MIT Biomedical Devices and Clinical Pharmacology & Therapeutics

Clinical Applications of Biomedical Microdevicesfor Controlled Drug DeliveryPablo Gurman, MD; Oscar R. Miranda, PhD; Kevin Clayton, BS; Yitzhak Rosen, MD;and Noel M. Elman, PhD

Abstract

Miniaturization of devices to micrometer and nanometer scales, combined with the use of biocompatibleand functional materials, has created new opportunities for the implementation of drug delivery systems.Advances in biomedical microdevices for controlled drug delivery platforms promise a new generation ofcapabilities for the treatment of acute conditions and chronic illnesses, which require high adherence totreatment, in which temporal control over the pharmacokinetic profiles is critical. In addition, clinicalconditions that require a combination of drugs with specific pharmacodynamic profiles and local deliverywill benefit from drug delivery microdevices. This review provides a summary of various clinical appli-cations for state-of-the-art controlled drug delivery microdevices, including cancer, endocrine and oculardisorders, and acute conditions such as hemorrhagic shock. Regulatory considerations for clinicaltranslation of drug delivery microdevices are also discussed. Drug delivery microdevices promise aremarkable gain in clinical outcomes and a substantial social impact. A review of articles covering the fieldof microdevices for drug delivery was performed between January 1, 1990, and January 1, 2014, usingPubMed as a search engine.

ª 2014 Mayo Foundation for Medical Education and Research n Mayo Clin Proc. 2014;nn(n):1-16

B iomedical microdevices are fabricateddevices with critical features on the orderof 1 to 100mm. Thesemicrodevices range

in complexity from simple microstructures suchas microchannels to more sophisticated micro-functional parts such as microtransducers andmicroelectromechanical systems (MEMS).1

These devices integrate mechanisms thatactivate a variety of physical signals to achievea specific function. For example, MEMS-basedinertial sensors transduce a mechanical signalinput to an electrical signal response. Currenttransducers are able to combine multiplephysical inputs with multiple output signals.

Biomedical microdevices present a varietyof key advantages for applications in healthcare owing to their (1) extremely small sizesproviding minimally invasive procedures, (2)low power consumption, (3) batch fabricationprocesses with high reproducibility, and (4)low cost per device, in conjunction with theirmultiple functionalities and compatibility withvery large-scale integration electronics.

These novel technologies have acceleratedthe development of a variety of micromedicaldevices, such as catheter pressure sensors,microelectronic components for pacemakers,

hand-held point-of-care diagnostic devices,and drug delivery systems, all of which haveprovided significant improvement over treat-ment possibilities for numerous chronic andnonchronic illnesses.1-4 Figure 1 shows a varietyof biomedical microdevices for several therapeu-tic applications.

Controlled drug delivery systems that arebased onmicrodevices contain structural micro-parts, such as microchannels and microreser-voirs, to store drugs. In addition, drug deliverysystems based on MEMS incorporate micro-transducers such as microactuators and micro-sensors, which improve the device capabilities.

Drug delivery devices based on MEMSprovide an opportunity for improved diag-nosis, monitoring, and treatment of numerousillnesses. The MEMS can deliver a variety ofdrugs, including drugs in combination, usinga single device. The MEMS drug delivery de-vices have the ability to control the rate ofdrug release to a target area. They can be pro-grammed for pulsatile or continuous deliveryand can release the drug locally, which in-creases treatment efficacy using a smalleramount of drug, reducing systemic concentra-tion levels1-6 and associated toxicity.

From the Institute forSoldier Nanotechnologies,Massachusetts Institute ofTechnology, Cambridge(P.G., O.R.M., K.C., Y.R.,N.M.E.); and Departmentof Materials Science,University of Texas atDallas, Richardson (P.G.).

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http://dx.doi.org/10.1016/j.mayocp.2014.10.003

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Finally, the scope of novel materials forbiomedical devices has expanded the potentialuse of biocompatible platforms with high biolog-ical performance, eg, less toxic and nonreactivedevices, enabling new therapeutic applications.

This review provides a summary of currentstate-of-the-art biomedical microdevices forcontrolled drug delivery and their correspond-ing clinical applications. The following sectionsdescribe passive and active delivery devicesbased on MEMS technology. Each section pro-vides a technical description of a microdevicefollowed by its suggested clinical application.The review continues with a summary of theregulatory strategies for obtaining Food andDrug Administration (FDA) approval for suchmicrodevices. Finally, a perspective on thefuture of these novel devices is presented.

DATA SOURCES AND SEARCHESA PubMed search between January 1, 1990, andJanuary 1, 2014, was performed. The searchterms were drug delivery AND MEMS, implant-able devices AND MEMS, control release ANDmicrochip, controlled release AND BioMEMS,

neural probes AND drug delivery, vaccines ANDmicroneedles, diabetes AND microneedles, intraoc-ular AND drug delivery devices, and inner earAND drug delivery AND microfluidics. Paperswere selected following the definition of micro-devices and MEMS. Selection also was per-formed with the aim of having examples ofdifferent types of microdevices (passive andactive, actuationmechanism, andmaterials). Ex-amples of different clinical applications for drugdelivery microdevices assisted in selecting pa-pers more close to the clinical application thanthose focused solely on fundamental science.Diagnostic microdevices were specificallyexcluded from the search.

PASSIVE DEVICESPassive biomedical microdevices for drug deliv-ery do not rely on an actuation mechanism oron monitoring for feedback. These devices arereservoir based, relying on mass transfer acrossa permeable membrane to deliver pharmaceu-tical drugs, the biodegradation of a hermeticmembrane, or a unique reservoir structure toachieve controlled release. The rate of releasecan be controlled by taking into account thefollowing design parameters: (1) the effectivepermeability of the membranes by fine-tuningstructural dimensions and materials (poresize, thickness), (2) the rate of degradation ofthe polymer contained on the membrane orin the reservoir, (3) the diffusivity propertiesof the drug, and (4) the osmotic pressure. Pas-sive delivery of drugs cannot be modified afterimplementation. Other passive-release devicesoperate based on actuation resulting fromin vivo conditions inside the body, such as pHor temperature, to accelerate degradation ofthe materials that encapsulate the pharmaceu-tical drugs. Typically, the controlled release isachieved by considering the pharmacokineticsof the selected drug for delivery. Design and ma-terial parameters are thereafter adjusted andselected during the design process to provide aconstant and superior pharmacokinetic perfor-mance, such as an improvement in treatmentefficacy duration over the typical half-life ofthe pharmaceutical drug. Existing passive-release devices, such as the fentanyl transdermalsystem (DURAGESIC; Janssen PharmaceuticalsInc) and the fluocinolone acetonide intravitrealimplant (Retisert; Bausch & Lomb Inc),are used for either short-term (3 days) or

ARTICLE HIGHLIGHTS

n Drug delivery systems can be classified as passive and active.Passive devices do not incorporate sensors and actuators fordrug delivery.

n Active microdevices include microelectromechanical systems(MEMS), which comprise microparts such as microchannels andmicrovalves and transducers, including microsensors andmicroactuators, integrated into a singular microdevice.

n Advantages of MEMS drug delivery systems include miniaturization,integration with microelectronics, actively controlled, low cost,multiple pharmacologic therapies in a single device, controlled overrelease rate, and in vivo long-term storage of drugs.

n The MEMS are being used for a variety of clinical conditions,including diabetes, neurologic disorders, inner ear diseases, andcancer.

n Fluzone is an example of a Food and Drug Administrationeapproved drug delivery microdevice for vaccine delivery.

n The MEMS drug delivery devices can be considered combina-tion products. Many combination products are considereddrugs, requiring a New Drug Application for Food and DrugAdministration approval.

MAYO CLINIC PROCEEDINGS

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long-term (2.5-3 years) continuous treatment ofdiseases. The lack of integrated electronics re-duces the complexity of these devices.7,8

HydrogelsImplantable devices based on environmentallysensitive hydrogels were developed forcontrolled release (Figure 2).9 The device ar-chitecture consists of a reservoir and a 100-mm-thick silicon membrane with orificesmeasuring 140 mm in diameter. Each orificecontains a support post in the center and istethered to confine the hydrogel to the mem-brane. The hydrogel is loaded around the

central support post such that the entire orificeis blocked by the hydrogel in the swollen state.

Under activation by chemical or physicalstimuli, the hydrogel shrinks and the drug isallowed to diffuse through the resultingorifice. The response of the hydrogel openingor closing is critical in controlling the rate ofdrug delivery. Additional control of drug de-livery can be gained by manipulating themembrane thickness, the size of the orifices,the support posts, and the tethers.

The temperature, pH, and glucose sensitivityof different hydrogels are some of the parametersthat provide additional control over activation.For example, N-isopropylacrylamide hydrogels

MicrochannelPt conducting wire

Neural probe withmicrochannels for drug

delivery (Figure 4)

MEMS Chip

Pump

Into innerear for intracochleardelivery

Cochlear implanted devicewith pump and electronic

control (Figure 10)

StratumcorneumEpidermis

Dermis

Subcutaneoustissue

Microneedles

Hypodermic needleDendritic cell

Langerhans cell

Microneedle transdermalpatch (Figure 8)

Drug solution Drug reservoir

Parylene cannulaIntocannula

Into eye

Eye wall

Electrolysis pumpPump outlet

Ocular device withelectrolysis pump (Figure 7)

MEMS delivery foremergency (Figure 6)

Microchip drugdelivery for

osteoporosis (Figure 5)Implantabe MEMS device for drug

delivery (Figure 9)

FIGURE 1. Technology map for applications of biomedical microdevices. MEMS ¼ microelectromechanical systems.

CLINICAL APPLICATIONS OF BIOMEDICAL MICRODEVICES

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were found to rapidly contract at 34�C, resultingin a sharp increase in the flow rate of the drugfrom 0 to approximately 1 mL/min. This typeof hydrogel exhibited a fast response to environ-mental conditions, contracting in 10 seconds at25�C and expanding back to close the orifice in20 seconds at 50�C. N-butan-2-ylbutan-2-amine/anodic alumina membrane hydrogelwas measured to respond to a change in pH of3.0 to 10.0 in 4 minutes, whereas for changesin glucose levels from 0 to 20 mmol/L, thishydrogel responded in 40 minutes.9

Clinical Application: DiabetesDiabetes represents a significant burden inhealth care as the number of people withtype 2 diabetes is increasing dramaticallyowing to a pandemic of obesity worldwide.10

One of the key issues in diabetes is adherencewith insulin administration. Adherence islimited owing to the frequent and uncomfort-able subcutaneous (SC) injections that the pa-tient needs to treat his or her diabetes.

Glucose-responsive hydrogels provide anopportunity for the controlled delivery of insu-lin. Incorporating more channels with varioustypes of hydrogels and channel sizes couldimprove control and treatment. Further workis needed to better understand and engineerresponse kinetics and the reliability of suchhydrogel-based devices for clinical applications.

Passive Nanochannel-Based Drug DeliveryDeviceA novel, high-throughput nanochannel drugdelivery system for the sustained delivery ofchemotherapeutics was developed and testedin vitro.11 The device was developed to beimplantable to improve patient adherence andquality of life by avoiding the need for repeatedadministrations and frequent visits to the clinic.The device passively controls the release ofdrugs by physical-electrostatic confinement.By manipulating the size of the nanochannels,zero-order release of chemotherapeutics wasachieved. The nanochannel membrane com-prises a silicon substrate reservoir and acapping layer. An array of 161 channels,measuring 200�200 mm and spaced by 50-mm-thick walls, makes up the membrane sur-face (Figure 3). It consists of 30-mm-widemicrochannels that connect the reservoir andthe capping layer. The nanochannels connectthe inlet and outlet channels at the interfaceof the silicon substrate and the capping layer.

Clinical Application: MelanomaMelanoma, a tumor originating from melano-cyte cells, represents the most aggressiveform of skin cancer, with 5-year survival of20% for advanced cases. Current pharmaco-logic therapies include the use of interferonalfa-2b as an adjuvant for stage III melanomas.Interferon alfa-2b is an immunomodulatorydrug that activates the immune system againstthe tumor, increasing patient relapse-free sur-vival. An important issue with interferonalfa-2b is its adverse effects. Interferon alfa-2b in high doses has been linked to hepatotox-icity and suicidal ideation.12

The use of implantable, controlled nano-channel delivery systems could potentiallyovercome some of the limitations associatedwith current therapies by decreasing theamount of drug that reaches the systemic cir-culation. This improvement could avoidadverse effects in healthy tissues while keepinghigh concentrations of interferon alfa-2b at thetargeted site where the tumor is located.

Clinical Application: Prostate CancerProstate cancer represents the sixth leadingcause of cancer death in men, with an incidenceof 233,000 new cases and 29,480 deaths in

Front sidetethers

Centraltethered post

Back sidetethers

Flow

Siliconmembrane

FIGURE 2. Schematic of silicon membrane withstructured orifices. From Sens Actuators B Chem,9

with permission.





2014.13 Leuprolide acetate is a syntheticanalogue of gonadotropin-releasing hormone.Gonadotropin-releasing hormone stimulatesthe release of follicle-stimulating hormone andluteinizing hormone, which promote the pro-duction of estrogen and testosterone. Testos-terone is metabolized in the interior of prostatecells to dihydrotestosterone, which upregulatescell proliferation, gene expression, and proteinsynthesis. It is thought that leuprolide acts as agonadotropin-releasing hormone analogue andwhen given continuously by the SC or intramus-cular route (leuprolide acetate could not beadministered orally because is a peptide) leadsto testosterone deprivation. Deprivation of pros-tate cells from testosterone would lead toapoptosis and cytoreduction of tumor volume.14

Interferon alfa-2b and leuprolide acetatewere chosen to test the nanochannel micro-chip delivery device. The release of interferonalfa-2b was tested using 20-nm membranesand was measured to be a mean � SD of29.7�1.5 mg/d for 6 days, which is in agree-ment with current maintenance doses of inter-feron alfa-2b used in patients with melanoma(10 million IU/m2 SC 3 times/wk; 10 millioninternational units ¼ 38 mg).

The release of leuprolide was tested using5- and 15-nm channels and was measured tobe zero order for the 5-nm channel at amean � SD rate of 100�10 mg/d for 3 days,which is close in agreement with current leu-prolide doses used in prostate cancer (250mg/d). By increasing reservoir sizes, thenanochannel-based delivery system has thepotential to achieve the current dose regimensused for interferon alfa-2b and leuprolide.

Multifunctional MEMS for Neural Recordingand Drug DeliveryMultifunctional MEMS for simultaneousrecording of neural activity and drug deliverywere developed (Figure 4).15,16 One such de-vice has been reported by Altuna et al16 basedon flexible microprobes made of SU-8. Thepolymer SU-8 was used as the structural mate-rial for the probes, with platinum for the elec-trodes. Tetrode-like probes with a singlemicrofluidic channel and linear probes with 2microfluidic channels were tested. Electrodesfor the tetrode-like probe were spaced 25 mmapart, with diameters of 20 mm to sense indi-vidual neuronal firing at the tip of the probe.

The microfluidic channel measured 50�20mm, with 3 outlet ports also near the tip of theprobe. In the linear probe, 8 electrodes werespaced 100 mm apart, allowing for sensing atdifferent depths of the brain. The 2microfluidicchannels measured 40�20 mm and had inde-pendent outlet ports. Both devices were 55mm thick. The tetrode-like probe was 90 mmwide, and the linear probe was 150 mm wide.

The probes were tested in vivo in anesthe-tized rats. The SU-8 linear probes were used todeliver kainate at the CA1 cell and dendriticlayers at a flow rate of 3 to 6 mL/min to induceseizures. Neuronal excitability was recordedagainst a control delivery of saline to confirm de-livery of the drug. The tetrode-like probe deliv-ered potassium at a high flow rate of 0.6 to 1.5mL/min to the CA1 cell layer. The probe wasable to record isolated neurons together withmulti-unit firing. Both probes had the abilityto measure ripples and spikes common duringlarge irregular brain activity at the CA1 cell layer.

Clinical Applications: Parkinson Disease andEpilepsyEffective treatments for neurologic diseases arestill lacking. Parkinson disease, the second most

FIGURE 3. Schematics (A, C, and D) and optical microscopy (B) of a passivenanochannel delivery system designed for drug release with zero-orderkinetics. The innovative architecture of the device includes macro-channels, microchannels, and nanochannels. M ¼ macrochannel; mO ¼microchannel outlet; mI ¼ microchannel inlet; n¼ nanochannel; w ¼supporting walls. From Pharm Res,11 with permission.



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common neurodegenerative disorder after Alz-heimer disease, has been managed for the pastfew decades with L-3,4-dihydroxyphenylalanine(L-DOPA).17 The L-DOPA is a precursor to dopa-mine, which is a neurotransmitter that is absentin the brain of patients with Parkinson disease

owing to the progressive loss of dopaminergicneurons. In the long-term, patients start to expe-rience L-DOPA adverse effects that deterioratetheir quality of life.

