WO2002089828A2

WO2002089828A2 - Interferons in the treatment of ischemia

Info

Publication number: WO2002089828A2
Application number: PCT/EP2002/003758
Authority: WO
Inventors: Yin Hwee Tan; Hubertus Schellekens
Original assignee: Institute Of Molecular And Cell Biology
Priority date: 2001-05-04
Filing date: 2002-04-04
Publication date: 2002-11-14
Also published as: WO2002089828A3; AU2002310922A1

Abstract

Ischemia may be prevented or treated by administering an interferon such as human β-interferon. The β-interferon may be a glycosylated β-interferon. The ischemia may be cerebral or myocardial ischemia. In this way, the condition of a patient suffering from ischemia may be improved.

Description

TREATMENT OF ISCHEMIA

Field of the Invention

This invention relates to the prophylaxis or treatment of ischemia.

Background to the Invention

Ischemia relates to reduced blood flow to a particular part of the body. It may be transient or prolonged and can lead to cell death in the affected area. The two most common types of ischemia are those that affect the brain or heart. Cerebral ischemia relates to a lack of nutrition in the brain resulting from insufficient cerebral blood flow. Some individuals may suffer transient ischemic attacks, which are often forerunners of stroke. Stroke is one of the leading causes of death worldwide, and is usually caused by ischemia rather than haemorrhage. Myocardial ischemia occurs when the heart muscle does not receive an adequate blood supply and is often a forerunner of myocardial infarction (heart attack). Ischemia can also occur in various other organs, such as the kidneys and the colon.

There are three main classes of human interferons, called alpha, beta and gamma interferons. There are at least 15 subtypes of alpha interferon (Henco K. et al, J. Mol. Biol. 185_* 227-260, 1985) and only one each of the beta and gamma interferons. Interferon-α is used in. the treatment of chronic viral infections. Among these hepatitis C virus (HCV) and hepatitis B virus (HBV) infections are the most important, and interferon-α is a standard treatment in HCV and HBV infections (Strannogard O. ejiFCC 3, 1999, www.ifce.ora).

Interferon-β is a broad spectrum anti viral. It also has a proven beneficial influence on the course of patients with the neuro-degenerative disease multiple sclerosis (MS), and has become a standard treatment of this disease (The Interferon β Multiple Sclerosis Study Group, Neurology 43, 655-661, 1993). The mechanisms behind the beneficial effects of mterferon-β in MS are not known. Gamma interferon is used in the treatment of diseases associated with defective cell-mediated immunity. In particular, gamma interferon has been approved for use in the treatment of clironic gra ulomatous disease, and good results have been obtained in severe mycobacterial infections and mycoses (Strannogard O._» ejiFCC. 3, 1999, www.ifc.org).

Summary of the Invention According to the present invention, there is provided the use of an interferon in the manufacture of a medicament for use in the prophylaxis or treatment of ischemia. The invention also provides: a method for the treatment of a patient who has suffered or is suffering from ischemia, which method comprises the step of administering to the patient a therapeutical iy effective amount of an interferon; an agent for treating ischemia comprising an interferon; an interferon for use in the treatment of ischemia; and products containing an interferon and a thrombolytic agent as a combined preparation for simultaneous, separate or sequential use in the treatment of ischemia.

Description of the Figures

Figure 1 shows a typical view of a tin-tagged clot (blood clot emulsified with tin to enhance visibility) embedded in the middle cerebral artery.

Figure 2 illustrates the post-surgery semi-paralytic condition of rabbits chosen for further analysis.

Figure 3 shows a representative brain slice following TTC staining, wherein the infarct region appears pale against the negatively TTC-stained tissue background.

Detailed Description of the Invention Interferons

The interferon for use in the present invention is an α, β or γ interferon, typically a human interferon, in particular the human interferon-β The term "interferon" includes fragments which have interferon activity and mutant forms of an interferon which retain interferon activity, For example, the sequence of an interferon α or β may have been modified to enhance activity or stability as reported in US-A-55S2824, US-A-5593667 or US-A-5594107. The interferon may have been purified from natural sources or may be a recombinant interferon. The human interferon β typically has a specific activity of from 4x 1 ( to

SxlO⁸, preferably from 4.8 10^s to 6.4x10^s IU per mg protein. Interferon α and interferon β specific activities are determined according to reference standards MRC 69/19 and Gb-23-902-531 respectively. Specific activity is determined according to a modification of the method of Armstrong, Applied Microbiology 21 , 723, 1971, in which 0.2 μg/ml of actinomycin D is included in the viral challenge and the viral induced cytopathic effect is read directly.

