George J. Shaw

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.

Serum cleaved tau does not predict postconcussion syndrome after mild traumatic brain injury.

OBJECTIVES Our objective was to determine if the biomarker for axonal injury, serum cleaved tau (C-tau), predicts postconcussion syndrome (PCS) in adults after mild traumatic brain injury (mTBI). METHODS C-tau was measured from blood obtained in the emergency department. Outcome was assessed at 3 months post injury using the Rivermead Postconcussion Symptoms Questionnaire and Acute Medical Outcomes SF-36v2 Health Survey (SF-36). RESULTS Of 50 patients, there were 15 patients with detectable levels of C-tau, 10 patients with abnormal findings on initial head computed tomography (CT) and 22 patients with PCS. One-third of patients with detectable C-tau and 14.3% of patients without detectable C-tau had abnormal findings on head CT (P = .143). Serum C-tau was not detected more frequently in patients with PCS than those without, neither for all patients (P = .115) nor the subgroup with negative head CT (P = .253). CONCLUSIONS C-tau is a poor predictor of PCS after mTBI regardless of head CT result.

Characterization of ultrasound propagation through ex-vivo human temporal bone.

Adjuvant therapies that lower the thrombolytic dose or increase its efficacy would represent a significant breakthrough in the treatment of patients with ischemic stroke. The objective of this study was to perform intracranial measurements of the acoustic pressure field generated by 0.12, 1.03 and 2.00-MHz ultrasound transducers to identify optimal ultrasound parameters that would maximize penetration and minimize aberration of the beam. To achieve this goal, in vitro experiments were conducted on five human skull specimens. In a water-filled tank, two unfocused transducers (0.12 and 1.03 MHz) and one focused transducer (2.00 MHz) were consecutively placed near the right temporal bone of each skull. A hydrophone, mounted on a micropositioning system, was moved to an estimated location of the middle cerebral artery (MCA) origin, and measurements of the surrounding acoustic pressure field were performed. For each measurement, the distance from the position of maximum acoustic pressure to the estimated origin of the MCA inside the skulls was quantified. The -3 dB depth-of-field and beamwidth in the skull were also investigated as a function of the three frequencies. Results show that the transducer alignment relative to the skull is a significant determinant of the detailed behavior of the acoustic field inside the skull. For optimal penetration, insonation normal to the temporal bone was needed. The shape of the 0.12-MHz intracranial beam was more distorted than those at 1.03 and 2.00 MHz because of the large aperture and beamwidth. However, lower ultrasound pressure reduction was observed at 0.12 MHz (22.5%). At 1.03 and 2.00 MHz, two skulls had an insufficient temporal bone window and attenuated the beam severely (up to 96.6% pressure reduction). For all frequencies, constructive and destructive interference patterns were seen near the contralateral skull wall at various elevations. The 0.12-MHz ultrasound beam depth-of-field was affected the most when passing through the temporal bone and showed a decrease in size of more than 55% on average. The speed of sound in the temporal bone of each skull was estimated at 1.03 MHz and demonstrated a large range (1752.1 to 3285.3 m/s). Attenuation coefficients at 1.03 and 2.00 MHz were also derived for each of the five skull specimens. This work provides needed information on ultrasound beam shapes inside the human skull, which is a necessary first step for the development of an optimal transcranial ultrasound-enhanced thrombolysis device.

Arrhenius temperature dependence of in vitro tissue plasminogen activator thrombolysis

Stroke is a devastating disease and a leading cause of death and disability. Currently, the only FDA approved therapy for acute ischemic stroke is the intravenous administration of the thrombolytic medication, recombinant tissue plasminogen activator (tPA). However, this treatment has many contraindications and can have dangerous side effects such as intra-cerebral hemorrhage. These treatment limitations have led to much interest in potential adjunctive therapies, such as therapeutic hypothermia (T <or 35 degrees C) and ultrasound enhanced thrombolysis. Such interest may lead to combining these therapies with tPA to treat stroke, however little is known about the effects of temperature on the thrombolytic efficacy of tPA. In this work, we measure the temperature dependence of the fractional clot mass loss Deltam(T) resulting from tPA exposure in an in vitro human clot model. We find that the temperature dependence is well described by an Arrhenius temperature dependence with an effective activation energy E(eff) of 42.0 +/- 0.9 kJ mole(-1). E(eff) approximates the activation energy of the plasminogen-to-plasmin reaction of 48.9 kJ mole(-1). A model to explain this temperature dependence is proposed. These results will be useful in predicting the effects of temperature in future lytic therapies.

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.

In vitro microscopic imaging of enhanced thrombolysis with 120-kHz ultrasound in a human clot model.

Substantial enhancement of recombinant tissue plasminogen activator (rt-PA) thrombolysis can be achieved with ultrasound, suggesting its use as an adjunctive treatment in thrombolytic therapy for stroke. A microscopic visualization method was used to measure the lysis of human whole-blood clots treated with human fresh frozen plasma (HFFP), rt-PA, and 120-kHz ultrasound for 30 min at T = 37 ° C. The clot-plasma interface was imaged using an inverted optical microscope and the thrombolytic front analyzed as a function of time. Ultrasound treatment significantly enhanced the mean lytic rate from 0.5 to 3.4 μm/min (a 580% change) compared with rt-PA treatment alone.

Thrombolytic effects of 120‐kHz and 1‐MHz ultrasound and tissue plasminogen activator on porcine whole blood clots

Stroke is the third leading cause of death and the leading cause of disability in the United States. For patients with ischemic stroke, the thrombolytic drug tissue plasminogen activator (tPA) is the only FDA‐approved treatment. To aid in the development of a stroke therapy, the synergistic thrombolytic effect of tissue plasminogen activator (t‐PA) and ultrasound was assessed in vitro in a porcine clot model. Whole blood clots were prepared from fresh porcine blood by aliquoting 1.5 ml into 8‐mm‐i.d. glass tubes, immersing the tubes in a 37 °C water bath for 3 h and storing the clots at 5 °C for at least 3 days prior to use in comparative ultrasound and t‐PA studies, which ensured complete clot retraction. The 120‐kHz or 1‐MHz ultrasound peak‐to‐peak pressure amplitude used for exposures was 0.35, 0.70, or 1.00 MPa. The range of duty cycles varied from 10% to 100% (continuous wave) and the pulse repetition frequency was 1.7 kHz. Clot mass loss was measured as a function of t‐PA concentration (withou...

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.