Epilepsy, a disorder characterized by un-controlled propagation of electrical stimuli inthe brain, has been managed with drugs thatreduce neuron excitability. Some types of epi-lepsy, however, remain refractory to drugs.Therefore, it is clear that current pharmaco-logic therapies alone have not reached anacceptable benefit for neurologic disordersrequiring additional intervention. Implantabledevices such as neural stimulators haveemerged as an attractive option for patientswith advanced Parkinson disease, refractoryepilepsy, and other neurologic conditions.18

Despite the aforementioned benefits, thesenovel delivery modalities need to overcome is-sues of poor biocompatibility, such as inflamma-tory response and fibrosis around the implant,which limit overall device performance. Forexample, neural probes have been found to elicitglial scar formation and neuronal loss during im-plantation, impairing device performance. Hav-ing an anti-inflammatory drug in the samedevice could decrease the inflammatory responseand, thus, the generation of fibrotic tissue (eg,glial scar formation) surrounding the implant,thus preserving functionality.19,20

Therefore, it is being realized that bycombining devices with drug therapies, it ispossible to maximize the benefits of both whileavoiding their adverse effects. This clinical needhas been met by using MEMS technologies, inwhich neural electrodes are being combinedwith microfluidic channels or microreservoirs.This combines the capability to record neuraldata and drug delivery.

ACTIVE DEVICESActive drug delivery devices use a variety ofmechanisms to release pharmaceutical drugsand provide an increased level of control.The MEMS devices have been developed usingdifferent actuation modalities, includingmicropumps based on gas pressure from elec-trolysis, integration of magnetic actuators, andelectrochemical and electrothermal actuationsystems. Active devices can be customized totreat a range of diseases requiring specificpharmacokinetic drug delivery profiles. More-over, as opposed to passive delivery systems,

Bonding pads

Microelectrode film

Microelectrodesites

Outlet

Microchannel

Molded PDMS film

Vacuum

A

B

C

MicrochannelPt conducting wire

FIGURE 4. Schematics (A and B) and optical picture (C) of a neural probewith drug delivery capabilities. A, Assembly of device components. B, Methodused to incorporate the drug into the microchannels. C, Optical picture of thefinished device and microchannels containing a dye solution. PDMS ¼ poly-dimethylsiloxane; Pt¼ platinum. From Sens Actuators A Phys,15 with permission.





MEMS can be activated and stopped at anytime after implantation.

Active devices commonly require minia-turized power electronics for actuation, typi-cally increasing the overall form factor,which is a key limiting factor in implantableapplications. Alternatively, telemetry systemsto transfer energy for activation can be adop-ted to overcome this limiting issue.

Electrothermally Actuated MEMS DrugDelivery MicrochipSantini et al21 developed a device for thecontrolled, pulsatile release of chemicals fromsingle or multiple reservoirs. The controlleddrug release was triggered by the applicationof an electric potential to burst sealing goldmembranes electrothermally. Drugs inside thereservoir were then free to diffuse to the tar-geted site. This original device had the func-tionality for complex release of kinetics byvarying the amount or substance type placedin each reservoir and varying the timing ofrelease.

This type of device would be able to deliverdrugs in a pulsatile manner. Since then, severalworks on active drug delivery devices based onMEMS have made substantial progress towardeffectively treating various ailments.

Clinical Application: OsteoporosisOsteoporosis is the progressive degradation ofbone architecture and loss of mass bone den-sity that leads to bone fragility that ultimatelyincreases the risk of fractures. Osteoporosis ismore common in postmenopausal women,who are at risk for lower levels of estrogens,which are known to be involved in bone for-mation. According to the National Institutefor Health and Clinical Excellence, 9 millionosteoporotic fractures occur annually in theworld.22

A microchip device containing parathyroidhormone (PTH) was developed for the treat-ment of osteoporosis23; PTH is known to stim-ulate bone formation by increasing osteoblastnumber and function.24 An implantablemicrochip device capable of releasing PTHwould prevent the need for frequent injectionsof PTH.

The first in-human testing performed inpostmenopausal women evaluated the in vivopharmacokinetic profile of a PTH-release

microchip (Figure 5) against standard SC in-jections.25 The microchip was implanted sub-cutaneously in the abdomen, and thepharmacokinetic profile was measured after afibrous capsule was formed around theimplant.

The rationale of the study was to deter-mine the pharmacokinetic performance ofthe microchip when it was surrounded by afibrous capsule as a result of the host responseto the implant. In addition, bone biomarkerswere measured to determine the effect onbone formation of PTH injections vs PTHreleased by the microchip. A safety laboratorypanel was performed to determine the safety ofthe microchip vs that of the SC injections.

Overall, the microchip was found to bebioequivalent to the SC injections even inthe presence of the fibrous capsule. Themicrochip was also found to be as safe as theSC injections based on a laboratory panel.25

FIGURE 5. A microchip drug delivery systemfor parathyroid hormone (PTH) release forosteoporosis treatment. The picture depicts thetitanium packaging used for carryng the micro-chip. The device has undergone first humantrials. From Sci Transl Med,25 with permission.



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A Rapid-Delivery Microchip for AcuteClinical ConditionsA microchip drug delivery system for rapid de-livery of vasopressin was developed by Elmanet al.26 The device consists of a membrane layer,an actuation layer, and a reservoir layer. Themembrane layer consists of a biocompatible sil-icon nitride film that serves as a hermetic seal forthe reservoirs. The actuation layer consists of 3microresistors. Heat is generated when a currentis passed through these microresistors. The heatserves to nucleate bubbles and dramatically in-crease internal pressure inside of the reservoir.This step leads to rupturing of the silicon nitridemembrane, followed by rapid release of thepharmaceutical drug, used as a bolus. A pictureof the device in action is shown in Figure 6.

Clinical Application: Hemorrhagic ShockHemorrhagic shock is an acute condition that canresult from severe traumatic injuries associatedwith massive bleeding loss, which if not treatedwithin seconds or minutes could result in perma-nent damage or death. In most cases, critical pa-tients do not have immediate access to a healthcare facility where basic measures to restore he-modynamic stability are available. These mea-sures include oxygenation; restoration ofintravascular volume with colloids, crystalloids,or blood products; and use of inotropic and vaso-pressor drugs. In settings with limited or no ac-cess to health care facilities, interventions toprevent massive hemorrhages include self-applied hemostatic dressings.

This approach, however, does not accountfor internal bleeding sites, which occasionallyare themain cause of death. During hemorrhagicshock, the massive loss of blood compromisesvital organ activity in the brain, heart, and kid-neys, among others. The natural response ofthe body to avert vital organ damage is to pro-duce vasoconstriction to restore arterial bloodpressure and cardiac output to the level requiredtomaintain adequate oxygenation of vital organswhile avoiding further blood loss.27

Vasopressin and inotropic agents representan important tool in the management of hem-orrhagic shock.28-30 This biomedical microde-vice was designed to be implanted in high-riskpatients to deliver vasopressin for the manage-ment of hemorrhagic shock in emergency andambulatory settings. Finally, other potentialuses of the rapid-delivery microchip includeacute medical conditions that require immedi-ate intervention, such as cardiovascular andneurologic emergencies.

Magnetically Controlled MEMS Drug DeliveryA magnetic actuator MEMS drug delivery devicewas developed for the controlled release of achemotherapeutic agent. The device wasdesigned to avoid the use of batteries, improvingform factor. The device consists of a microreser-voir sealed by a thin magnetic membrane com-posite consisting of elastic polydimethylsiloxanematerial integrated with iron oxide nanoparticles.An external magnetic field applied by a neodym-ium iron boron permanentmagnet creates a forcethat allows the magnetic membrane to deflect.This process builds up pressure inside the reser-voir, enabling the drug to diffuse out through alaser-drilled micron-sized aperture.

On-demand release profiles can be createdfor optimal treatment using this device. Withno actuation, the mean � SD release of thedrug was measured to be 0.053�0.014 ng/min. With actuation of the membrane by appli-cation of a 255-mT magnetic field, the mean �SD release rate increased to 160�10.2 ng peractuation. The release rate exhibited sustaineddelivery for more than 35 days.31

Clinical Application: CancerDocetaxel was selected as a test drug to studythe device release profile. Docetaxel is an anti-neoplastic agent that disrupts the mitotic spin-dle, causing cell death; it is used for the

FIGURE 6. Pulsatile controlled delivery profileof a microelectromechanical systems devicewith a thermally induced actuator releasing drugout of a reservoir for emergency applications.From Biomed Microdevices,26 with permission.





treatment of a variety of tumors, such as breastcancer.32 An important issue in antineoplasticdrugs is to achieve maximum selectivity be-tween cancer cells and healthy cells byincreasing the local concentration of the drugwhile decreasing systemic drug biodistribution,avoiding exposure of healthy tissues.33 Thiscould be accomplished using a magneticallyactuated MEMS device that could release thedrug locally on demand.

In vitro drug-release experiments usingcell culture demonstrated that freshly prepareddocetaxel solutions and docetaxel from the de-vice described previously herein were found tohave comparable effects on target cells.Further development is still required beforeclinical translation.

Micropump MEMS-Based Drug DeliveryDevicesA refillable intraocular MEMS drug delivery de-vice was developed that uses a micropump foractuation. The device was designed to deliverdrugs from a 54-mL reservoir by sending thedrug through a cannula and past a 1-way checkvalve incorporated at the end of the cannula(Figure 7).34 A dose of medication is dispensedfrom the device via an electrolysis micropump.The device is intended to be implanted underthe conjunctiva, with the cannula pointinginto the anterior chamber of the eye.

Electrolysis of water is triggered by anapplied voltage, producing oxygen andhydrogen gases. These gases result in an internalpressure that forces the drug out of the reservoir.For driving currents ranging from 5 mA to 1.25mA, the flow rate of drug increased linearly from5 to 439 mL/min. Under normal and abnormalback pressures, the device was able to release1500 and 1300 nL/min, respectively, with adriving current of 200 mA. Silicone rubber wasselected as the reservoir material and was foundto be capable of resealing without leakage afterrepeated refills via a non-coring needle.

Replenish Inc further developed a similarsystem called the Ophthalmic MicroPumpSystem. Two types of micropump systemswere developed: an anterior micropump anda posterior micropump. Both devices use awireless programmer and charger for controlof drug delivery. A flow sensor controls theflow rate through a feedback loop, allowingthe dispensing of nanoliter volume of drugs.

The final piece of the system is a separate con-sole unit to refill the implant with drug.35

Clinical Application: Ocular DisordersTraditional ocular drug treatments, such as oraldrugs and eyedrops, require significant overdosebecause less than 5% of the drug is able to passthe physiologic barriers and reach the site of ac-tion.36 The overdose needed to achieve thera-peutic concentrations results in potentialsystemic adverse effects. A variety of passive im-plants were developed to overcome this issue.Current passive intraocular implants dependon polymer degradation to release the drugand have no control over the drug-release pro-file, which could lead to subtherapeutic orsupratherapeutic (toxic) drug concentrations.

Using the electrolysis micropump, it ispossible to circumvent these limitations byproviding the drug locally and controllingthe pharmacokinetic profiles. Release profilescan be programmed by adjusting the currentapplied to the electrolysis pump. The abilityof this device to be refilled makes it attractivefor long-term treatment of ocular diseases ofthe posterior segment, such as age-relatedmacular degeneration.

The anterior micropump developed byReplenish Inc was adapted to address disor-ders of the anterior chamber (glaucoma),whereas the posterior micropump was adapt-ed to address disorders of the posterior cham-ber (retina disorders).

Transdermal MEMS Microneedle PatchArray Delivery SystemResearchers developed a wearable patch basedon a microneedle array for the transdermal

Drug solution Drug reservoir

Parylene cannula

Intocannula

Into eye

Eye wall

Electrolysis pump

Pump outlet

FIGURE 7. Cross section of the ocular device illustrating the pump andcannula. From Sens Actuators A Phys,34 with permission.



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delivery of macromolecular drugs. Micronee-dles provide painless administration becausethey are designed to penetrate through thestratum corneum (the outer layer of the skin)without reaching the nerve terminals locateddeeper in the skin.

Studies have reported a strong correlationbetween microneedle length and pain percep-tion, although other features, such as drug vol-ume and number of microneedles, have alsobeen associated with pain development duringmicroneedle insertion.37,38 Figure 8 comparesinjection depth and physiologic impact be-tween an array of microneedles and a hypo-dermic needle.39

A device was developed consisting of 400-mm-long microneedles that are insertedthrough the outermost layer of the skin, result-ing in pain-free drug delivery. The whole de-vice consists of an array of 25 microneedles,each with 300-mm through-holes on a 4�4-mm cross section. A thermally expandable sil-icone composite is layered below the reservoir.A printed circuit board with heaters to expandthe silicone composite layer into the reservoirlayer was designed to perform controlledrelease of the drug through the microneedles.The amount of power applied to the electricalcopper heaters controls the amount of

expansion and, therefore, the flow rate of thedrug. The microneedles use side openings toallow an incredibly sharp apex to avoid coringof tissue during its therapeutic application.40

Clinical Application: DiabetesThe reduction of frequent SC injections of insu-lin can improve adherence to insulin therapy indiabetic patients. Moreover, emulating thephysiologic release of insulin by the pancreasis a highly desirable feature. In this regard,the transdermal microneedle MEMS array pro-vides painless administration (improving pa-tient adherence) and control over the flowrate that mimics the kinetics release of insulinby the pancreas (improving efficiency whileavoiding adverse effects).

Furthermore, a transdermal patch is aneasy-to-use device compared with current insu-lin SC injections. The device was tested in vivoon diabetic rats. With applied power of 150 to450 mW, the device was measured to dispense0.1 to 300 mL/h of insulin (a vial of insulin con-tains 100 IU/mL; therefore, 0.1 mL¼0.01 IUand 300 mL¼30 IU). Based on the pancreaticsecretion of insulin (1 IU/h), it is likely thatthe operational space of the micropump iswell suited to replicate the physiologic insulinproduction by the pancreas.

Further work is needed to determine theoptimal response of the thermally expandablematerial to allow for a precisely defined lowflow rate with no leakage. In this study, anexternal power source was used, but amicro-sized battery for practical use could betested in future work.

Clinical Application: VaccinesVaccines have greatly reduced the incidence ofseveral infectious diseases and represent one ofthe most cost-effective interventions in healthcare.41 Therefore, adherence with vaccineadministration has an important role in publichealth. Microneedle technologies for vaccinescan provide painless vaccines, improving pa-tient acceptability and adherence. This isparticularly relevant because most vaccinesare administered to pediatric populations.42,43

Moreover, it is expected that painless vac-cines could also improve adherence in theadult population, eg, tetanus vaccine. Anotherimportant advantage of transdermal micronee-dles over intramuscular vaccines is the

Stratumcorneum

Epidermis

Dermis

Subcutaneoustissue

Microneedles

Hypodermic needle

Dendritic cell

Langerhans cell

FIGURE 8. Schematic comparing a traditional hypodermic needle with amicroneedle array. Note how the microneedle array reaches the dermis,where Langerhans cells are found, and does not reach the subcutaneustissue, rich in nervous terminals. Both properties make microneedles a veryattractive option for vaccine delivery systems. From Clin Exp Vaccine Res,39

with permission.





possibility of stimulating antigen-presentingcells, located in the skin, to improve antigentransport to lymph nodes, which enhancesthe immune response. Microneedles alsocould overcome the technical problems relatedto intradermal vaccines (eg, poor reproduc-ibility over the injection site and the need totrain health care personnel).

Electrothermal MEMS Drug Delivery DeviceA MEMS-based intracranial drug delivery de-vice has been developed and tested for thetreatment of malignant brain tumors(Figure 9).44 Passive-release implants havedemonstrated some effectiveness, but incorpo-rating active MEMS to gain more control overthe release kinetics could improve efficacy anddecrease toxicity.

The MEMS drug delivery device consistedof an injection-molded liquid crystal polymerreservoir measuring 3.7�3.2�2.2 mm andcontaining a total drug payload of 10 mg oftemozolomide. A 300-mm-thick silicon micro-chip sits on top of a 200-mm lip on the interiorreservoir walls. The silicon microchip containsthree 300�300-mm suspended silicon nitridemembranes, which provides an effective,biocompatible barrier to diffusion.

The actuation mechanism relies on usingresistive heating to melt a metallic fuse thatsits on top of the silicon nitride membranes.Titanium and gold layers are deposited ontop of the silicon nitride membrane and areshaped into thin metallic fuses by using photo-lithography followed by wet etching. The fuseis melted using resistive heating by applyingan electrical pulse. This burst results in amembrane fracture and release of the reservoircontent. Each membrane can be designed tobe independently opened by varying the thick-ness of the gold and titanium layers or thewidth of the fuse to require more or less resis-tive heating. This allows for a variable drug-release profile.

Clinical Application: GlioblastomaGlioblastoma is a devastating type of humancancer with mean survival of 12 months andsurvival of less than 5% after 5 years.45,46 Avariety of pharmacologic therapies have beenexplored, with very poor clinical outcomes.47

A major challenge in drug delivery to the brainis circumventing or passing the blood-brain

barrier (BBB). The BBB is the separation ofthe vasculature system from the brain.48

The BBB maintains brain homeostasis byrestricting the transport of molecules presentin the circulatory system to and from thebrain. This is achieved by the unique charac-teristics of the brain microvasculature thatpossess endothelial cells connected by verytight junctions. These tight junctions impedethe passage of large macromolecules from theblood to the brain.