The interferon is preferably obtainable by the methodology of WO 96/30531. The interferon is thus obtainable by a process comprising eulturing mammalian cells trans fected with a nucleic acid vector comprising: (i) a coding sequence which encodes the interferon and which is operably linked to a promoter capable of directing expression of the coding sequence in mammalian ceils in the presence of a heavy metal ion; (ii) a first selectable marker sequence which comprises a metallothioncin gene and which is operably linked to a promoter capable of directing expression of the metallolhionem gene in the cells in the presence of a heavy metal ion; and

(iii) a second selectable marker sequence which comprises a two gene and which is operably linked to a promoter capable of directing expression of the neo gene in the cells; under conditions that allow expression of the coding sequence; and recovering the interferon thus produced.

The transfected mammalian cells may be cells of a human or animal cell line. They maybe BHK, COS, Vero, human fibroblasloid such as CIO, HeLa, or human lymphoblastoid cells or cells of a human tumour cell line. Preferably, however, the ceils are CHO cells, particularly wild-type CHO cells. Desirably, transfected cells will have all or part of such a vector integrated into their genomes. Such cells are preferred because they give stable expression of the coding sequence contained in the vector. Preferably, one or more copies of the entire vector will be integrated, with cells having multiple integrated copies of the vector, for example from 20 to 100 copies or more, being particularly preferred because these cells give a high stable level of expression of the coding sequence contained in the vector.

However, cells having less than complete sections of the vectors integrated into their genomes can be employed if they are functionally equivalent to cells having the entire vector integrated into their genomes, in the sense that the integrated sections of the vector enable the cell to express the coding sequence and to be selected for by the use of heavy metals. Thus, cells exhibiting partial integration of a vector may be employed if the integrated element or elements include the coding sequence operably linked to its associated promoter and the metallothionein marker sequence operably linked to its associated promoter, Any promoter capable of enhancing expression in a mammalian cell in the presence of a heavy metal ion such as Cd²⁺, Cu ⁺ and Zn^2** may be operably linked to the interferon coding sequence. A suitable promoter is a metallothionein gene promoter. The mouse metallot onem gene I (mMTl) promoter is preferred,

Suitable promoter/enhancer combinations for the coding sequence include the mMTl promoter flanked upstream with a mouse sarcoma virus (MSV) enhancer (MS V-mMTl) and a Rons sarcoma virus ( SV) enhancer upstream of a mouse mammary tumour vims (MMTV) promoter. MSV-mMTl is preferred.

As far as the first selectable marker sequence is concerned, any promoter capable of enhancing expression in a mammalian cell in the presence of a heavy metal ion such as Cd²⁺, Cu^2K and Zn^2"1 may be operably linked to the metallothionein gene such as a human metallothionein gene. Preferably, the marker sequence gene is a human metallothionein gene, such as die human metallothionein gene DLA, which has its own promoter.

The second selectable marker sequence is a neo gene. More than one type of this gene exists in nature: any specific neo gene can be used in a vector of the invention. One preferred neo gene is the E.coU neo gene.

The promoter for the neo gene is capable of directing expression of the gene in a mammalian cell. Suitable promoters are the cytomegalovirus (CMV) early promoter, the SV40 promoter, the mouse mammary tumour virus promoter, the human elongation factor 1 α-P promoter (EF-lα-P), the SRα promoter and a metallothionein gene promoter such as mMTl . The promoter may also be capable of expressing the neo gene in bacteria such as E.colt in which a vector may be constructed,

The interferon coding sequence (i) and the marker sequences (ii) and (iii) are thus each operably linked to a promoter capable of directing expression of the relevant sequence. The term "operably linked" refers to a juxtaposition wherein the promoter and the coding/marker sequence are in a relationship permitting the coding/marker sequence to be expressed under the control of the promoter, Thus, there may be elements such as 5* non-coding sequence between the promoter and coding/marker sequence. Such sequences can be included in the construct if they enhance or do not impair the correct control of the coding/marker sequence by the promoter.

