrombolytic drug tissue plasminogen activator (tPA). Real‐time thrombolysis of blood clots exposed to tPA‐incorporating ELIP (t‐ELIP) was monitored using video microscopy with an inverted optical microscope. Human whole blood clots on silk sutures were exposed to tPA alone (3.15 micrograms/ml), t‐ELIP alone (3.15 micrograms/ml), t‐ELIP and 120 kHz ultrasound (0.18 MPa peak negative pressure, 1.667 kHz pulse repetition frequency, 50% duty cycle), or tPA and ultrasound, for 30 min. The extent of thrombolysis was determined by assessing clot width as a function of time, using a time‐lapse microscopic imaging technique. The average percent change in clot width at 30 min for clots treated with t‐ELIP alone exceeded tPA alone (22.8% vs. 15.6%, respectively). Thrombolytic efficacy was similar for either tPA or t‐ELIP exposed to 120 kHz ultrasound. Thus, the thrombolytic drug could be effectively r...

A Novel Tool for Evaluation of Mild Traumatic Brain Injury Patients in the Emergency Department: Does Robotic Assessment of Neuromotor Performance Following Injury Predict the Presence of Postconcussion Symptoms at Follow-up?

OBJECTIVES Postconcussion symptoms (PCS) are a common complication of mild traumatic brain injury (TBI). Currently, there is no validated clinically available method to reliably predict at the time of injury who will subsequently develop PCS. The purpose of this study was to determine if PCS following mild TBI can be predicted during the initial presentation to an emergency department (ED) using a novel robotic-assisted assessment of neurologic function. METHODS All patients presenting to an urban ED with a chief complaint of head injury within the preceding 24 hours were screened for inclusion from March 2013 to April 2014. The enrollment criteria were as follows: 1) age of 18 years or greater, 2) ability and willingness to provide written informed consent, 3) blunt head trauma and clinical diagnosis of isolated mild TBI by the treating physician, and 4) blood alcohol level of <100 mg/dL. Eligible mild TBI patients were enrolled and their neuromotor function was assessed in the ED using a battery of five tests that cover a range of proprioceptive, visuomotor, visuospatial, and executive function performance metrics. At 3 weeks postinjury, participants were contacted via telephone to complete the Rivermead Post-Concussion Symptoms Questionnaire to assess the presence of significant PCS. RESULTS A total of 66 mild TBI patients were enrolled in the study with 42 of them completing both the ED assessment and the follow-up; 40 patients were included in the analyses. The area under the receiver operating characteristic curve (AUC) for the entire test battery was 0.72 (95% confidence interval [CI] = 0.54 to 0.90). The AUC for tests that primarily measure visuomotor and proprioceptive performance were 0.80 (95% CI = 0.65 to 0.95) and 0.71 (95% CI = 0.53 to 0.89), respectively. CONCLUSIONS The robotic-assisted test battery has the ability to discriminate between subjects who developed PCS and those who did not. Additionally, poor visuomotor and proprioceptive performance were most strongly associated with subsequent PCS.

Combination Treatment with rt-PA is More Effective than rt-PA Alone in an in Vitro Human Clot Model

Incidence of intra-cranial hemorrhage linked to treatment of ischemic stroke with recombinant tissue plasminogen activator (rt-PA) has led to interest in adjuvant therapies such as ultrasound (US) or plasminogen, to enhance rt-PA efficacy and improve patient safety. High-frequency US (∼MHz) such as 2-MHz transcranial Doppler (TCD) has demonstrated increased recanalization in situ. Low-frequency US (∼kHz) enhanced thrombolysis (UET) has demonstrated higher lytic capabilities but has been associated with incidence of intracerebral hemorrhage in some clinical trials. In vitro studies using plasminogen have shown enhancement of lysis. This study compared rt-PA-induced lysis using adjuvant therapies, with 120-kHz or 2-MHz pulsed US, or plasminogen, in an in vitro human whole blood clot model. Blood was drawn from 30 subjects after local institutional approval. Clots were exposed to rt-PA at concentrations of 0 to 3.15 μg/ml. Clots were exposed to rt-PA alone (rt-PA) or in combination with plasminogen (Plg), 120-kHz US (120-kHz), or 2-MHz US (2-MHz). Thrombolytic efficacy was determined by assessing the percent fractional clot loss (FCL) at 30 minutes using microscopic imaging. There was no enhancement of lysis for combination therapy with [rt-PA]=0 μg/ml. Adding rt- PA increased lysis for all groups. As [rt-PA] increased, lysis tended to increase for 120-kHz and Plg (FCL: from 50% to 70%, 120-kHz; 65% to 83%, Plg) but not for 2-MHz (58% to 52%). Lytic efficacy in combination therapy depends on rt- PA concentration and the adjuvant therapy type. For non-zero rt-PA concentrations, all combination therapies produced more lysis than rt-PA alone.