To circumvent the BBB, local implantsthat release drugs directly in the brain weredeveloped and commercialized.49 Althoughcommercial polymeric implants already exist,survival rates are poor and new approachesare needed. By using active implantablemicrochips, a multitarget approach using acombination of drugs with controllable phar-macokinetics could lead to better clinicaloutcomes.

It is important to note that active devicesthat require frequent drug refilling or powersource exchange are not suitable alternativesfor MEMS implanted in the central nervoussystem owing to the implicit requirementfor repeated neurosurgical procedures.Repeated neurosurgical procedures maylead to a variety of serious complications inthe central nervous system. Therefore,several design considerations for implantableMEMS drug delivery systems must beconsidered owing to the unique anatomicaland physiologic features of the central ner-vous system.

The electrothermal MEMS described previ-ously herein was tested in vitro and in vivo viaintracranial implantation in rats. In vitro tests

FIGURE 9. Microelectromechanical systems (MEMS) drug delivery devicefor the treatment of glioblastoma. Assembled MEMS device (A) andcomputer-aided design model of the reservoir (B). From Biomaterials,44

with permission.



11



confirmed that more membranes beingopened leads to more rapid drug release.With 3 membranes activated, the release ratewas measured at 0.3 mg/h, and the mean �SD total release was 90%�3.2% in 30 hours.The release rate and mean � SD total releasedecreased to 0.136 mg/h and 82%�1.9%,respectively, in 60 hours for 2 membranesactivated; further decreases to 0.007 mg/hand 60%�12%, respectively, in 800 hourswas observed for 1 membrane activated.

Implantation and activation of the devicewas found to be effective in increasing survivaltime of 9-L glioblastoma rats. Activation of all3 membranes in the device on the day of im-plantation was the most effective. This deviceshowed improved efficacy via control of drugpharmacokinetics, but further studies areneeded to determine optimal release ratesand timing.

Microfluidic Hydraulic MEMS-Based DrugDelivery DevicesThe MEMS devices for drug delivery to the in-ner ear were developed using microfluidics(Figure 10).50-52 A microcannula connectedto a closed microfluidic circuit allows fluidto flow in and out of the cochlea. Differencesin the micron-sized tubing used for the outletand inlet loops results in discharge andrecharge of fluid on the order of seconds andminutes, respectively.

As the solution is continuously pumped inand out of the cochlea and mixed with peri-lymph, dilution of a dissolved compound resultsin net delivery. The first and second generationsof devices use micropumps, and the third gener-ation uses a reciprocating delivery system tocontrol fluid flow. Reciprocating delivery in-volves infusing and drawing the same volumeof liquid, resulting in zero net volume transfer.

MEMS chip

Fluidicchannel

Drug loading port

Infusewithdraw port

Displacementdiaphragm andchamber

MEMS chip

PumpInto inner ear forintracochleardelivery

Fill port

~13 mm

FIGURE 10. Schematic diagram describing a cochlear microfluidic delivery device to prevent sensorineuralhearing loss. The miniaturized device comprises several components, including a microfluidic chip, tubingand cannula for delivery, and electronic circuitry and a battery to power the device. Device dimensions are5.5�4.0�3.8 cm. The device operates under the principle of reciprocating delivery, used for drug deliveryinto small and sensitive regions of the body, such as the cochlea, where a volume of drug is infused whilethe same amount of liquid is withdrawn, keeping constant the volume in the cochlear space and allowinghigher instantaneous flow rates. MEMS ¼ microelectromechanical systems.





This technique is suitable for small spaces whereoverall volume is limited, such as delivery ofdrugs in the cochlea.

Biological back pressures in the cochlea wereconfirmed to have no noticeable effect ondischarge. The distribution of agents in the co-chlea was tested using 6,7-dinitroquinoxaline-2,3-dione to alter the generation of compoundaction potential. In vitro and in vivo studies inguinea pigs found increases in the compoundaction potential threshold, indicating effectivedrug penetration.

Clinical Application: Inner Ear DisordersInner ear disorders comprise a variety of clinicalconditions affecting the inner ear structure orthe auditory nerve. The inner ear anatomy in-volves the cochlea and the vestibular system.The cochlea is responsible for transducing soundwaves into electrical impulses that are trans-ported through the auditory nerve to the regionin the brain responsible for audition perception.

Disorders that affect either the sensing(cochlear) or transducing (auditory nerve)component of the auditory system are knownas sensorineural hearing loss (SNHL). It is esti-mated that SNHL affects nearly 250 millionpeople worldwide.52 Disorders affecting theinner ear include infectious diseases (eg,congenital rubella and congenital cytomegalo-virus), genetic disorders (such as mutations onthe gene for myosin VIIa, a protein found inthe stereocilia), and sensing elements of thehair cells located in the cochlea.

Other causes include trauma due to long-term exposure to loud sounds and drugs suchas aminoglycosides.53 The physiopathology ofSNHL involves damage to and death of thehair cells located in the corti organ (a region ofthe cochlea that contains hair cells and auditoryneurons). Hair cells are a specialized type of cellthat contain stereocilia, a type of organelle thatin response to acoustic waves opens ionic chan-nels, resulting in depolarization of hair cellmembranes. This leads to the release of neuro-transmitters, which transport action potentialsalong the auditory nerve to the regions of thebrain responsible for auditory function.

The development of the cochlear implanthas been a great achievement to restore hearingto people with deafness.54 Cochlear implantsaim to stimulate ganglion cells. With the contin-uous degeneration of these cells as a result of

infectious, traumatic, or genetic disorders,cochlear implants lose their efficacy. Therefore,drug delivery devices such as the reciprocatingmicropumps described previously herein repre-sent a novel and promising modality forrestoring auditory perception. These devicesmay allow delivery of neurotropic factors withzero net volume transfer, thus maintaining intra-cochlear pressure constant and preserving thesensing elements of the cochlea.55

REGULATORY PROCESS FOR CLINICALTRANSLATIONTo date, there are a few examples of MEMS formedical applications approved by the FDA,including the CardioMEMS wireless pressuresensor (St Jude Medical, Inc), the i-STATpoint-of-care blood analyzer device (AbbottLaboratories), and Fluzone (Sanofi PasteurInc), an influenza vaccine based on micronee-dles.56-60 Several of the MEMS drug deliverydevices described previously herein have notbeen approved by the FDA for clinical use. Itis possible, however, based on previous tech-nologies, such as prefilled syringes (a deviceprefilled with a drug) and the case of Fluzone(which was approved under a Biologics LicenseApplication), to describe a potential regulatorypathway for future drug delivery microdevices.

First, MEMS drug delivery systems involve atleast 2 components: a device and a drug. If theMEMS device incorporates the drug into the finalpackaged product (it is expected, owing to theirsmall size, that the device and the drug will becopackaged in a single product), they will beconsidered combination products.61,62 Second,according to the FDA Office of CombinationProducts, because the drug incorporated intothe device provides the main mechanism of ac-tion (the therapeutic effect is due to the drug;the device only releases the drug), the system isconsidered a drug. Drug products are subjectedto premarket approval through a New DrugApplication (NDA) submission or an AbbreviatedNDA (ANDA) submission.61,63 As mentionedpreviously, Fluzone was approved under a Bio-logics License Application, which is similar toan NDA.60

An NDA requires a complete descriptionof the manufacturing process and preclinicaland clinical studies with the device to establishsafety and effectiveness. When the drug beingused in the device has already been approved,



13



an ANDA might be required. An ANDA is lessstringent than an NDA, demanding only bio-equivalence studies to establish a similar phar-macokinetic profile with existing devices orformulations using the same drug.4

The future of drug delivery microdevices ispromising. Their novelty, their complexity,and the fact that they are implantable, however,will make regulatory approval a challengingendeavor.

PERSPECTIVEBiomedical microdevices for controlled drug de-livery represent the next generation of deliverymodalities that combine miniaturization, lowcost, batch manufacturability and reproduc-ibility, and integration with very large-scale inte-gration electronics, allowing programmabilityand active control over drug release. The currentdevelopment of drug delivery microdevices is atan early stage, and most of the technologies arestill in the proof-of-concept stage.

There are a few examples of successful clin-ical translation of biomedical microdevices, suchas the clinical use of vaccinemicroneedles. Thereare several reasons that some of themicrodevicesare still in the drug delivery pipeline.

From a clinical standpoint, there must be aclear and identified unmet clinical need wherecurrent solutions are still lacking. Even if theclinical need exists and is identified, many appli-cations (eg, infectious diseases) demand largedrug payloads that cannot be accommodatedwith microdevices or that would require peri-odic refilling. Moreover, bringing these devicesto the market entails a very high-risk endeavor.

Finally, regulatory issues could also pose asignificant barrier for bringing microedrugdelivery devices to the market. Some recentinitiatives at the FDA, such as the Center forDevices and Radiological Health Medical Inno-vation Initiative, potentially will help ensure afaster transition of novel biomedical microde-vices into the market.

CONCLUSIONRecent advances in drug delivery devices thatuse biomedical microdevices for controlled de-livery promise improved treatment for a vari-ety of acute and chronic illnesses. Passivedevices operate by releasing the pharmaceu-tical drugs from reservoirs through permeablestructures, which can also be degraded by

environmental triggers, such as pH and os-motic forces, to regulate the release rate.

Active devices require power to actuate apart that releases the drug after the device isdeployed. The release profile of the drug canbe actively controlled after the device has beenimplanted. Passive and active devices can beused as part of minimally invasive proceduresand have the ability to deliver drugs with a pre-cise pharmacokinetic profile, enhancing the effi-cacy and decreasing the toxicity of the drugbeing used.

These devices offer a range of clinical ap-plications in which tailored pharmacokinetics,local release, and high adherence are prerequi-sites. These clinical conditions include cancer,endocrine disorders, and ocular diseases,among many others. Drug delivery devicesrepresent a novel technology but face a varietyof regulatory challenges.

Further understanding of biocompatiblematerials, alternative techniques for drug releaseactuation, and closed-loop microdevices willenhance the capability of microdevices for clin-ical drug delivery. Microdevices for drug deliv-ery represent the next generation of platformsfor more accurate and efficient drug deliverysystems that will enable new therapeutic modal-ities. These novel platforms promise to increasepatient adherence and overall significantlyimprove treatment outcomes.

Abbreviations and Acronyms: ANDA = AbbreviatedNew Drug Application; BBB = blood-brain barrier; FDA =Food and Drug Administration; L-DOPA = L-3,4-dihydroxyphenylalanine; MEMS = microelectromechanicalsystems; NDA = New Drug Application; PTH = parathyroidhormone; SC = subcutaneous; SNHL = sensorineuralhearing loss

Grant Support: This work was supported by the US ArmyResearch Office via the Institute for Soldier Nanotechnol-ogies at Massachusetts Institute of Technology (contractW911NF-07-D-0004).

Correspondence: Address to Noel M. Elman, PhD, Institutefor Soldier Nanotechnologies, Massachusetts Institute ofTechnology, 500 Technology Square, Cambridge, MA02139 ([email protected]).

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Recombinant Tissue Plasminogen Activators(rtPA): A ReviewP Gurman1,3, OR Miranda1, A Nathan1,2, C Washington1, Y Rosen1 and NM Elman1

INTRODUCTIONAcute ischemic stroke (AIS), acute myocardial infarction(AMI), and pulmonary embolism (PE) represent main causesof morbidity and mortality worldwide.1 These clinical condi-tions result from an imbalance of the hemostatic system, lead-ing to thrombosis. Recombinant tissue plasminogenactivators (rtPAs) are used in patients with AIS, AMI, and PEto treat thrombus. This review focuses on the pharmacologyand clinical applications of rtPAs, and therapeutic strategiesto improve thrombolytic therapy.

PHYSIOPATHOLOGY OF HEMOSTASIS: THROMBOSIS ANDFIBRINOLYSISThe hemostatic system is a combination of biochemical and cel-lular events occurring in the blood of arteries and veins designedto maintain the blood in a fluid state (fibrinolytic system) andprevent blood loss upon the injury of a blood vessel wall (coagula-tion system).2,3

Primary hemostasis results from small injuries to blood vesselsthat result in vasoconstriction and platelet activation, aggregation,and adhesion to the subendothelium of the damaged vessel wall,resulting in a platelet clot. Secondary hemostasis refers to thereinforcement of the platelet plug formed during primary hemo-stasis, through conversion of the soluble protein fibrinogen intoan insoluble meshwork of fibrin. This process is carried out bythe coagulation system in response to a larger vessel injury. Thecoagulation system is a complex mechanism involving coagulationfactors, a number of plasma proteins, which work in a coordi-nated fashion to generate fibrin that together with the plateletclot becomes a consolidated thrombus. The interested reader isreferred to the literature2–6 for a comprehensive review of thehemostatic system and mechanisms of thrombogenesis.Fibrinolysis is one of the components of the hemostatic system

that functions to counteract the coagulation process and dissolveinsoluble fibrin clots. The fibrinolytic system is a proteolytic enzy-matic process that consists of an inactive proenzyme, plasminogen,which has the ability to be converted to the active enzyme,

plasmin, by tissue plasminogen activator (tPA). Structurally, tPA isa 70 kDa globular protein with serine proteinase activity, consist-ing of five domains including finger (F domain), epidermal growthfactor (E domain), two kringle domains (K1 and K2), and the pro-tease region (P domain). While the finger domains and the secondkringle domain are involved in fibrin binding, the F and Edomains are involved in tPA clearance by the liver, while the prote-ase region displays plasminogen-specific proteolytic activity.7,8 tPAis synthesized primarily by endothelial cells.9

Plasminogen belongs to a class of proteins known as zymogens.These proteins are present in fibrin and remain in an inactiveform until activated via hydrolysis, a kinase coupled reaction, or achange in configuration. Specifically, tPA binds to fibrin in athrombus and converts the entrapped plasminogen to plasmin,thereby initiating local fibrinolysis. tPA has the property of fibrin-enhanced conversion of plasminogen to plasmin. It produces lim-ited conversion of plasminogen in the absence of fibrin. Plasmin isinactivated by alpha-2 antiplasmin, a serine protease inhibitor. tPAcan be deactivated by a tissue plasminogen activator inhibitorknown as PAI-1. In this manner, the fibrinolytic process is a tightlyregulated system, designed to avoid systemic fibrinolysis, and thusexcessive bleeding. Figure 1 summarizes the mechanism of actionof tPA and fibrinolysis inhibitors present in the plasma.10,11

Under certain conditions, however, the fibrinolytic system canbe bypassed by procoagulation states, such as alterations in bloodflow or blood constituents, promoting the development of athrombus, as shown in Figure 2.12 In these situations, externalintervention with synthetic tPA agents may be necessary. Thesesynthetic forms of tPA are known as recombinant tissue activa-tors, rtPAs, or thrombolytics.

THROMBOLYTIC THERAPYGeneral considerationsPharmacokinetics. All thrombolytic agents are administeredintravenously (i.v.). Intraarterial thrombolysis (IAT) has emergedas a potential strategy for thrombolysis in patients who do notmatch inclusion criteria for i.v. therapy such as time window or

1Institute for Soldier Nanotechnologies, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; 2Sackler Faculty of Medicine, Tel-AvivUniversity, Ramat Aviv, Israel; 3Department of Materials Science and Bioengineering, University of Texas at Dallas, Richardson, Texas, USA. Correspondence:N Elman ([email protected])

Received 25 August 2014; accepted 4 November 2014; advance online publication 31 January 2015. doi:10.1002/cpt.33

274 VOLUME 97 NUMBER 3 | MARCH 2015 | www.wileyonlinelibrary/cpt

REVIEWS

with large vessel occlusions. Although a number of clinical studieshave been performed to determine whether IAT could offer analternative to the i.v. thrombolysis, further large, prospective,randomized clinical trials comparing IAT with standard i.v. ther-apy will be needed to demonstrate a clinical advantage of IATover i.v. thrombolysis.13

Indications for thrombolytic therapy. Thrombolytics have beenapproved by the US Food and Drug Administration (FDA) forclinical use in the treatment of AIS, AMI, and PE, as shown inFigure 3.14

Contraindications for thrombolytic therapy. Contraindications inthe use of thrombolytics include: serious gastrointestinal bleedingduring the last 3 months; surgery within 10 days including organbiopsy, puncture of noncompressible vessels, serious trauma, andcardiopulmonary resuscitation; history of hypertension (diastolicpressure >110 mmHg); active bleeding; previous cerebrovascularaccident or active intracranial process; aortic dissection and acutepericarditis.15

Side effects of thrombolytic therapy. Bleeding is the major risk ofthrombolytic therapy, particularly intracranial hemorrhage. Thecauses of bleeding result from systemic activation of plasmin out-side the thrombus that leads to systemic fibrinolysis. This mightbe attributed to the fact that under physiological conditions theconcentration of tPA around the fibrin clot (5–10 ng/mL) makesthe systemic conversion of plasminogen to plasmin unlikely.When external administration of rtPAs becomes necessary, how-ever, the plasma concentration of rtPAs could rise to 300–3,000

ng/mL, increasing the chances of a hyper fibrinolytic state result-ing in hemorrhage.15 Risk factors associated with intracranialhemorrhage during thrombolytic therapy include patients age

Figure 1 Schematic representation of the mechanism of action of tPA. Plasminogen is converted to the proteolytic enzyme plasmin by tissue-type plas-minogen activator (tPA). tPA can be inhibited by tissue plasminogen activator inhibitor or PAI-1. Free plasmin in the blood is rapidly inactivated by a2-antiplasmin, but plasmin generated at the fibrin surface is partially protected from inactivation. [Color figure can be viewed in the online issue, which isavailable at wileyonlinelibrary.com.]