The vector may be a DNA or RNA vector, preferably a DNA vector. Typically, the vector is a plasmid. Each of the sequences (i) to (iii) will typically be associated with other elements that control their expression. In relation to each sequence, the following elements are generally present, usually in a 5' to 3' arrangement: a promoter for directing expression of the sequence and optionally a regulator of the promoter, a translational start codon, the coding/marker sequence, a polyadenylation signal and a transcriptional terminator. Further, the vector typically comprises one or more origins of replication, for example a bacterial origin of replication, such as the pBR322 origin, that allows replication in. bacterial cells. Alternatively or additionally, one or more eukaryotic origins of replication may be included in the vector so that replication is possible in, for example yeast cells and/or mammalian cells. The vector may also comprise one or more introns or other non-coding sequences 3' or 5* to the coding sequence or to one or more of the marker sequences. Such non-coding sequences may be derived from any organism, or may be synthetic in nature. Thus, they may have any sequence, Such sequences may be included if they enhance or do not impair correct expression of the coding sequence or marker sequences.

The transfected cells are typically cultured in the presence of a heavy metal ion selected from Cd^21", Cu^2"" and Zn²⁺, particularly in an amount which is not toxic to the cells. That can lead to higher expression of the desired interferon. The concentration of the heavy metal ion in the culture medium is typically from 100 to 200 μM. Cells may therefore be cultured in the presence of from 100 to 200 μM of a heavy metal ion selected from Cd^2÷, Cu^2" and Zn ^h, for example from 130 to 170 μM of the heavy metal ion. A useful concentration is about 150 μM, particularly when the heavy metal ion is Zn^2i".

The interferon that is produced may be recovered by any suitable means and the method of recovery may vary depending on, for example, the type of cells employed and the culture conditions thai have been used. Desirably, the interferon produced will be purified after recovery. Substantially pure interferon can thus be obtained.

The human β-interferon provided by WO 96/30531 has a high degree of sialylation. Like natural human β-interferon produced by primary diploid human fibroblasts, it is well glycosylated. However, it has a higher bioavailability than the natural β-interferon or recombinant β-interferon produced in E.coH (betaseron).

The higher bioavailability of the β-interferon can be characterised. When 1.5 x 10⁶ IU of the interferon is injected subcutaneously into the back of a rabbit of about 2 kg: (a) ≥ 128 IU/ml of Ore interferon is detectable m the serum of the rabbit after 1 hour, and/or (b) ≥ 64 IU/ml of the interferon is detectable in the serum of the rabbit after 5 hours.

The maximum level of interferon is typically observed after 1 hour. According to (a), therefore, 128 to 256 IU/ml such as 140 to 190 IU/ml of the interferon may be detectable in the rabbit serum after 1 hour. After 5 hours according to (b), ≥ 70 IU/ml such as ≥ 80 IU/ml of the interferon may be detectable in the rabbit serum. Typically according to (b), an amount of interferon in the range of 64 to

128 IU/ml such as 80 to 110 IU/ml can be detected.

Additionally or alternatively, the human interferon β can be characterised by its specific activity. It can have a specific activity from 4.8 x 10^s to 6.4 x 10⁸ IU per mg equivalent of bovine serum albumin protein, as noted above. The specific activity may be from 5 x 10⁸ to 6 x 10^s, for example from 5.2 x 10^s to 5.8 x 10⁸ such as from 5.3 x 10^s to 5.5 x 1 ^s, IU per mg equivalent of bovine serum albumin protein, The human interferon β may also be characterised by one or more of the following properties:

1. The interferon β typically has an apparent molecular weight of 26,300 as determined by 15% sodium dodeeyi sulphate-polyaerylamide gel electrophoresis (SDS-PAGE). 2. When injected as a neat intravenous bolus into a rabbit, the half life of the interferon is typically in the range of from 12 to 15 in such as about 1354 min. The bolus is injected into the rabbit ear vein and blood samples are withdrawn from the rabbit ear artery. Rabbit serum is assayed for the antiviral activity of the interferon according to the modification of the method of Armstrong (1971).