Randomized Controlled Noninferiority Trial Comparing Daptomycin to Vancomycin for the Treatment of Complicated Skin and Skin Structure Infections in an Observation Unit

BACKGROUND Incidence of methicillin-resistant Staphylococcus aureus (MRSA) is increasing in complicated skin and skin structure infection (cSSSI) presenting to emergency departments (EDs). Treatment is heterogeneous and can require inpatient admission to an observation unit (OU). Vancomycin is commonly used in the OU for treatment, but increasing MRSA resistance to vancomycin suggests the need for alternatives. Daptomycin is an alternative but it is not known how it compares with vancomycin. OBJECTIVE This study tested the hypothesis that daptomycin is noninferior to vancomycin for the treatment of cSSSI in an OU, using a relative risk (RR) of 1.3 as the noninferiority limit. METHODS Subjects admitted to an ED-based OU with a diagnosis of cSSSI were eligible. Consenting subjects were randomized 1:1 to intravenous (i.v.) vancomycin at 15 mg/kg dosing every 12 h or i.v. daptomycin at 4 mg/kg once. Subjects were followed until they met objective criteria for discharge home or hospital admission. Discharged patients were prescribed 10-14 days of oral cephalexin and trimethoprim-sulfamethoxazole, or clindamycin if allergic to either of these medications. The primary endpoint was meeting objective discharge criteria with no change in antibiotic therapy or return to the ED for the same cellulitis within 30 days of OU discharge. RESULTS There were 100 patients enrolled. RR for satisfying the endpoint was 1.07 (95% confidence interval 0.58-1.98) for daptomycin compared with vancomycin. Hospital admission rates were 36% and 32% for daptomycin and vancomycin treatment, respectively. CONCLUSION Daptomycin was not inferior to vancomycin in the treatment of cSSSI in an OU.

Integration of New Technology for Research in the Emergency Department: Feasibility of Deploying a Robotic Assessment Tool for Mild Traumatic Brain Injury Evaluation

The objective of this paper is to demonstrate the effective deployment of a robotic assessment tool for the evaluation of mild traumatic brain injury (mTBI) patients in a busy, resource-constrained, urban emergency department (ED). Methods: Functional integration of new robotic technology for research in the ED presented several obstacles that required a multidisciplinary approach, including participation from electrical and computer engineers, emergency medicine clinicians, and clinical operations staff of the hospital. Our team addressed many challenges in deployment of this advanced technology including: 1) adapting the investigational device for the unique clinical environment; 2) acquisition and maintenance of appropriate testing space for point-of-care assessment; and 3) dedicated technical support and upkeep of the device. Upon successful placement of the robotic device in the ED, the clinical study required screening of all patients presenting to the ED with complaints of head injury. Eligible patients were enrolled and tested using a robot-assisted test battery. Three weeks after the injury, patients were contacted to complete follow-up assessments. Results: Adapting the existing technology to meet anticipated physical constraints of the ED was performed by engineering a mobile platform. Due to the large footprint of the device, it was frequently moved before ultimately being fully integrated into the ED. Over 14 months, 1423 patients were screened. Twenty-eight patients could not be enrolled because the device was unavailable due to operations limitations. Technical problems with the device resulted in failure to include 20 patients. A total of 66 mTBI patients were enrolled and 42 of them completed both robot-assisted testing and follow-up assessment. Successful completion of screening and enrollment demonstrated that the challenges associated with integration of investigational devices into the ED can be effectively addressed through a collaborative patient-oriented research model. Conclusion: Effective deployment and use of new robotic technology for research in an urban academic ED required significant planning, coordination, and collaboration with key personnel from multiple disciplines. Clinical Impact: A pilot clinical study on mTBI patients using the robotic device provided useful data without disrupting the ED workflow. Integration of this technology into the ED serves as an important step toward pursing active clinical research in an acute care setting.