Figure 2 Schematic depicting the evolution of a thrombus in the vascula-ture system. The thrombogenic process involves activation, aggregation,and adhesion of platelets to the subendothelium, precipitation of fibrino-gen into a fibrin meshwork, and subsequent trapping of red blood cells.[Color figure can be viewed in the online issue, which is available atwileyonlinelibrary.com.]

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>70 years old and those patients who had taken aspirin beforestarting thrombolytic therapy.16

Thrombolytic agentsSignificant advances in thrombolytic therapies have been madesince the 1980s. Since 2010 several thrombolytics have beendeveloped. Currently, there are five principal thrombolytic agentsapproved for clinical use: 1) recombinant tissue plasminogen acti-vators (rtPAs) including alteplase, reteplase, and tenecteplase; 2)streptokinase (SK); and 3) urokinase (UK).17–26

SK is a bacterial product and thus antigenic, resulting in theproduction of antibodies that preclude repeat doses of SK. Inaddition, SK is nonfibrin-selective.27 UK has been shown to bemore expensive than alteplase and has suffered from manufactur-ing shortfalls.28 For these reasons, rtPAs are among the mostwidely adopted thrombolytic drugs in the clinical setting for themanagement of thrombolytic diseases.Therefore, this review will focus on the pharmacology and clin-

ical applications of rtPAs. Table 1 summarizes key pharmacologi-cal and nonpharmacological thrombolytic therapies.

Table 1 Summary of pharmacological and nonpharmacological approaches in thrombolytic therapies

Drug name Advantages Limitations Stage of development

Streptokinase First thrombolytic discovered Allergenic FDA-approved (streptase)

Urokinase Second thrombolytic discovered � Expensive� Manufacturing issues

Withdrawn from the market in1999. Reintroduced in 2002(abbokinase)

Alteplase, Reteplase,Tenecteplase

Current standards for Stroke, AMI, and PE � Poor selectivity towards fibrin� Long infusion time (alteplase)� Neurotoxicity

FDA-approved (activase,retavase, TNKase)

Desmoteplase � Potential use after 6 hoursstroke onset� Long half-life allowing singlebolus administration� High fibrin selectivity� Lack of neurotoxicity

Under clinical developmentthe DIAS-2 clinical trial hasdemonstrated higher mortality rateswith higher doses, withoutclinical improvement

Clinical trials: DIAS-3, DIAS-4studies (ongoing)

Mechanical thrombectomy Can be performed in patients wherertPAs are contraindicated

More clinical trials needed to assessclinical outcome as endpoints

MERCI retriever FDA-approved

Mechanical thrombectomy Successful recanalization demonstratedafter 8 hours of the onset of stroke

� Cost (requires interventionalneurologist and angiography team)� Equipment is expensive� Careful selection of patients is needed

Trevo stent retrieverFDA-approved

AMI, acute myocardial infarction; PE, pulmonary embolism; FDA, Food and Drug Administration; DIAS, Desmoteplase in Acute Ischemic Stroke Trial.

Figure 3 FDA-approved uses of rtPAs. (A) Acute ischemic stroke. (B) Pulmonary embolism (PE). (C) Acute myocardial infarction (AMI). [Color figure canbe viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Recombinant tissue plasminogen activators (rtPAs)rtPAs are produced using genetic engineering techniques throughmutations in the DNA sequence of native tPA. These new thera-peutic agents exhibit longer half-lives than native tPA, allowingconvenient bolus dosing, enhanced fibrin specificity, and higherresistance to inactivation by PAI-1. Three tPA analogs areapproved in the United States for use as therapeutic agents inthrombotic disorders including: 1) alteplase, 2) tenecteplase, and3) reteplase. Desmoteplase, a fourth recombinant form of tPA, iscurrently being tested in clinical trials.29,30 In this section, keyfeatures of tPA analogs are summarized.Alteplase (Activase) is synthesized using the complementary

natural cDNA sequence of native tPA. Alteplase is administeredi.v. in patients experiencing AIS, AMI, or PE. Alteplase is admin-istrated in a single i.v. bolus and then in a 3-hour or 90-minute(accelerated) infusion regime. Circulating fibrinogen levelsdecrease about 16% to 36% when 100 mg of alteplase isadministered.Alteplase has a half-life of 4–8 minutes, requiring long infu-

sion times to achieve recanalization of occluded arteries. Theliver mediates clearance of alteplase from the plasma. The mostfrequent adverse reaction to alteplase in all approved indicationsis bleeding.Alteplase has been associated with neurotoxic properties.31

This is because alteplase has been shown to activate matrix metal-loproteinases (MMP), resulting in breakdown of the blood–brainbarrier (BBB) with an increased risk of cerebral hemorrhage andedema. In addition, alteplase has been shown to interact with N-methyl-D-aspartate (NMDA) receptors and elicit calcium excito-toxicty and cell death. The failure of alteplase to achieve rapidreperfusion, the increased risk of cerebral hemorrhage, and itspotential neurotoxicity has led to the development of newerthrombolytic agents, as described below.Reteplase is another recombinant form of tissue plasminogen

activator. Reteplase is composed of the second kringle domainand protease domain of native tPA and is normally used forpatients who experience AMI. It has a longer half-life than alte-plase (13–16 minutes), which makes reteplase easier to adminis-ter than alteplase, allowing a double bolus injection (secondinjection given 30 minutes after the first injection) and thusavoiding the longer infusion times needed for alteplase.Reteplase has been shown to possess similar specificity

towards fibrin but with lower binding affinity than alteplase.This property allows reteplase to penetrate the thrombus moreefficiently and improve the reperfusion time in occluded arteriescompared to alteplase. Clinical trials comparing the efficacy andsafety of both thrombolytics in AMI, however, did not find asignificant difference in mortality rates between either agent.The liver and kidneys mediate reteplase clearance from plasma,another difference with alteplase, which is cleared mainly by theliver. Similarly, the most common adverse effect of reteplase isexcessive bleeding.Tenecteplase was designed by multiple point mutations of the

native tPA DNA sequence resulting in a molecule with longerhalf-life (20–24 minutes compared to 5–10 minutes), enhancedfibrin specificity, and increased resistance to PAI-1 when com-

pared to alteplase. Tenecteplase is approved for the treatment ofAMI. Tenecteplase can be administered in a single i.v. bolus over5 seconds, which was demonstrated to provide similar efficacy toa 90-minute infusion of alteplase.A recent phase IIb randomized controlled trial comparing tenec-

teplase vs. alteplase has shown better reperfusion rates as measuredby magnetic resonance imaging (MRI), as well as better clinicaloutcomes after 24 hours of administration of the drugs, without asignificant difference in intracranial hemorrhage between thegroups. Tenecteplase is cleared from the plasma by the liver.Desmoteplase is a recombinant form of native tPA derived

from a chemical found in the saliva of vampire bats with similarstructure to native tPA. Desmoteplase has a half-life of 4 hoursand higher selectivity for fibrin than alteplase. Due to its highfibrin specificity that avoids systemic activation of plasminogen,and the lack of neurotoxic effects, researchers have sought toreplace alteplase by desmoteplase.Desmoteplase alpha I (DSPA a1) exhibits the most favorable

profile based on preliminary biochemical and pharmacologicalanalysis and therefore has been chosen for most clinical studies.DSPA a1 shares 70% structural homology with native tPA, butthey differ in their proteolytic activities. Currently, a phase IIIclinical trial, the Desmoteplase in Acute Ischemic Stroke Trial(DIAS-4), is being conducted to study the efficacy and safety of asingle i.v. bolus of 90 lg/kg dose of desmoteplase given between3–9 hours after the onset of AIS. Table 2 summarizes the keypharmacological features of rtPAs

CURRENT CLINICAL USES OF rtPAsAcute ischemic stroke (AIS)Stroke remains as the main cause of disability and the third causeof mortality in industrialized nations. Stroke can be divided intoischemic and hemorrhagic. Ischemic stroke accounts for 85% ofcases of stroke while hemorrhagic stroke accounts for 15% of thecases of stroke.32 Ischemic stroke results from a region of thebrain that becomes hypoperfused as a consequence of theobstruction of a vessel with a thrombus or embolus. Diagnosis ofischemic stroke requires computed tomography (CT) that mustbe performed as soon as a stroke is suspected, as shown inFigure 4A. While management of hemorrhagic stroke remainselusive, ischemic stroke can be managed pharmacologically ormechanically.33 One of the drugs that has demonstrated more suc-cess in the management of acute ischemic stroke is alteplase. Alte-plase has been shown to be effective for the treatment of acuteischemic stroke and was approved for this use in the US in 1996.

Clinical trials to assess the optimal therapeutic window for rtPAs inAIS. Traditionally, thrombolytic therapies have been shown to beeffective within the first 3 hours after the onset of stroke symp-toms.34,35 Recent studies, however, have suggested that somertPAs could also be effective up to 4.5 hours after the onset ofthe symptoms.36,37 Furthermore, ongoing clinical trials are study-ing the effectiveness of desmoteplase within 3–9 hours of theonset of the symptoms of AIS.Determining the optimal therapeutic window for adminis-

tration of rtPAs after the onset of AIS results is critical for an

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effective treatment and will provide an opportunity to broadenthe inclusion criteria for rtPAs therapies and thus benefitmore patients previously excluded from rtPAs treatments. Abrief description of some of the clinical trials designed to studythe best therapeutic window for rtPAs therapy is detailedbelow.

The National Institute for Neurological Disorders and StrokeStudy (NIDDS): rtPAs within 3 hours of symptoms onsetOne of the key clinical studies assessing the effectiveness of tPAwithin 3 hours of the onset of stroke was the National Institutefor Neurological Disorders and Stroke study (NINDS). Thisstudy demonstrated early improvement of neurological symptoms

Table 2 Summary of key pharmacological features of rtPAs

Drug name Pharmacokinetics Fibrin selectivity Clinical use/FDA status Study

Alteplase Single bolus followed by90 minutes to 3 hours infusionHalf-life: 4–8 minutesClearance mediated by liver

11 Stroke, AMIPE (FDA-approved)(activase)

NIDDS, ECASS-3, IST-3Wardlaw et al.

Goldhaber et al.

Yamasawa et al.

Dong et al.

GUSTO

Reteplase Double bolus injection(2nd injection given30 minutes after 1st injection)Half-life: 13–16 minutes

Clearance mediated byliver and kidneys

1 AMI (FDA-approved)(retavase)

GUSTO-3

Tenecteplase Single bolus injection given in 5 secondsHalf-life: 20–24 minutesClearance mediated by liver

111 AMI (FDA-approved)(TNKase)

ASENT-2

Desmoteplase Single bolusHalf-life: 4 hours

111111 Stroke(phase III clinical trial)

DIAS-4

AMI, acute myocardial infarction; FDA, Food and Drug Administration; PE, pulmonary embolism; NIDDS, National Institute of Neurological Disorders and Stroke Trial;ECASS, European Cooperative Acute Stroke Study; IST, International Stroke Trial 3; GUSTO, Global Utilization of Streptokinase and Tissue plasminogen activator foroccluded coronary arteries.

Figure 4 (A) CT images of the brain depicting a thrombus formation in the brain vasculature system. The hyperdense image shown at the right repre-sents a clot formation in the M1 segment of the middle cerebral artery (MCA), while the loss of gray-white differentiation in the insular cortex representsan infarct secondary to the thrombus (courtesy of Dr Eble, Dept. of Medical Imaging, University of Arizona). (B) Axial image from a contrast-enhanced CTof the chest demonstrating a saddle embolus in the main pulmonary artery with extension into the lobar arterial branches bilaterally (courtesy of Dr Oliva,Dept. of Medical Imaging, University of Arizona).

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after 24 hours of symptoms onset and improved clinical out-comes after 3 months. The study also showed an increased inci-dence of intracerebral hemorrhage in the tPA group compared tothe placebo group.

European Cooperative Acute Stroke Study III (ECASS-3 trial):evaluation of alteplase within 3–4.5 hours of the onset of AISsymptomsIn the ECASS-3 study, the safety and efficacy of alteplase admin-istered between 3–4.5 hours after the onset of stroke symptomswas evaluated. ECASS III demonstrated an improvement in clini-cal outcomes in a 90-day period in the groups treated with tPAcompared with the placebo group. Safety analysis showed that, inspite of the fact that intracranial hemorrhage was more fre-quently found in patients receiving alteplase compared to patientsreceiving placebo, the overall mortality did not change signifi-cantly between the groups. A meta-analysis by Lansberg et al.36

involving the clinical studies ECASS-1, ECASS-2, and ECASS-3and the Alteplase Thrombolysis for Acute NoninterventionalTherapy in Ischemic Stroke (ATLANTIS), concluded that rtPAstherapy between 3–4.5 hours after the onset of AIS was associ-ated with increased chances of favorable outcomes withoutadversely affecting mortality.

The International Stroke Trial (IST-3): alteplase within 6 hoursof the onset of AIS symptoms onsetThe International Stroke Trial (IST-3) was a multicenter,randomized, open-label study designed to study whether morepatients could benefit from thrombolytic therapy by extendingthe therapeutic window for rtPAs administration beyond 6 hoursafter the onset of the AIS. Patients were allocated to either rtPAor placebo. The primary endpoint of the study was the propor-tion of patients alive and independent after 6 months.The study confirmed the need for an early intervention of

thrombolytic therapy after the onset of stroke symptoms,although it also provided data that some patients might benefitfrom rtPAs administration even 6 hours after the onset of thesymptoms of stroke. The study also provided data that justify theuse of rtPA in patients older than 80 years and did not supportany restriction in the use of rtPA according to stroke severity.The study also encouraged that more controlled clinical trials areneeded to evaluate the efficacy of rtPAs beyond 4.5 hours of theonset of stroke symptoms.

DIAS-4 trial: desmoteplase within 3–9 hours of the onset ofAIS symptomsDIAS-4 is an ongoing randomized, double-blind, parallel groupplacebo-controlled phase III study to determine the efficacy andsafety of desmoteplase vs. placebo within 3–9 hours of the onsetof AIS symptoms. The study started in 2009 and is expected tobe completed by 2015.

SYSTEMATIC REVIEW AND META-ANALYSIS OF rtPAs INAISA recent systematic review and meta-analysis of clinical trialsinvolving i.v. rtPAs vs. placebo was conducted by Wardlaw

et al.38 The meta-analysis included 12 randomized controlled tri-als totaling 7,012 patients with i.v. rtPAs vs. placebo (includingNIDDS, ECASS III, and IST-3) administrated up to 6 hours ofthe development of stroke symptoms. In terms of the optimaltherapeutic window for rtPAs therapy, the meta-analysis con-cluded that rtPAs should be initiated as early as possible, but didnot rule out the possibility that some patients might benefit fromrtPAs therapy even 6 hours after the onset of ischemic stroke.The study reported that interindividual variability could

explain why some individuals might benefit even after 6 hours ofthe onset of stroke while others do not. In addition, the meta-analysis also concluded that the reason that later treatment (after6 hours) could not benefit more patients than early treatment(between 3–6 hours) might be attributed to less benefit in tissueto salvage rather than increased odds of intracranial hemorrhage.

Acute myocardial infarctionAcute myocardial infraction (AMI) is a life-threatening condi-tion that results from oxygen deprivation to the heart tissue(ischemia) that occurs when a coronary artery becomes occluded.AMI produces the death of myocardial tissue affecting cardiacoutput with a drop in blood pressure. The drop in blood pressureincreases sympathetic reflexes leading to vasoconstriction, whichdecreases coronary flow hindering even more cardiac contractil-ity, ultimately leading to cardiogenic shock and death. Therefore,AMI must be treated immediately to save as much tissue as possi-ble before cardiogenic shock can occur.Current approaches in the management of AMI aim at restor-

ing the blood flow to myocardial cells in the shortest time possi-ble. These include minimally invasive procedures such aspercutaneous coronary interventions (PCI), and pharmacologicaltherapies such as rtPAs.39 Due to the increasing importance ofprehospital management of AMI in terms of mortality gain withdecreasing delay time to reperfusion after the onset of the symp-toms, prehospital thrombolysis became a very important thera-peutic tool.Prehospital thrombolysis offers several advantages, such as

immediate access (only 25% of US hospitals provide primaryangioplasty and the delay time to receive PCI treatment cannotexceed 90 minutes), and the possibility to add adjunctive pharma-cotherapy.40 Alteplase, reteplase, and tenecteplase are the throm-bolytics approved for the management of AMI in the US.Tenecteplase has found a place in the management of AMI

during prehospital management, before PCI is available.41 TheASSENT-2 clinical trial demonstrated that tenecteplase was simi-lar to alteplase in terms of mortality rate, with an additionaladvantage in terms of major bleeding and reduced rate of conges-tive failure.41 Additional advantages of tenecteplase over alteplaseinclude its ease of administration (single bolus), its higher affinityto fibrin than alteplase, its 80 times less inhibition by PAI-1 thanalteplase, and that it is not affected by nitrates, as seems to occurwith alteplase.It is important to note that, although PCI has been demon-

strated to be superior to fibrinolysis therapy in reducing mortalityin patients with ST elevation, there seems to be a synergisticeffect when PCI is performed after thrombolytic therapy.