3, The antiviral activity of the interferon in a human hepatoblastoma cell line (HepG2) is at least equal to and, typically, about 1.5 times the activity of natural interferon β from primary diploid human fibroblast cells. The interferon is also about 2.2 times more effective than bctaseron in protecting Hep2 cells against a viral challenge. Antiviral activity is again determined according to the modified method of Armstrong (1971). Actinomycin D was omitted in the antiviral determination in HepG2 cells. The oligosaccharides associated with the interferon β of the invention may also characterise the interferon β. The interferon β carries oligosaccharides which can be characterised by one or more of the following features: 1. Neutral (no acidic substituents): 5 to 15%, preferably about 10% or lower.

Acidic : 95 to 85%, preferably about 90% or higher.

2. The total desiaϊylated oligosacchari.de pool is heterogeneous with at least six distinct structural components present in the pool.

3. Matri -Assisted Laser Desorption lonϊsation - Time of Flight (MALDI-TOF) mass spectrometry and high resolution gel permeation chromatography data are summarised as follows:

The carbohydrate moiety of the human interferon β of WO 96/30531 consists of bi-₅ tri- and tetra-antennary complex type N-l inked oligosaccharides. These oligosaccharides contain repeating lactosamine(s). About 20 to 50%, for example 20 to 30%, 30 to 40% or 35 to 50%, of the oligosaccharides are bi-antennary oligosaccharides. About 30 to 65%, for example from 40 to 60% or 50 to 60%, of the oligosaccharides are tri-antennary oligosaccharides. About 2 to 15%, for example from 2 to 8%, 4 to 10% or 5 to 15%, of the oligosaccharides are tetra- antennary oligosaccharides. Percentages are calculated by weight of total anaiysable oligosaccharide content.

Therapeutic uses The interferons are used to treat ischemia, particularly in humans. One or more interferons may be used to improve the condition of a patient who has suffered, is suffering or it at risk of suffering from ischemia, In particular, an interferon may be used in the treatment of cerebral or myocardial ischemia.

An interferon may be used to treat a transient cerebral ischemic attack, cerebral ischemia or a stoke or cerebral infarction. For example, one or more interferons may be used in the treatment of thromboembolic stroke. Cerebral ischemia relates to a lack of nutrition in the brain resulting from insufficient cerebral blood flow (CBF). The consequences of cerebral ischemia depend on the degree and duration of reduced CBF. Normal CBF is 50~55ml/100gm/mtn. If the CBF drops below 18, electrical activity ceases. If the CBF drops below 12, neuronal metabolism stops. The range of CBF between 12 and 18 has been called the "ischemic penumbra" because the neuronal damage is mild and reversible if flow is restored within a few hours. Clinically, a window of opportunity is available to intervene therapeutically and to prevent the ischemic brain tissue from going on to infarction. According to the invention, therefore, the interferon will be administered typically when the CBF is below about 55ml/100gm/mm, for example below 18. The interferon is preferably administered to a patient when their CBF is 10 or above, more preferably when the CBF is 12 or above.

An interferon may also be used to treat myocardial ischemia, myocardial reperfusion injury or acute myocardial infarction. Myocardial ischemia occurs when tlie heart muscle does not receive an adequate blood supply and is thus deprived of necessary levels of oxygen and nutrients. The most common cause of myocardial ischemia is atherosclerosis, causing blockages in the blood vessels (coronary arteries) that provide blood flow to the heart muscle. The ischemia may be attributable to low cardiac output, angina, clot formation or an arterial spasm. Acute myocardial infarction has a pathological progression that bears resemblance to that of cerebral infarction. An initial loss of blood supply results in reversible cellular damage to myocytes. However, a major mode of cellular insult occurs during reperfusion. Although a necessary event for the preservation of reversibly injured myocardium, reperfusion is associated with additional injury.

The interferon may be administered to humans in various manners such as orally, intracranially, intravenously, intramuscularly, intraperitoneally, intranasally, intrademally, and subcutaneously. The particular mode of administration and dosage regimen will be selected by the attending physician, taking into account a number of factors including the age, weight and condition of the patient.