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Goodman and Cantor reviewed the clinical studies comparingearly fibrinolytic therapy with PCI in patients entering the emer-gency room with AMI with ST elevation.42

These studies, which include the “TRANSFER”AMI trial(Trial of Routine Angioplasty and Stenting after Fibrinolysis toEnhance Reperfusion in Acute Myocardial Infarction), haveshown a benefit in administering early fibrinolytic therapy fol-lowed by PCI.42

Pulmonary embolismPulmonary embolism (PE) is one of the manifestations of venousthromboembolism together with thrombophlebitis and deep veinthrombosis. PE can range from asymptomatic findings to life-threatening clinical entities (massive pulmonary embolism) withmortality rates of up to 65%. PE refers to the migration of embolito the lung capillaries leading to pulmonary complications includ-ing a decrease in gas exchange from the lungs to the systemic cir-culation and pulmonary edema, leading to a fall in oxygensaturation in the blood, difficulty breathing, and increase in heartrate.PE increases pulmonary artery pressure affecting the right ven-

tricle. In severe cases, PE leads to a failure of the left ventriclewith cardiogenic shock. Figure 4B shows a CT image of PE ofthe main pulmonary artery and lobar arterial branches.Fibrinolytics have an important role in pulmonary embolism

by dissolving the thrombus and releasing the pressure on the pul-monary arteries, improving cardiac output. Pioneer studies con-ducted by Buneameux and Goldhaber and coworkersdemonstrated the usefulness of intravenous tPA for the manage-ment of this serious condition.43–50

Yamasawa et al.47 compared the effect on oxygen pressure andlung perfusion of tPA vs. heparin alone in 45 patients, demon-strating a clinical benefit of tPA vs. heparin in these parameters.Meneveau49 highlighted the need for thrombolytic therapy as a

first-line treatment in patients presenting pulmonary embolismwith signs of cardiogenic shock.Wan et al.50 reported a meta-analysis of randomized controlled

trials comparing heparin alone vs. thrombolytic therapy. Themeta-analysis showed that no clear benefit of using thrombolytictherapy was observed. However, subgroup analysis showed thatthose patients with massive pulmonary embolism at risk of deathhave a benefit from thrombolytics compared with heparin alone.

NOVEL USES OF rtPAsFrostbiteFrostbite has been defined as the freezing of tissues resultingfrom exposure of intact skin to temperatures below the freezingpoint. This traumatic freezing leads to devastating ischemic-baseddamage to distal extremities, which in severe cases leads to ampu-tation of devitalized tissue.51

The damage caused by frostbite can be divided into two sepa-rate categories: mechanical and ischemic. Mechanical damagerefers to the injury caused by the formation of ice crystals,whereas ischemic damage refers to injury caused by thrombosis.Intravenous rtPAs administration, together with heparin and

aspirin, has been suggested as a pharmacological approach in the

management of frostbite. tPA has been recommended to beadministered as fast as possible after exposure to freezing temper-ature. It is thought that tPA would limit the formation of micro-vascular thrombus, avoiding reperfusion injuries.52

The Wilderness Medical Society (WMS)53 developed one ofthe most detailed guidelines on frostbite management. This isbecause frostbite tends to occur in wild environments and reportsof cases in urban settings is very limited. The strongest level ofclinical evidence provided to date in the use of tPA for frostbiteis detailed below.Twomey et al.54 performed an open label study to assess the

safety and efficacy of rtPA for treating severe frostbite. Patientsincluded in the study had suffered severe frostbite, exhibited noimprovement with rewarming, lacked Doppler pulses in distallimb or digits, and had no perfusion indicated by the use of atechnetium (Tc) 99m three-phase bone scan.Six patients received intra-arterial rtPA, while 13 patients were

treated with intravenous tPA. In addition, all patients of thestudy received i.v. heparin. Patients who did not respond tothrombolytic therapy had more than 24 hours exposure to cold,warm ischemia greater than 6 hours, or multiple freeze-thawcycles. Twomey et al. saw a reduction in digits requiring amputa-tions from a predicted, at-risk number of 174 to only 33 digitsamputated (in 18 patients) after treatment with i.v. rtPA. Theauthors indicated that i.v. rtPA with heparin after rapid rewarm-ing was safe and reduced significantly the amount of predicteddigit amputations.In another open-label study conducted by Cauchy et al.,55

three treatment regimens were randomly assigned to 47 patientswith severe frostbite. The regimens assigned randomly includedeither of the following therapies for 8 days: aspirin plus buflo-medil, aspirin plus prostacyclin, or aspirin plus iloprost alongwith recombinant rtPA for the first day as the additive therapy.Cauchy et al. indicated that adding rtPA should be based on acase-by-case basis, depending on severity level (at least stage 4),presence of trauma such as head trauma, related contraindica-tions, and amount of time since rewarming. Cauchy et al. didnot rule out the possible additive effects of rtPA in selectedpatients.

Submacular hemorrhageA randomized clinical trial to treat submacular hemorrhagecaused by wet macular degeneration, a degenerative disorder ofthe retina, is currently being performed using rtPAs and perfluor-opropane (C3F8). The rationale of using tPA is to help to dis-solve the clot formed during the hemorrhage, while C3F8 is usedto shift the clot away from the central region of the retina (mac-ula) where high resolution vision is achieved.56

Pediatric empyemaEmpyema represents a complication of pneumonia where liquidand pus are accumulated in the pleural cavity. In children, theincidence of this condition has been increasing in the last years,requiring prolonged hospitalization for recovery. In order toshorten the hospitalization times, a new therapeutic modalityinvolving rtPAs is being explored.

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A double-blind, randomized clinical trial using rtPAs andDNAse is ongoing to investigate the effect of rtPAs and DNAsefor the treatment of pediatric empyema. It is thought that tPAwill break down the organized pus while DNAse will decreasethe viscosity of the pus, thus improving the drainage of the liquidentrapped in the pleural space, leading to a faster clinicalrecovery.57

PeritonitisRecent literature supports the potential benefits of tPA in pre-venting abscess formation after surgical treatment of peritonitis.58

A pilot clinical study is being conducted to investigate the safetyof intraperitoneal administration of tPA and DNase includingadverse events such as bleeding and pain, and biochemicalmarkers of inflammation. The study is being conducted inpatients undergoing peritoneal dialysis who suffer fromperitonitis.59

NOVEL THERAPEUTIC STRATEGIES FOR rtPAsIn 1996 the FDA approved the first recombinant form of nativetPA (alteplase) for the treatment of AIS. In the following decade,new generations of rtPAs were developed and commercialized.Despite the wide adoption of rtPAs in the management ofthrombotic disorders, there are still a number of limitations, suchas a short time therapeutic window, a low rate of arterial recanali-zation, a substantial risk of intracranial hemorrhage, and numer-ous contraindications that limit their use to selected patients.60

All these factors have contributed in the last decade to the devel-opment of novel delivery systems to improve the safety and effec-tiveness of thrombolytic therapy.A number of approaches have been explored to improve the

interaction of tPA with fibrin molecules and activate plasmino-gen to plasmin locally without activating plasmin systemically,the major cause of bleeding during thrombolytic therapy. In thefollowing, a brief description of new therapeutic modalities fortPA delivery is described.

Superparamagnetic nanoparticlesSuperparamagnetic nanoparticles are nanoparticles that can bemagnetized when exposed to an external magnetic field andbecome demagnetized when the magnetic field is removed. Mostmagnetic nanoparticles used for medical applications are ironoxides such as ferrite or magnetite, which allow their surfacefunctionalization with different types of biomolecules.61

Magnetic nanoparticles containing tPA have been developedto improve the localization of tPA molecules in the thrombuswhile avoiding the interaction of tPA with healthy tissues,decreasing the life-threatening side effects (hemorrhage) associ-ated with tPA therapy. When an external magnetic field isapplied at the site of the thrombus, the magnetic particles loadedwith tPA are attracted by the magnetic field, improving the pene-tration of tPA molecules into the thrombus. Furthermore, bylocalizing tPA molecules inside the thrombus, it is possible todecrease the dosage of tPA, improving the safety of the therapy.In this regard, Chen et al.62 developed a nanocarrier delivery

system consisting of a superparamagnetic iron oxide core and an

SiO2 shell functionalized with tPA molecules. The nanoparticleplatform (SiO2-MNP) was prepared by the sol-gel method; 94%of rtPAs was attached to the carrier with 86% full retention offibrinolytic activity.A reduction in clot lysis time and penetration of SiO2-MNP

into blood clots were observed and confirmed under magneticguidance from microcomputed tomography analysis. Therefore,by conjugating rtPAs to an SiO2-MNP surface, a new form ofthrombolytic drug therapy with improved efficacy was demon-strated, resulting in a promising therapeutic modality for thetreatment of thrombotic disorders.62

Gelatin-zinc-tPA complexA formulation comprising tissue-type plasminogen activator(tPA), basic gelatin, and zinc ions, has been developed.63 The sys-tem operates reversibly by inhibiting tPA activity in the presenceof Zn ions. Upon application of ultrasound waves in the range of1 MHz, tPA activity is restored. The system represents a first steptowards the realization of tPA switchable drug delivery systems,where the activity of tPA could be switched “on” and “off” in thepresence of ultrasound waves.

Albumin-rtPA nanoparticlesA nanoparticle system made of rtPA and an anionic peptide elec-trostatically bonded to a protamine-albumin complex was devel-oped. The system maintains rtPA as inactive due to sterichindrance produced by albumin molecules avoiding the contactof rtPA with proteases in the plasma. rtPA is then transportedthrough the circulation in inactive form and becomes activatedupon contact with heparin molecules.64

BioMEMSMEMS drug delivery devices (BioMEMS) have emerged as a newmodality for delivery of drugs. The advantages of BioMEMSinclude active control over drug release, local delivery of drugsavoiding potential systemic toxicity, multiple pharmacotherapiesin a single device, and storage of unstable drugs in powder form,opening the possibility of in vivo reconstitution of drugs. Thepossibility of storage of lyophilized drugs and local delivery areattractive features for delivery of rtPAs. For example, since rtPAshave a very short half-life, a locally implantable BioMEMS couldincrease the local concentration of rtPAs molecules at the site ofaction before they become metabolized systemically.Clinical conditions where rtPAs are being used require imme-

diate intervention to prevent serious sequel. Elman et al.described a rapid MEMS drug delivery device for delivery inambulatory emergency care.65 The microdevice consists of threelayers: 1) a sealing membrane layer, 2) a reservoir layer, and 3) anactuation layer consisting of microresistors. The microresistorsheat the liquid contained in the reservoir layer generating bubblesthat jet the drug outside of the reservoirs, bursting the sealingmembranes, and releasing the drug immediately.Finally, as has been demonstrated with MEMS technology, it

can be envisioned that MEMS drug delivery devices with rtPAswill operate by a closed-loop feedback, or by remote activationfrom the hospital, increasing the chances of survival.66

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Ultrasound-based thrombolysisUltrasound-based thrombolysis represents an emerging technol-ogy that is gaining increasing attention for the management ofstroke. It is thought that ultrasound could improve the transportof rtPAs molecules to the thrombus, increasing fibrinolyticactivity.In addition, mechanical effects such as acoustic cavitation have

been postulated as one of the potential mechanisms involved inimproved thrombolysis using ultrasound.

Ultrasound-based thrombolysis device. One of the key limitationsin ultrasound-based thrombolytic therapies is operator depend-ency, which limits the wide adoption of the technology, as well ashindering the development of new clinical trials due to the needof highly trained operators. In order to address this issue, anoperator-independent ultrasound therapeutic device for the treat-ment of stroke in patients receiving rtPA has been developed.67

The ultrasound device includes a multiple transducer transcra-nial head frame comprising broadband transducers placed at thetemporal and the suboccipital regions. Each transducer isdesigned to address a specific arterial segment and only one trans-ducer is operating at a certain time, avoiding exposure levelsabove FDA-mandated ultrasound exposure limits. A computer-controlled ultrasound generator–receiver system energizes thetransducers. A safety trial was conducted with 15 healthy volun-teers and the platform is currently being tested in phase II andIII clinical trials to assess the overall safety and effectiveness ofthis device against current operator-dependent systems.67

Echogenic liposomes for ultrasound-based thrombolysis. A noveldiagnostic-therapeutic modality (“theranostics”) based on echo-genic liposomes has been described by Laing et al.68 The systemcomprises echogenic liposomes that work as both contrast agentsand drug-delivery vectors. The echogenicity of liposomes allowsusing them as contrast agents for molecular imaging during throm-bolytic therapy. The drug-delivery component is based on tPA

molecules entrapped inside the liposomes, with the domains of thetPA molecule that interact with fibrin exposed at the liposome sur-face. When the liposomes reach the fibrin located in the thrombus,the liposomes release the tPA molecules, avoiding broad biodistri-bution of tPA, which leads to unwanted side effects.Based on preclinical studies using the aortic rabbit thrombus

model, as shown in Figure 5, the researchers provided evidencethat echogenic liposomes loaded with rtPA can be a promisingtheranostic modality that allows direct visualization and targetdelivery of thrombolytics, improving the clinical safety and effi-cacy of rtPAs. Table 3 summarizes novel therapeutic strategiesbased on rtPAs.

MONITORING THROMBOLYTIC THERAPY IN STROKEThrombolytic therapy in stroke with rtPAs remains a challengedue to the short therapeutic window and unpredicted outcomes,such as hemorrhagic transformation. MRI and CT represent twocurrent imaging modalities that have been widely adopted toassist in early stroke diagnosis and therapeutic decision-making.69

These techniques provide the first critical distinction betweenischemic and hemorrhagic stroke, thus excluding patients withhemorrhagic stroke from thrombolytic therapy.These techniques, however, suffer from several limitations such

as limited availability, cost, and image resolution problems to dis-criminate between stroke and clinical entities with stroke-likesymptoms such as migraine, epilepsy, and structural brain lesions(CT). In recent years, there has been an intensive search of tech-niques other than neuroimaging that could provide a high speci-ficity and high sensitivity diagnosis of stroke. In addition, strokediagnosis in the prehospital setting, where MRI or CT are notavailable, has prompted the search for alternative techniques.This search has been focused not only on stroke diagnosis, butalso on prognosis and therapeutic monitoring. These techniqueswould eventually allow the physician to perform critical decisionsto improve clinical outcomes to a magnitude not achievabletoday.In this regard, significant efforts have been dedicated to iden-

tify disease biomarkers and biomarkers for patient selection andstratification. Disease biomarkers can provide information aboutstroke progression and stroke severity scales, providing a powerfultool in the differential diagnosis between healthy and strokepatients, and among stroke patients, between patients with ische-mic stroke, and patients with hemorrhagic stroke.Stroke biomarkers will be critical for the selection of patients

who would benefit from rtPAs, or those patients who may sufferfrom hemorrhagic transformation after rtPA therapy has beenimplemented. Diagnosis via biomarkers will allow a more cost-effective, prehospital management of stroke without the need ofneuroimaging techniques, further reducing the time betweenstroke onset and treatment.To date, however, only a few biomarkers have been supported

by statistical analysis, including C-reactive protein (CRP), P-selectin, homocysteine, and gliar fibrillary acid protein (GFAP)(diagnostic biomarkers); and glucose, glutamate, D-dimer, andfibrinogen (prognostic biomarkers). Above all, high glucose levelsat hospital admission have shown to be a stronger predictor of

Figure 5 Schematic of the rabbit aortic thrombus model used to studythrombolytic drugs. An ultrasound probe is introduced near the location ofthe thrombus to induce ultrasound thrombolysis using echogenic lipo-somes loaded with thrombolytic agents (reproduced with permission fromElsevier, ref. 68). [Color figure can be viewed in the online issue, which isavailable at wileyonlinelibrary.com.]

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poor prognosis and hemorrhage transformation after rtPA ther-apy, and is perhaps the biomarker with the highest chance of clin-ical translation.70 For example, a patient who is admitted to theemergency room with high glucose levels might not benefit fromrtPAs therapy. Instead, another thrombolytic modality, such asmechanical thrombectomy, can provide better clinical outcomeswithout detrimental side effects.Biomarkers could not only provide benefits for clinical man-

agement of stroke, but also assist during the drug discovery, pre-clinical and clinical testing of novel thrombolytic drugs.71

Biomarkers used during the drug development process could pro-vide the information required to determine if a potential molecu-lar target plays a role in stroke physiopathology and to determinethe physical interaction of such targets with the drug being inves-tigated. During preclinical development, pharmacokinetic (PK)and pharmacodynamic (PD) biomarkers would define the phar-macokinetic and pharmacodynamic implications of the interac-tion between the candidate drug and its molecular target. Duringclinical research, biomarkers for patient selection and stratifica-tion would avoid several types of bias related to poor patientselection.New imaging modalities could also provide effective monitor-

ing systems during the pharmacological treatment in the clinicalsetting. For example, ultrasound imaging combined withultrasound-induced thrombolysis is a promising therapeuticmodality that allows real-time monitoring of thrombolytic ther-

apy.72 Furthermore, the use of ultrasound imaging combinedwith echogenic delivery systems such as echogenic liposomesloaded with new drug candidates could provide valuable informa-tion to validate novel thrombolytic agents suitable for clinicaltranslation during the drug development process.Finally, genetic biomarkers such as single nucleotide polymor-

phisms (SNPs) could open a new era in thrombolytic therapy bymeans of personalized therapy. The development of an SNP testin patients with stroke could provide a powerful tool to selectthose patients who would benefit from rtPA (patient selectionand stratification biomarkers). In this regard, in a prospectiveclinical study conducted in 497 patients treated with rtPAs,Fernandez-Cadenas et al. identified three SNPs associated withrecanalization rates after rtPA therapy including the interleukin-1 gene (IL1B) and von Willlebrand factor gene (VWF). Accord-ing to the authors, these SNPs could influence rtPA efficacythrough modulation of coagulation factors activity.73

PERSPECTIVERecombinant tissue plasminogen activators (rtPAs) have emergedas a powerful pharmacotherapy for the management of AIS,AMI, and PE. Moreover, an increasing number of clinical studiesare reporting novel potential therapeutic applications for thesedrugs. Thrombolytic therapies, however, suffer from a short ther-apeutic window, serious side effects, and several contraindications.

Table 3 Novel therapeutic strategies for rtPAs therapyTechnology Advantages Limitations Development stage References

Superparamagneticnanoparticles

� Decrease drug dosage� Decrease side effects dueto broad biodistribution

Loss of tPA activity whenimmobilized in nanoparticlesurface� Change on magnetic proper-ties upon particlefunctionalization

Preclinical 61,62

Zn-gelatin complex Switchable system. tPA activ-ity controlled by ultrasound

� Early stage of development� Need clinical validation ofswitchable capacity

Preclinical 63

Albumin-tPA nanoparticles Prodrug. tPA remains inactiveuntil reach the target

� Early stage of development� Potential interference ofplasma proteins

In vitro 64

BioMEMS � Actively controlled� Local release� Batch fabrication� Integration with electronics� In vitro reconstitutionpossible

� Small payload� Surgical procedure forimplantation might be needed

First in human testing 65,66

Ultrasound-basedthrombolysis device

� Improve thrombolysis bymechanical cavitation andimproved transport of tPAmolecules to fibrin� Operator independent� Advanced stage of develop-ment (phase II, phase III clini-cal trials)

� Optimal acoustic exposureparameters not yetdetermined� Variability between subjects

Phase II, III clinical trials 67

Echogenic liposomes � Combined diagnostics andtherapeutics� Lower dose of tPA required

Early stage of development Preclinical 68

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Therefore, there is a critical need to develop new therapeutic strat-egies to improve thrombolytic therapies.New therapeutic modalities such as ultrasound-based delivery,

magnetic nanoparticles, and BioMEMS are promising technolo-gies, although they are at a very early stage of development andwill require further clinical trials to demonstrate their utility.Meanwhile, other interventions based on education of patients,relatives, and the public in early recognition of symptoms of AIS,AMI, and PE, rapid transport to the hospital, and proper triageassessment in the hospital will contribute to improve the clinicaloutcomes for these devastating conditions.

CONCLUSIONThrombolytic diseases are the leading cause of death in the world.Tissue plasminogen activator (tPA), a protein present in theblood and a component of the fibrinolytic system, is responsiblefor degradation of thrombus formed during the coagulation pro-cess, which normally protects against bleeding when a blood ves-sel is damaged. tPA has now been developed synthetically usinggenetic engineering techniques for the management of pathologi-cal conditions including acute ischemic stroke, acute myocardialinfarction, and pulmonary embolism. In these clinical disordersthe coagulation processes bypass the fibrinolytic system capacityto maintain the blood in a liquid state, resulting in thrombus for-mation, vessel obstruction, and, ultimately, ischemia and tissueinfarct. These synthetic forms of tPA known as recombinant tis-sue plasminogen activators or rtPAs have improved some of thefeatures of native tPA, including increase in the half-life andresistance to plasma inhibitors, leading to improved therapeuticregimes. Novel clinical applications of rtPAs are being exploredincluding frostbite, submacular hemorrhage, and pediatric empy-ema, while novel therapeutic technologies such as ultrasound,nanoparticles. and BioMEMS are being investigated. Therefore,it is foreseen that in the near future rtPAs will positively affect anumber of clinical conditions, if their safety issues can be over-come and their benefits fully exploited.

ACKNOWLEDGMENTSThe authors thank T. Hunter, J. Eble, and I. Oliva for contributions withthe CT images. This research work was supported by the US ArmyResearch Office via the Institute for Soldier Nanotechnologies (ISN) atMIT (contract: W911NF-07-D-0004).

CONFLICT OF INTERESTThe authors declared no conflict of interest.

VC 2015 American Society for Clinical Pharmacology and Therapeutics

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Recombinant Tissue Plasminogen Activators(rtPA): A ReviewP Gurman1,3, OR Miranda1, A Nathan1,2, C Washington1, Y Rosen1 and NM Elman1

INTRODUCTIONAcute ischemic stroke (AIS), acute myocardial infarction(AMI), and pulmonary embolism (PE) represent main causesof morbidity and mortality worldwide.1 These clinical condi-tions result from an imbalance of the hemostatic system, lead-ing to thrombosis. Recombinant tissue plasminogenactivators (rtPAs) are used in patients with AIS, AMI, and PEto treat thrombus. This review focuses on the pharmacologyand clinical applications of rtPAs, and therapeutic strategiesto improve thrombolytic therapy.

PHYSIOPATHOLOGY OF HEMOSTASIS: THROMBOSIS ANDFIBRINOLYSISThe hemostatic system is a combination of biochemical and cel-lular events occurring in the blood of arteries and veins designedto maintain the blood in a fluid state (fibrinolytic system) andprevent blood loss upon the injury of a blood vessel wall (coagula-tion system).2,3

Primary hemostasis results from small injuries to blood vesselsthat result in vasoconstriction and platelet activation, aggregation,and adhesion to the subendothelium of the damaged vessel wall,resulting in a platelet clot. Secondary hemostasis refers to thereinforcement of the platelet plug formed during primary hemo-stasis, through conversion of the soluble protein fibrinogen intoan insoluble meshwork of fibrin. This process is carried out bythe coagulation system in response to a larger vessel injury. Thecoagulation system is a complex mechanism involving coagulationfactors, a number of plasma proteins, which work in a coordi-nated fashion to generate fibrin that together with the plateletclot becomes a consolidated thrombus. The interested reader isreferred to the literature2–6 for a comprehensive review of thehemostatic system and mechanisms of thrombogenesis.Fibrinolysis is one of the components of the hemostatic system

that functions to counteract the coagulation process and dissolveinsoluble fibrin clots. The fibrinolytic system is a proteolytic enzy-matic process that consists of an inactive proenzyme, plasminogen,which has the ability to be converted to the active enzyme,

plasmin, by tissue plasminogen activator (tPA). Structurally, tPA isa 70 kDa globular protein with serine proteinase activity, consist-ing of five domains including finger (F domain), epidermal growthfactor (E domain), two kringle domains (K1 and K2), and the pro-tease region (P domain). While the finger domains and the secondkringle domain are involved in fibrin binding, the F and Edomains are involved in tPA clearance by the liver, while the prote-ase region displays plasminogen-specific proteolytic activity.7,8 tPAis synthesized primarily by endothelial cells.9

Plasminogen belongs to a class of proteins known as zymogens.These proteins are present in fibrin and remain in an inactiveform until activated via hydrolysis, a kinase coupled reaction, or achange in configuration. Specifically, tPA binds to fibrin in athrombus and converts the entrapped plasminogen to plasmin,thereby initiating local fibrinolysis. tPA has the property of fibrin-enhanced conversion of plasminogen to plasmin. It produces lim-ited conversion of plasminogen in the absence of fibrin. Plasmin isinactivated by alpha-2 antiplasmin, a serine protease inhibitor. tPAcan be deactivated by a tissue plasminogen activator inhibitorknown as PAI-1. In this manner, the fibrinolytic process is a tightlyregulated system, designed to avoid systemic fibrinolysis, and thusexcessive bleeding. Figure 1 summarizes the mechanism of actionof tPA and fibrinolysis inhibitors present in the plasma.10,11

Under certain conditions, however, the fibrinolytic system canbe bypassed by procoagulation states, such as alterations in bloodflow or blood constituents, promoting the development of athrombus, as shown in Figure 2.12 In these situations, externalintervention with synthetic tPA agents may be necessary. Thesesynthetic forms of tPA are known as recombinant tissue activa-tors, rtPAs, or thrombolytics.

THROMBOLYTIC THERAPYGeneral considerationsPharmacokinetics. All thrombolytic agents are administeredintravenously (i.v.). Intraarterial thrombolysis (IAT) has emergedas a potential strategy for thrombolysis in patients who do notmatch inclusion criteria for i.v. therapy such as time window or

1Institute for Soldier Nanotechnologies, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; 2Sackler Faculty of Medicine, Tel-AvivUniversity, Ramat Aviv, Israel; 3Department of Materials Science and Bioengineering, University of Texas at Dallas, Richardson, Texas, USA. Correspondence:N Elman ([email protected])

Received 25 August 2014; accepted 4 November 2014; advance online publication 31 January 2015. doi:10.1002/cpt.33


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with large vessel occlusions. Although a number of clinical studieshave been performed to determine whether IAT could offer analternative to the i.v. thrombolysis, further large, prospective,randomized clinical trials comparing IAT with standard i.v. ther-apy will be needed to demonstrate a clinical advantage of IATover i.v. thrombolysis.13

Indications for thrombolytic therapy. Thrombolytics have beenapproved by the US Food and Drug Administration (FDA) forclinical use in the treatment of AIS, AMI, and PE, as shown inFigure 3.14

Contraindications for thrombolytic therapy. Contraindications inthe use of thrombolytics include: serious gastrointestinal bleedingduring the last 3 months; surgery within 10 days including organbiopsy, puncture of noncompressible vessels, serious trauma, andcardiopulmonary resuscitation; history of hypertension (diastolicpressure >110 mmHg); active bleeding; previous cerebrovascularaccident or active intracranial process; aortic dissection and acutepericarditis.15

Side effects of thrombolytic therapy. Bleeding is the major risk ofthrombolytic therapy, particularly intracranial hemorrhage. Thecauses of bleeding result from systemic activation of plasmin out-side the thrombus that leads to systemic fibrinolysis. This mightbe attributed to the fact that under physiological conditions theconcentration of tPA around the fibrin clot (5–10 ng/mL) makesthe systemic conversion of plasminogen to plasmin unlikely.When external administration of rtPAs becomes necessary, how-ever, the plasma concentration of rtPAs could rise to 300–3,000

ng/mL, increasing the chances of a hyper fibrinolytic state result-ing in hemorrhage.15 Risk factors associated with intracranialhemorrhage during thrombolytic therapy include patients age

Figure 1 Schematic representation of the mechanism of action of tPA. Plasminogen is converted to the proteolytic enzyme plasmin by tissue-type plas-minogen activator (tPA). tPA can be inhibited by tissue plasminogen activator inhibitor or PAI-1. Free plasmin in the blood is rapidly inactivated by a2-antiplasmin, but plasmin generated at the fibrin surface is partially protected from inactivation. [Color figure can be viewed in the online issue, which isavailable at wileyonlinelibrary.com.]

Figure 2 Schematic depicting the evolution of a thrombus in the vascula-ture system. The thrombogenic process involves activation, aggregation,and adhesion of platelets to the subendothelium, precipitation of fibrino-gen into a fibrin meshwork, and subsequent trapping of red blood cells.[Color figure can be viewed in the online issue, which is available atwileyonlinelibrary.com.]

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>70 years old and those patients who had taken aspirin beforestarting thrombolytic therapy.16

Thrombolytic agentsSignificant advances in thrombolytic therapies have been madesince the 1980s. Since 2010 several thrombolytics have beendeveloped. Currently, there are five principal thrombolytic agentsapproved for clinical use: 1) recombinant tissue plasminogen acti-vators (rtPAs) including alteplase, reteplase, and tenecteplase; 2)streptokinase (SK); and 3) urokinase (UK).17–26

SK is a bacterial product and thus antigenic, resulting in theproduction of antibodies that preclude repeat doses of SK. Inaddition, SK is nonfibrin-selective.27 UK has been shown to bemore expensive than alteplase and has suffered from manufactur-ing shortfalls.28 For these reasons, rtPAs are among the mostwidely adopted thrombolytic drugs in the clinical setting for themanagement of thrombolytic diseases.Therefore, this review will focus on the pharmacology and clin-

ical applications of rtPAs. Table 1 summarizes key pharmacologi-cal and nonpharmacological thrombolytic therapies.

Table 1 Summary of pharmacological and nonpharmacological approaches in thrombolytic therapies

Drug name Advantages Limitations Stage of development

Streptokinase First thrombolytic discovered Allergenic FDA-approved (streptase)

Urokinase Second thrombolytic discovered � Expensive� Manufacturing issues

Withdrawn from the market in1999. Reintroduced in 2002(abbokinase)

Alteplase, Reteplase,Tenecteplase

Current standards for Stroke, AMI, and PE � Poor selectivity towards fibrin� Long infusion time (alteplase)� Neurotoxicity

FDA-approved (activase,retavase, TNKase)

Desmoteplase � Potential use after 6 hoursstroke onset� Long half-life allowing singlebolus administration� High fibrin selectivity� Lack of neurotoxicity

Under clinical developmentthe DIAS-2 clinical trial hasdemonstrated higher mortality rateswith higher doses, withoutclinical improvement

Clinical trials: DIAS-3, DIAS-4studies (ongoing)

Mechanical thrombectomy Can be performed in patients wherertPAs are contraindicated

More clinical trials needed to assessclinical outcome as endpoints

MERCI retriever FDA-approved

Mechanical thrombectomy Successful recanalization demonstratedafter 8 hours of the onset of stroke

� Cost (requires interventionalneurologist and angiography team)� Equipment is expensive� Careful selection of patients is needed

Trevo stent retrieverFDA-approved

AMI, acute myocardial infarction; PE, pulmonary embolism; FDA, Food and Drug Administration; DIAS, Desmoteplase in Acute Ischemic Stroke Trial.