The pharmaceutical compositions that contain the interferon as an active principal will normally be formulated with an appropriate pharmaceutically acceptable carrier or diluent depending upon the particular mode of administration being used. For instance, parenteral formulations are usually mjectable fluids that use pharmaceutically and physiologically acceptable fluids such as physiological saline, balanced sail solutions, or the like as a vehicle. Oral formulations, on the other hand, may be solids, e.g. tablets or capsules, or liquid solutions or suspensions. The amount of interferon that is given to a patient will depend upon a variety of factors including the condition being treated, the nature of the patient under treatment and the severity of the condition under treatment. Typically, a patient will be given a dose of 0.01 to 50 million IU of an interferon, preferably 0.1 to 20 million IU, more preferably 0.5 to 10 million IU. The timing of administration of the interferon should be determined by medical personnel, depending on whether the use is prophylactic or to treat ischemia. Generally administration should occur as soon as possible after an ischemic episode has been detected or is suspected of having taken place, for example within 12 hours of such an episode. The interferon is preferably administered within 6 hours of an ischemic event or episode, more preferably within 3 hours of the onset of ischemia. An initial administration may be followed by repeated dosages of 0. 1 to 50 million IU, preferably 0,1 to 20 million IU, more preferably 0.5 to 10 million IU of the interferon every 12 to 24 hours for up to 7, 14, 30 or 45 days.

The interferon may be given alone or in combination with a tliromboiytic treatment, depending on the clinical situation. For example, the interferon may be administered in combination with tissue plasminogen activator (tPA). The interferon and the tliromboiytic agent may be administered simultaneously, separately or sequentially. For example, the interferon and the tliromboiytic agent may be administered within 24 hours, within 12 hours, within 6 hours or within 1 hour of each other. The interferon and the thrombolytic agent may also be administered in a combined preparation. The following Example illustrates the invention:

Example

1. Materials and Methods

Surgical Procedure

New Zealand White rabbits weighing 2.5-3.2 kg were employed in this study, Cerebral ischemia was induced with a protocol adapted from published work (Lew S.M et al., Brain Res. Bull. 48, 325-331, 1999) with some modifications. Rabbits were tranquillized with Hypnorm* (0.2ml/kg). A 1 cm³ autologous blood sample was drawn from the ear vein for clot preparation. It was mixed well with 50mg tin powder (Sigma, US) and slowly injected into a polyethylene (PE-90) tubing previously flushed with tlirombin (1000 U/ml, Sigma, USA). The subsequent clot was cut into a 1.5 cm segment for embolization. Anaesthesia was maintained by intravenous injection with a mixture containing equal parts of ketamine (lOOmg/ml) and diazepara (10mg/2ml, F. Hoffman-La Roche Ltd, Basel). Repeated small boluses were administered throughout the experiment as needed in response to evidence of discomfort. Once adequate anaesthesia was established, a midline neck incision was made and the common (CCA), internal (ICA) and external carotid arteries (ECA) were isolated. Temporary aneurysm clips were placed on the CCA, and proximal ECA and ICA. An arteriotomy was then made in the CCA distal to the CCA aneurysm clip, and the artery cannulated with the PE tubing containing a 1.5cm-long segment of the autologous clot. The clot-containing tubing was then advanced to the origin of the ICA. The ICA clip was then momentarily opened, and the clot embolus was injected into the ICA under direct microscopic visualization. The temporary clip was then reapplied. The PE-90 catheter was removed and the arteriotomy immediately repaired with interrupted 10-0 nylon sutures (B.Braun, Germany), after which the ECA, ICA and CCA clips were sequentially removed.

Glycρferpn^te Treatment and Neurological Assessment

Glycoferon^® was administered in treated animals according to the following regime. "Pre~treatment" rabbits received 1X10⁷ IU Glycoferon* subcutaneously 4 h before clot placement and 0.5X10⁷ IU Glycoferon^'® within 30 minutes after. "Post- treatment" rabbits received 1X10⁷ IU Glycoferon* subcutaneously immediately after clot placement and 0,5X10⁷ IU Glycoferon*4h later. Glycoferon^® is a glycosylated human β interferon prepared according to Example 4 of WO96/30531. Control rabbits were similarly operated on with clot placement but received nothing. The subcutaneous mode of administration was used as this allowed much better sustenance of serum Glycoferon^® levels. Rabbits with an obvious neurological deficit (semi-paralysis or circling motion) that persisted after 24 h were used for further analysis.

Analysis of infarct volume

Rabbits were euthanized after 24 h and the brains harvested. The position of the clot was noted. Brains were cut into 2 mm slices and incubated with 1.5% buffered triphenyltetrazolium chloride (TTC) to delineate the infarct region.