Figure 3 FDA-approved uses of rtPAs. (A) Acute ischemic stroke. (B) Pulmonary embolism (PE). (C) Acute myocardial infarction (AMI). [Color figure canbe viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Recombinant tissue plasminogen activators (rtPAs)rtPAs are produced using genetic engineering techniques throughmutations in the DNA sequence of native tPA. These new thera-peutic agents exhibit longer half-lives than native tPA, allowingconvenient bolus dosing, enhanced fibrin specificity, and higherresistance to inactivation by PAI-1. Three tPA analogs areapproved in the United States for use as therapeutic agents inthrombotic disorders including: 1) alteplase, 2) tenecteplase, and3) reteplase. Desmoteplase, a fourth recombinant form of tPA, iscurrently being tested in clinical trials.29,30 In this section, keyfeatures of tPA analogs are summarized.Alteplase (Activase) is synthesized using the complementary

natural cDNA sequence of native tPA. Alteplase is administeredi.v. in patients experiencing AIS, AMI, or PE. Alteplase is admin-istrated in a single i.v. bolus and then in a 3-hour or 90-minute(accelerated) infusion regime. Circulating fibrinogen levelsdecrease about 16% to 36% when 100 mg of alteplase isadministered.Alteplase has a half-life of 4–8 minutes, requiring long infu-

sion times to achieve recanalization of occluded arteries. Theliver mediates clearance of alteplase from the plasma. The mostfrequent adverse reaction to alteplase in all approved indicationsis bleeding.Alteplase has been associated with neurotoxic properties.31

This is because alteplase has been shown to activate matrix metal-loproteinases (MMP), resulting in breakdown of the blood–brainbarrier (BBB) with an increased risk of cerebral hemorrhage andedema. In addition, alteplase has been shown to interact with N-methyl-D-aspartate (NMDA) receptors and elicit calcium excito-toxicty and cell death. The failure of alteplase to achieve rapidreperfusion, the increased risk of cerebral hemorrhage, and itspotential neurotoxicity has led to the development of newerthrombolytic agents, as described below.Reteplase is another recombinant form of tissue plasminogen

activator. Reteplase is composed of the second kringle domainand protease domain of native tPA and is normally used forpatients who experience AMI. It has a longer half-life than alte-plase (13–16 minutes), which makes reteplase easier to adminis-ter than alteplase, allowing a double bolus injection (secondinjection given 30 minutes after the first injection) and thusavoiding the longer infusion times needed for alteplase.Reteplase has been shown to possess similar specificity

towards fibrin but with lower binding affinity than alteplase.This property allows reteplase to penetrate the thrombus moreefficiently and improve the reperfusion time in occluded arteriescompared to alteplase. Clinical trials comparing the efficacy andsafety of both thrombolytics in AMI, however, did not find asignificant difference in mortality rates between either agent.The liver and kidneys mediate reteplase clearance from plasma,another difference with alteplase, which is cleared mainly by theliver. Similarly, the most common adverse effect of reteplase isexcessive bleeding.Tenecteplase was designed by multiple point mutations of the

native tPA DNA sequence resulting in a molecule with longerhalf-life (20–24 minutes compared to 5–10 minutes), enhancedfibrin specificity, and increased resistance to PAI-1 when com-

pared to alteplase. Tenecteplase is approved for the treatment ofAMI. Tenecteplase can be administered in a single i.v. bolus over5 seconds, which was demonstrated to provide similar efficacy toa 90-minute infusion of alteplase.A recent phase IIb randomized controlled trial comparing tenec-

teplase vs. alteplase has shown better reperfusion rates as measuredby magnetic resonance imaging (MRI), as well as better clinicaloutcomes after 24 hours of administration of the drugs, without asignificant difference in intracranial hemorrhage between thegroups. Tenecteplase is cleared from the plasma by the liver.Desmoteplase is a recombinant form of native tPA derived

from a chemical found in the saliva of vampire bats with similarstructure to native tPA. Desmoteplase has a half-life of 4 hoursand higher selectivity for fibrin than alteplase. Due to its highfibrin specificity that avoids systemic activation of plasminogen,and the lack of neurotoxic effects, researchers have sought toreplace alteplase by desmoteplase.Desmoteplase alpha I (DSPA a1) exhibits the most favorable

profile based on preliminary biochemical and pharmacologicalanalysis and therefore has been chosen for most clinical studies.DSPA a1 shares 70% structural homology with native tPA, butthey differ in their proteolytic activities. Currently, a phase IIIclinical trial, the Desmoteplase in Acute Ischemic Stroke Trial(DIAS-4), is being conducted to study the efficacy and safety of asingle i.v. bolus of 90 lg/kg dose of desmoteplase given between3–9 hours after the onset of AIS. Table 2 summarizes the keypharmacological features of rtPAs

CURRENT CLINICAL USES OF rtPAsAcute ischemic stroke (AIS)Stroke remains as the main cause of disability and the third causeof mortality in industrialized nations. Stroke can be divided intoischemic and hemorrhagic. Ischemic stroke accounts for 85% ofcases of stroke while hemorrhagic stroke accounts for 15% of thecases of stroke.32 Ischemic stroke results from a region of thebrain that becomes hypoperfused as a consequence of theobstruction of a vessel with a thrombus or embolus. Diagnosis ofischemic stroke requires computed tomography (CT) that mustbe performed as soon as a stroke is suspected, as shown inFigure 4A. While management of hemorrhagic stroke remainselusive, ischemic stroke can be managed pharmacologically ormechanically.33 One of the drugs that has demonstrated more suc-cess in the management of acute ischemic stroke is alteplase. Alte-plase has been shown to be effective for the treatment of acuteischemic stroke and was approved for this use in the US in 1996.

Clinical trials to assess the optimal therapeutic window for rtPAs inAIS. Traditionally, thrombolytic therapies have been shown to beeffective within the first 3 hours after the onset of stroke symp-toms.34,35 Recent studies, however, have suggested that somertPAs could also be effective up to 4.5 hours after the onset ofthe symptoms.36,37 Furthermore, ongoing clinical trials are study-ing the effectiveness of desmoteplase within 3–9 hours of theonset of the symptoms of AIS.Determining the optimal therapeutic window for adminis-

tration of rtPAs after the onset of AIS results is critical for an

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effective treatment and will provide an opportunity to broadenthe inclusion criteria for rtPAs therapies and thus benefitmore patients previously excluded from rtPAs treatments. Abrief description of some of the clinical trials designed to studythe best therapeutic window for rtPAs therapy is detailedbelow.

The National Institute for Neurological Disorders and StrokeStudy (NIDDS): rtPAs within 3 hours of symptoms onsetOne of the key clinical studies assessing the effectiveness of tPAwithin 3 hours of the onset of stroke was the National Institutefor Neurological Disorders and Stroke study (NINDS). Thisstudy demonstrated early improvement of neurological symptoms

Table 2 Summary of key pharmacological features of rtPAs

Drug name Pharmacokinetics Fibrin selectivity Clinical use/FDA status Study

Alteplase Single bolus followed by90 minutes to 3 hours infusionHalf-life: 4–8 minutesClearance mediated by liver

11 Stroke, AMIPE (FDA-approved)(activase)

NIDDS, ECASS-3, IST-3Wardlaw et al.

Goldhaber et al.

Yamasawa et al.

Dong et al.

GUSTO

Reteplase Double bolus injection(2nd injection given30 minutes after 1st injection)Half-life: 13–16 minutes

Clearance mediated byliver and kidneys

1 AMI (FDA-approved)(retavase)

GUSTO-3

Tenecteplase Single bolus injection given in 5 secondsHalf-life: 20–24 minutesClearance mediated by liver

111 AMI (FDA-approved)(TNKase)

ASENT-2

Desmoteplase Single bolusHalf-life: 4 hours

111111 Stroke(phase III clinical trial)

DIAS-4

AMI, acute myocardial infarction; FDA, Food and Drug Administration; PE, pulmonary embolism; NIDDS, National Institute of Neurological Disorders and Stroke Trial;ECASS, European Cooperative Acute Stroke Study; IST, International Stroke Trial 3; GUSTO, Global Utilization of Streptokinase and Tissue plasminogen activator foroccluded coronary arteries.

Figure 4 (A) CT images of the brain depicting a thrombus formation in the brain vasculature system. The hyperdense image shown at the right repre-sents a clot formation in the M1 segment of the middle cerebral artery (MCA), while the loss of gray-white differentiation in the insular cortex representsan infarct secondary to the thrombus (courtesy of Dr Eble, Dept. of Medical Imaging, University of Arizona). (B) Axial image from a contrast-enhanced CTof the chest demonstrating a saddle embolus in the main pulmonary artery with extension into the lobar arterial branches bilaterally (courtesy of Dr Oliva,Dept. of Medical Imaging, University of Arizona).

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after 24 hours of symptoms onset and improved clinical out-comes after 3 months. The study also showed an increased inci-dence of intracerebral hemorrhage in the tPA group compared tothe placebo group.

European Cooperative Acute Stroke Study III (ECASS-3 trial):evaluation of alteplase within 3–4.5 hours of the onset of AISsymptomsIn the ECASS-3 study, the safety and efficacy of alteplase admin-istered between 3–4.5 hours after the onset of stroke symptomswas evaluated. ECASS III demonstrated an improvement in clini-cal outcomes in a 90-day period in the groups treated with tPAcompared with the placebo group. Safety analysis showed that, inspite of the fact that intracranial hemorrhage was more fre-quently found in patients receiving alteplase compared to patientsreceiving placebo, the overall mortality did not change signifi-cantly between the groups. A meta-analysis by Lansberg et al.36

involving the clinical studies ECASS-1, ECASS-2, and ECASS-3and the Alteplase Thrombolysis for Acute NoninterventionalTherapy in Ischemic Stroke (ATLANTIS), concluded that rtPAstherapy between 3–4.5 hours after the onset of AIS was associ-ated with increased chances of favorable outcomes withoutadversely affecting mortality.

The International Stroke Trial (IST-3): alteplase within 6 hoursof the onset of AIS symptoms onsetThe International Stroke Trial (IST-3) was a multicenter,randomized, open-label study designed to study whether morepatients could benefit from thrombolytic therapy by extendingthe therapeutic window for rtPAs administration beyond 6 hoursafter the onset of the AIS. Patients were allocated to either rtPAor placebo. The primary endpoint of the study was the propor-tion of patients alive and independent after 6 months.The study confirmed the need for an early intervention of

thrombolytic therapy after the onset of stroke symptoms,although it also provided data that some patients might benefitfrom rtPAs administration even 6 hours after the onset of thesymptoms of stroke. The study also provided data that justify theuse of rtPA in patients older than 80 years and did not supportany restriction in the use of rtPA according to stroke severity.The study also encouraged that more controlled clinical trials areneeded to evaluate the efficacy of rtPAs beyond 4.5 hours of theonset of stroke symptoms.

DIAS-4 trial: desmoteplase within 3–9 hours of the onset ofAIS symptomsDIAS-4 is an ongoing randomized, double-blind, parallel groupplacebo-controlled phase III study to determine the efficacy andsafety of desmoteplase vs. placebo within 3–9 hours of the onsetof AIS symptoms. The study started in 2009 and is expected tobe completed by 2015.

SYSTEMATIC REVIEW AND META-ANALYSIS OF rtPAs INAISA recent systematic review and meta-analysis of clinical trialsinvolving i.v. rtPAs vs. placebo was conducted by Wardlaw

et al.38 The meta-analysis included 12 randomized controlled tri-als totaling 7,012 patients with i.v. rtPAs vs. placebo (includingNIDDS, ECASS III, and IST-3) administrated up to 6 hours ofthe development of stroke symptoms. In terms of the optimaltherapeutic window for rtPAs therapy, the meta-analysis con-cluded that rtPAs should be initiated as early as possible, but didnot rule out the possibility that some patients might benefit fromrtPAs therapy even 6 hours after the onset of ischemic stroke.The study reported that interindividual variability could

explain why some individuals might benefit even after 6 hours ofthe onset of stroke while others do not. In addition, the meta-analysis also concluded that the reason that later treatment (after6 hours) could not benefit more patients than early treatment(between 3–6 hours) might be attributed to less benefit in tissueto salvage rather than increased odds of intracranial hemorrhage.

Acute myocardial infarctionAcute myocardial infraction (AMI) is a life-threatening condi-tion that results from oxygen deprivation to the heart tissue(ischemia) that occurs when a coronary artery becomes occluded.AMI produces the death of myocardial tissue affecting cardiacoutput with a drop in blood pressure. The drop in blood pressureincreases sympathetic reflexes leading to vasoconstriction, whichdecreases coronary flow hindering even more cardiac contractil-ity, ultimately leading to cardiogenic shock and death. Therefore,AMI must be treated immediately to save as much tissue as possi-ble before cardiogenic shock can occur.Current approaches in the management of AMI aim at restor-

ing the blood flow to myocardial cells in the shortest time possi-ble. These include minimally invasive procedures such aspercutaneous coronary interventions (PCI), and pharmacologicaltherapies such as rtPAs.39 Due to the increasing importance ofprehospital management of AMI in terms of mortality gain withdecreasing delay time to reperfusion after the onset of the symp-toms, prehospital thrombolysis became a very important thera-peutic tool.Prehospital thrombolysis offers several advantages, such as

immediate access (only 25% of US hospitals provide primaryangioplasty and the delay time to receive PCI treatment cannotexceed 90 minutes), and the possibility to add adjunctive pharma-cotherapy.40 Alteplase, reteplase, and tenecteplase are the throm-bolytics approved for the management of AMI in the US.Tenecteplase has found a place in the management of AMI

during prehospital management, before PCI is available.41 TheASSENT-2 clinical trial demonstrated that tenecteplase was simi-lar to alteplase in terms of mortality rate, with an additionaladvantage in terms of major bleeding and reduced rate of conges-tive failure.41 Additional advantages of tenecteplase over alteplaseinclude its ease of administration (single bolus), its higher affinityto fibrin than alteplase, its 80 times less inhibition by PAI-1 thanalteplase, and that it is not affected by nitrates, as seems to occurwith alteplase.It is important to note that, although PCI has been demon-

strated to be superior to fibrinolysis therapy in reducing mortalityin patients with ST elevation, there seems to be a synergisticeffect when PCI is performed after thrombolytic therapy.

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Goodman and Cantor reviewed the clinical studies comparingearly fibrinolytic therapy with PCI in patients entering the emer-gency room with AMI with ST elevation.42

These studies, which include the “TRANSFER”AMI trial(Trial of Routine Angioplasty and Stenting after Fibrinolysis toEnhance Reperfusion in Acute Myocardial Infarction), haveshown a benefit in administering early fibrinolytic therapy fol-lowed by PCI.42

Pulmonary embolismPulmonary embolism (PE) is one of the manifestations of venousthromboembolism together with thrombophlebitis and deep veinthrombosis. PE can range from asymptomatic findings to life-threatening clinical entities (massive pulmonary embolism) withmortality rates of up to 65%. PE refers to the migration of embolito the lung capillaries leading to pulmonary complications includ-ing a decrease in gas exchange from the lungs to the systemic cir-culation and pulmonary edema, leading to a fall in oxygensaturation in the blood, difficulty breathing, and increase in heartrate.PE increases pulmonary artery pressure affecting the right ven-

tricle. In severe cases, PE leads to a failure of the left ventriclewith cardiogenic shock. Figure 4B shows a CT image of PE ofthe main pulmonary artery and lobar arterial branches.Fibrinolytics have an important role in pulmonary embolism

by dissolving the thrombus and releasing the pressure on the pul-monary arteries, improving cardiac output. Pioneer studies con-ducted by Buneameux and Goldhaber and coworkersdemonstrated the usefulness of intravenous tPA for the manage-ment of this serious condition.43–50

Yamasawa et al.47 compared the effect on oxygen pressure andlung perfusion of tPA vs. heparin alone in 45 patients, demon-strating a clinical benefit of tPA vs. heparin in these parameters.Meneveau49 highlighted the need for thrombolytic therapy as a

first-line treatment in patients presenting pulmonary embolismwith signs of cardiogenic shock.Wan et al.50 reported a meta-analysis of randomized controlled

trials comparing heparin alone vs. thrombolytic therapy. Themeta-analysis showed that no clear benefit of using thrombolytictherapy was observed. However, subgroup analysis showed thatthose patients with massive pulmonary embolism at risk of deathhave a benefit from thrombolytics compared with heparin alone.

NOVEL USES OF rtPAsFrostbiteFrostbite has been defined as the freezing of tissues resultingfrom exposure of intact skin to temperatures below the freezingpoint. This traumatic freezing leads to devastating ischemic-baseddamage to distal extremities, which in severe cases leads to ampu-tation of devitalized tissue.51

The damage caused by frostbite can be divided into two sepa-rate categories: mechanical and ischemic. Mechanical damagerefers to the injury caused by the formation of ice crystals,whereas ischemic damage refers to injury caused by thrombosis.Intravenous rtPAs administration, together with heparin and

aspirin, has been suggested as a pharmacological approach in the

management of frostbite. tPA has been recommended to beadministered as fast as possible after exposure to freezing temper-ature. It is thought that tPA would limit the formation of micro-vascular thrombus, avoiding reperfusion injuries.52

The Wilderness Medical Society (WMS)53 developed one ofthe most detailed guidelines on frostbite management. This isbecause frostbite tends to occur in wild environments and reportsof cases in urban settings is very limited. The strongest level ofclinical evidence provided to date in the use of tPA for frostbiteis detailed below.Twomey et al.54 performed an open label study to assess the

safety and efficacy of rtPA for treating severe frostbite. Patientsincluded in the study had suffered severe frostbite, exhibited noimprovement with rewarming, lacked Doppler pulses in distallimb or digits, and had no perfusion indicated by the use of atechnetium (Tc) 99m three-phase bone scan.Six patients received intra-arterial rtPA, while 13 patients were

treated with intravenous tPA. In addition, all patients of thestudy received i.v. heparin. Patients who did not respond tothrombolytic therapy had more than 24 hours exposure to cold,warm ischemia greater than 6 hours, or multiple freeze-thawcycles. Twomey et al. saw a reduction in digits requiring amputa-tions from a predicted, at-risk number of 174 to only 33 digitsamputated (in 18 patients) after treatment with i.v. rtPA. Theauthors indicated that i.v. rtPA with heparin after rapid rewarm-ing was safe and reduced significantly the amount of predicteddigit amputations.In another open-label study conducted by Cauchy et al.,55

three treatment regimens were randomly assigned to 47 patientswith severe frostbite. The regimens assigned randomly includedeither of the following therapies for 8 days: aspirin plus buflo-medil, aspirin plus prostacyclin, or aspirin plus iloprost alongwith recombinant rtPA for the first day as the additive therapy.Cauchy et al. indicated that adding rtPA should be based on acase-by-case basis, depending on severity level (at least stage 4),presence of trauma such as head trauma, related contraindica-tions, and amount of time since rewarming. Cauchy et al. didnot rule out the possible additive effects of rtPA in selectedpatients.

Submacular hemorrhageA randomized clinical trial to treat submacular hemorrhagecaused by wet macular degeneration, a degenerative disorder ofthe retina, is currently being performed using rtPAs and perfluor-opropane (C3F8). The rationale of using tPA is to help to dis-solve the clot formed during the hemorrhage, while C3F8 is usedto shift the clot away from the central region of the retina (mac-ula) where high resolution vision is achieved.56

Pediatric empyemaEmpyema represents a complication of pneumonia where liquidand pus are accumulated in the pleural cavity. In children, theincidence of this condition has been increasing in the last years,requiring prolonged hospitalization for recovery. In order toshorten the hospitalization times, a new therapeutic modalityinvolving rtPAs is being explored.

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A double-blind, randomized clinical trial using rtPAs andDNAse is ongoing to investigate the effect of rtPAs and DNAsefor the treatment of pediatric empyema. It is thought that tPAwill break down the organized pus while DNAse will decreasethe viscosity of the pus, thus improving the drainage of the liquidentrapped in the pleural space, leading to a faster clinicalrecovery.57

PeritonitisRecent literature supports the potential benefits of tPA in pre-venting abscess formation after surgical treatment of peritonitis.58

A pilot clinical study is being conducted to investigate the safetyof intraperitoneal administration of tPA and DNase includingadverse events such as bleeding and pain, and biochemicalmarkers of inflammation. The study is being conducted inpatients undergoing peritoneal dialysis who suffer fromperitonitis.59

NOVEL THERAPEUTIC STRATEGIES FOR rtPAsIn 1996 the FDA approved the first recombinant form of nativetPA (alteplase) for the treatment of AIS. In the following decade,new generations of rtPAs were developed and commercialized.Despite the wide adoption of rtPAs in the management ofthrombotic disorders, there are still a number of limitations, suchas a short time therapeutic window, a low rate of arterial recanali-zation, a substantial risk of intracranial hemorrhage, and numer-ous contraindications that limit their use to selected patients.60

All these factors have contributed in the last decade to the devel-opment of novel delivery systems to improve the safety and effec-tiveness of thrombolytic therapy.A number of approaches have been explored to improve the

interaction of tPA with fibrin molecules and activate plasmino-gen to plasmin locally without activating plasmin systemically,the major cause of bleeding during thrombolytic therapy. In thefollowing, a brief description of new therapeutic modalities fortPA delivery is described.

Superparamagnetic nanoparticlesSuperparamagnetic nanoparticles are nanoparticles that can bemagnetized when exposed to an external magnetic field andbecome demagnetized when the magnetic field is removed. Mostmagnetic nanoparticles used for medical applications are ironoxides such as ferrite or magnetite, which allow their surfacefunctionalization with different types of biomolecules.61

Magnetic nanoparticles containing tPA have been developedto improve the localization of tPA molecules in the thrombuswhile avoiding the interaction of tPA with healthy tissues,decreasing the life-threatening side effects (hemorrhage) associ-ated with tPA therapy. When an external magnetic field isapplied at the site of the thrombus, the magnetic particles loadedwith tPA are attracted by the magnetic field, improving the pene-tration of tPA molecules into the thrombus. Furthermore, bylocalizing tPA molecules inside the thrombus, it is possible todecrease the dosage of tPA, improving the safety of the therapy.In this regard, Chen et al.62 developed a nanocarrier delivery

system consisting of a superparamagnetic iron oxide core and an

SiO2 shell functionalized with tPA molecules. The nanoparticleplatform (SiO2-MNP) was prepared by the sol-gel method; 94%of rtPAs was attached to the carrier with 86% full retention offibrinolytic activity.A reduction in clot lysis time and penetration of SiO2-MNP

into blood clots were observed and confirmed under magneticguidance from microcomputed tomography analysis. Therefore,by conjugating rtPAs to an SiO2-MNP surface, a new form ofthrombolytic drug therapy with improved efficacy was demon-strated, resulting in a promising therapeutic modality for thetreatment of thrombotic disorders.62

Gelatin-zinc-tPA complexA formulation comprising tissue-type plasminogen activator(tPA), basic gelatin, and zinc ions, has been developed.63 The sys-tem operates reversibly by inhibiting tPA activity in the presenceof Zn ions. Upon application of ultrasound waves in the range of1 MHz, tPA activity is restored. The system represents a first steptowards the realization of tPA switchable drug delivery systems,where the activity of tPA could be switched “on” and “off” in thepresence of ultrasound waves.

Albumin-rtPA nanoparticlesA nanoparticle system made of rtPA and an anionic peptide elec-trostatically bonded to a protamine-albumin complex was devel-oped. The system maintains rtPA as inactive due to sterichindrance produced by albumin molecules avoiding the contactof rtPA with proteases in the plasma. rtPA is then transportedthrough the circulation in inactive form and becomes activatedupon contact with heparin molecules.64

BioMEMSMEMS drug delivery devices (BioMEMS) have emerged as a newmodality for delivery of drugs. The advantages of BioMEMSinclude active control over drug release, local delivery of drugsavoiding potential systemic toxicity, multiple pharmacotherapiesin a single device, and storage of unstable drugs in powder form,opening the possibility of in vivo reconstitution of drugs. Thepossibility of storage of lyophilized drugs and local delivery areattractive features for delivery of rtPAs. For example, since rtPAshave a very short half-life, a locally implantable BioMEMS couldincrease the local concentration of rtPAs molecules at the site ofaction before they become metabolized systemically.Clinical conditions where rtPAs are being used require imme-

diate intervention to prevent serious sequel. Elman et al.described a rapid MEMS drug delivery device for delivery inambulatory emergency care.65 The microdevice consists of threelayers: 1) a sealing membrane layer, 2) a reservoir layer, and 3) anactuation layer consisting of microresistors. The microresistorsheat the liquid contained in the reservoir layer generating bubblesthat jet the drug outside of the reservoirs, bursting the sealingmembranes, and releasing the drug immediately.Finally, as has been demonstrated with MEMS technology, it

can be envisioned that MEMS drug delivery devices with rtPAswill operate by a closed-loop feedback, or by remote activationfrom the hospital, increasing the chances of survival.66

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Ultrasound-based thrombolysisUltrasound-based thrombolysis represents an emerging technol-ogy that is gaining increasing attention for the management ofstroke. It is thought that ultrasound could improve the transportof rtPAs molecules to the thrombus, increasing fibrinolyticactivity.In addition, mechanical effects such as acoustic cavitation have

been postulated as one of the potential mechanisms involved inimproved thrombolysis using ultrasound.

Ultrasound-based thrombolysis device. One of the key limitationsin ultrasound-based thrombolytic therapies is operator depend-ency, which limits the wide adoption of the technology, as well ashindering the development of new clinical trials due to the needof highly trained operators. In order to address this issue, anoperator-independent ultrasound therapeutic device for the treat-ment of stroke in patients receiving rtPA has been developed.67

The ultrasound device includes a multiple transducer transcra-nial head frame comprising broadband transducers placed at thetemporal and the suboccipital regions. Each transducer isdesigned to address a specific arterial segment and only one trans-ducer is operating at a certain time, avoiding exposure levelsabove FDA-mandated ultrasound exposure limits. A computer-controlled ultrasound generator–receiver system energizes thetransducers. A safety trial was conducted with 15 healthy volun-teers and the platform is currently being tested in phase II andIII clinical trials to assess the overall safety and effectiveness ofthis device against current operator-dependent systems.67

Echogenic liposomes for ultrasound-based thrombolysis. A noveldiagnostic-therapeutic modality (“theranostics”) based on echo-genic liposomes has been described by Laing et al.68 The systemcomprises echogenic liposomes that work as both contrast agentsand drug-delivery vectors. The echogenicity of liposomes allowsusing them as contrast agents for molecular imaging during throm-bolytic therapy. The drug-delivery component is based on tPA

molecules entrapped inside the liposomes, with the domains of thetPA molecule that interact with fibrin exposed at the liposome sur-face. When the liposomes reach the fibrin located in the thrombus,the liposomes release the tPA molecules, avoiding broad biodistri-bution of tPA, which leads to unwanted side effects.Based on preclinical studies using the aortic rabbit thrombus

model, as shown in Figure 5, the researchers provided evidencethat echogenic liposomes loaded with rtPA can be a promisingtheranostic modality that allows direct visualization and targetdelivery of thrombolytics, improving the clinical safety and effi-cacy of rtPAs. Table 3 summarizes novel therapeutic strategiesbased on rtPAs.

MONITORING THROMBOLYTIC THERAPY IN STROKEThrombolytic therapy in stroke with rtPAs remains a challengedue to the short therapeutic window and unpredicted outcomes,such as hemorrhagic transformation. MRI and CT represent twocurrent imaging modalities that have been widely adopted toassist in early stroke diagnosis and therapeutic decision-making.69

These techniques provide the first critical distinction betweenischemic and hemorrhagic stroke, thus excluding patients withhemorrhagic stroke from thrombolytic therapy.These techniques, however, suffer from several limitations such

as limited availability, cost, and image resolution problems to dis-criminate between stroke and clinical entities with stroke-likesymptoms such as migraine, epilepsy, and structural brain lesions(CT). In recent years, there has been an intensive search of tech-niques other than neuroimaging that could provide a high speci-ficity and high sensitivity diagnosis of stroke. In addition, strokediagnosis in the prehospital setting, where MRI or CT are notavailable, has prompted the search for alternative techniques.This search has been focused not only on stroke diagnosis, butalso on prognosis and therapeutic monitoring. These techniqueswould eventually allow the physician to perform critical decisionsto improve clinical outcomes to a magnitude not achievabletoday.In this regard, significant efforts have been dedicated to iden-

tify disease biomarkers and biomarkers for patient selection andstratification. Disease biomarkers can provide information aboutstroke progression and stroke severity scales, providing a powerfultool in the differential diagnosis between healthy and strokepatients, and among stroke patients, between patients with ische-mic stroke, and patients with hemorrhagic stroke.Stroke biomarkers will be critical for the selection of patients

who would benefit from rtPAs, or those patients who may sufferfrom hemorrhagic transformation after rtPA therapy has beenimplemented. Diagnosis via biomarkers will allow a more cost-effective, prehospital management of stroke without the need ofneuroimaging techniques, further reducing the time betweenstroke onset and treatment.To date, however, only a few biomarkers have been supported

by statistical analysis, including C-reactive protein (CRP), P-selectin, homocysteine, and gliar fibrillary acid protein (GFAP)(diagnostic biomarkers); and glucose, glutamate, D-dimer, andfibrinogen (prognostic biomarkers). Above all, high glucose levelsat hospital admission have shown to be a stronger predictor of

Figure 5 Schematic of the rabbit aortic thrombus model used to studythrombolytic drugs. An ultrasound probe is introduced near the location ofthe thrombus to induce ultrasound thrombolysis using echogenic lipo-somes loaded with thrombolytic agents (reproduced with permission fromElsevier, ref. 68). [Color figure can be viewed in the online issue, which isavailable at wileyonlinelibrary.com.]

REVIEWS


poor prognosis and hemorrhage transformation after rtPA ther-apy, and is perhaps the biomarker with the highest chance of clin-ical translation.70 For example, a patient who is admitted to theemergency room with high glucose levels might not benefit fromrtPAs therapy. Instead, another thrombolytic modality, such asmechanical thrombectomy, can provide better clinical outcomeswithout detrimental side effects.Biomarkers could not only provide benefits for clinical man-

agement of stroke, but also assist during the drug discovery, pre-clinical and clinical testing of novel thrombolytic drugs.71

Biomarkers used during the drug development process could pro-vide the information required to determine if a potential molecu-lar target plays a role in stroke physiopathology and to determinethe physical interaction of such targets with the drug being inves-tigated. During preclinical development, pharmacokinetic (PK)and pharmacodynamic (PD) biomarkers would define the phar-macokinetic and pharmacodynamic implications of the interac-tion between the candidate drug and its molecular target. Duringclinical research, biomarkers for patient selection and stratifica-tion would avoid several types of bias related to poor patientselection.New imaging modalities could also provide effective monitor-

ing systems during the pharmacological treatment in the clinicalsetting. For example, ultrasound imaging combined withultrasound-induced thrombolysis is a promising therapeuticmodality that allows real-time monitoring of thrombolytic ther-

apy.72 Furthermore, the use of ultrasound imaging combinedwith echogenic delivery systems such as echogenic liposomesloaded with new drug candidates could provide valuable informa-tion to validate novel thrombolytic agents suitable for clinicaltranslation during the drug development process.Finally, genetic biomarkers such as single nucleotide polymor-

phisms (SNPs) could open a new era in thrombolytic therapy bymeans of personalized therapy. The development of an SNP testin patients with stroke could provide a powerful tool to selectthose patients who would benefit from rtPA (patient selectionand stratification biomarkers). In this regard, in a prospectiveclinical study conducted in 497 patients treated with rtPAs,Fernandez-Cadenas et al. identified three SNPs associated withrecanalization rates after rtPA therapy including the interleukin-1 gene (IL1B) and von Willlebrand factor gene (VWF). Accord-ing to the authors, these SNPs could influence rtPA efficacythrough modulation of coagulation factors activity.73

PERSPECTIVERecombinant tissue plasminogen activators (rtPAs) have emergedas a powerful pharmacotherapy for the management of AIS,AMI, and PE. Moreover, an increasing number of clinical studiesare reporting novel potential therapeutic applications for thesedrugs. Thrombolytic therapies, however, suffer from a short ther-apeutic window, serious side effects, and several contraindications.

Table 3 Novel therapeutic strategies for rtPAs therapyTechnology Advantages Limitations Development stage References

Superparamagneticnanoparticles

� Decrease drug dosage� Decrease side effects dueto broad biodistribution

Loss of tPA activity whenimmobilized in nanoparticlesurface� Change on magnetic proper-ties upon particlefunctionalization

Preclinical 61,62

Zn-gelatin complex Switchable system. tPA activ-ity controlled by ultrasound

� Early stage of development� Need clinical validation ofswitchable capacity

Preclinical 63

Albumin-tPA nanoparticles Prodrug. tPA remains inactiveuntil reach the target

� Early stage of development� Potential interference ofplasma proteins

In vitro 64

BioMEMS � Actively controlled� Local release� Batch fabrication� Integration with electronics� In vitro reconstitutionpossible

� Small payload� Surgical procedure forimplantation might be needed

First in human testing 65,66

Ultrasound-basedthrombolysis device

� Improve thrombolysis bymechanical cavitation andimproved transport of tPAmolecules to fibrin� Operator independent� Advanced stage of develop-ment (phase II, phase III clini-cal trials)

� Optimal acoustic exposureparameters not yetdetermined� Variability between subjects

Phase II, III clinical trials 67

Echogenic liposomes � Combined diagnostics andtherapeutics� Lower dose of tPA required

Early stage of development Preclinical 68

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Therefore, there is a critical need to develop new therapeutic strat-egies to improve thrombolytic therapies.New therapeutic modalities such as ultrasound-based delivery,

magnetic nanoparticles, and BioMEMS are promising technolo-gies, although they are at a very early stage of development andwill require further clinical trials to demonstrate their utility.Meanwhile, other interventions based on education of patients,relatives, and the public in early recognition of symptoms of AIS,AMI, and PE, rapid transport to the hospital, and proper triageassessment in the hospital will contribute to improve the clinicaloutcomes for these devastating conditions.

CONCLUSIONThrombolytic diseases are the leading cause of death in the world.Tissue plasminogen activator (tPA), a protein present in theblood and a component of the fibrinolytic system, is responsiblefor degradation of thrombus formed during the coagulation pro-cess, which normally protects against bleeding when a blood ves-sel is damaged. tPA has now been developed synthetically usinggenetic engineering techniques for the management of pathologi-cal conditions including acute ischemic stroke, acute myocardialinfarction, and pulmonary embolism. In these clinical disordersthe coagulation processes bypass the fibrinolytic system capacityto maintain the blood in a liquid state, resulting in thrombus for-mation, vessel obstruction, and, ultimately, ischemia and tissueinfarct. These synthetic forms of tPA known as recombinant tis-sue plasminogen activators or rtPAs have improved some of thefeatures of native tPA, including increase in the half-life andresistance to plasma inhibitors, leading to improved therapeuticregimes. Novel clinical applications of rtPAs are being exploredincluding frostbite, submacular hemorrhage, and pediatric empy-ema, while novel therapeutic technologies such as ultrasound,nanoparticles. and BioMEMS are being investigated. Therefore,it is foreseen that in the near future rtPAs will positively affect anumber of clinical conditions, if their safety issues can be over-come and their benefits fully exploited.

ACKNOWLEDGMENTSThe authors thank T. Hunter, J. Eble, and I. Oliva for contributions withthe CT images. This research work was supported by the US ArmyResearch Office via the Institute for Soldier Nanotechnologies (ISN) atMIT (contract: W911NF-07-D-0004).

CONFLICT OF INTERESTThe authors declared no conflict of interest.

VC 2015 American Society for Clinical Pharmacology and Therapeutics

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