Photographs of the slices were taken using a digital camera (Olympus Optics Co.) and the infarct volume was calculated with appropriate compensations using the

Micro Image Lite 4.0 image analysis software. 2, Results

A rabbit model for thromboembolic stroke

In deciding on a model animal we took into consideration our finding that Glycoferon*¹ has a distinct antiviral effect in rabbit kidney R 13 cells, an indication that the rabbit type I interferon receptor could recognize the human ligand to elicit downstream responses. Rodent models were ruled out on the basis that Glycoferon^** did not elicit a significant antiviral response in rodent cell lines.

Induction of thromboembolic stroke by the introduction of an autologous blood clot into the middle cerebral artery provided a moderately reproducible model (Figure 1). Animals that satisfied all four criteria below are included in the final analysis. These are: 1) clear neurological defects after surgery; 2) survival after 24 hours; 3) clot discemable in middle cerebral artery; and 4) a clear subcortical infarct as revealed by TTC staining. About 30% of rabbits operated upon survived after 24h. A clear neurological defect persisted in about 50% of these (Figure 2). A representative brain slice following TTC staining is shown in Figure 3.

Glycoferon treatment reduces the infarct volume

Glycoferon*^* treated rabbits had consistently lower infarct volume compared to untreated rabbits, The average infarct volume of pre-treated rabbits was 46.3 +/- 9.3 mm³ (n=4), and that of post-treatment rabbits was 40.0 +/- 23.1 mm³ (n=4). Both are significantly lower (ρ=0.003 and p=0.004, respectively) than the average infarct volume of control (untreated) rabbits (121.6 +/- 32.9 mm³, n=5) as assessed using the student's T-test. The results are summarised in Table 1.

Table 1. A summary of the infarct volumes of control, pre-treatment and post-treatment rabbits that satisfied all primary assessment criteria and are included in the final analysis. Statistical outliers were excluded based on Chauvenet's criteria.

Human interferon-β has a 10-fold lower efficacy in terms of antiviral effect in rabbit cells as compared to human cells and tliere is a certain time period required to achieve maximum serum Glycoferon* concentration upon subcutaneous administration. In both the pre- and post-treatment regimes, Glyeo feron ^f* treatment confers a beneficial outcome to stroke, manifested in this case by the reduction of infarct volume. The mere fact that the beneficial effect of Glycoferon^® is already obvious by direct measurement of infarct volume is encouraging. The beneficial effect of Glycoferon*^'' is expected to become even more significant In more refined assessments, including alternative models of transient, global ischemia and detailed neurological examination of animals recovering from stroke. Furthermore, the effect of Glycoferon⁶ would be expected to be even greater when used in human subjects, because human type I interferon receptors have a higher affinity for the ligand.

Interferon-β has been shown to be a relatively safe and non-toxic therapeutic agent in aπti- viral treatment and MS trials.

Claims

1. Use of an interferon in the manufacture of a medicament for use in the prophylaxis or treatment of ischemia.

2. Use according to claim 1 , wherein the interferon is a human interferon,

3. Use according to claim I or 2, wherein the interferon is human β- interferon,

4. Use according to any of the preceding claims, wherein the interferon is a glycosylated human β-interferon,

5. Use according to any of the preceding claims, wherein the interferon is used in combination with tissue plasrainogen activator (tPA).

6. Use according to any of the preceding claims, wherein the ischemia is cerebral ischemia.

7. Use according to claim 6, wherein the cerebral ischemia is associated with stroke or cerebral infarction.

8. Use according to claim 1, wherein the stroke is a thromboembolic stroke.

9, Use according to any of claims 1 to 5, wherein the ischemia is myocardial ischemia.

10. Use according to claim 9, wherein the myocardial ischemia is associated with myocardial reperfusion injury or acute myocardial infarction.

11. A method for the treatment of a patient who has suffered, is suffering from or is at risk of suffering from ischemia, which method comprises the step of administering to the patient a therapeutically effective amount of an interferon.

12. An agent for preventing or treating ischemia comprising an interferon,

13. An interferon for use in the prophylaxis or treatment o ischemia,

14. Products containing an interferon and a thrombolytic agent as a combined preparation for simultaneous, separate or sequential use in the prophylaxis or treatment of ischemia,

15, A product according to claim 14, wherein the tliromboiytic agent is tissue plasminogen activator (tPA).