Novel applications for tissue plasminogen activators

Abstract

Provided is a pharmaceutical composition with which it is possible to prevent the development of late-onset cerebral ischemia and obtain a prognosis-improving effect by performing active hematoma removal using a tissue plasminogen activator. This pharmaceutical composition contains a tissue plasminogen activator as an active ingredient and is for use in hematoma removal therapy in the cerebral cistern to remove a dislodged hematoma by suction by using the tissue plasminogen activator in an aqueous solution state as a cleansing agent, said therapy being performed at the same time as treatment of a cerebral aneurysm by means of craniotomy with clipping during the acute phase of a subarachnoid hemorrhage associated with a ruptured cerebral aneurysm.

Description

Technical Field

[0001] The present invention relates to a novel use of tissue plasminogen activator.

Background Art

[0002] Eighty-five percent of the causes of subarachnoid hemorrhage are ruptured cerebral aneurysms. Approximately 20% of subarachnoid hemorrhage cases die suddenly at an early stage during onset, and the total mortality rate reaches 25 - 50% including subsequent deaths. The mortality rate due to subarachnoid hemorrhage accounts for only 4.4% of all stroke deaths, but it occurs in a younger generation compared to other strokes. In particular, more than 25% of stroke death patients under 65 years old are due to subarachnoid hemorrhage.

[0003] As factors that deteriorate function and life prognosis in subarachnoid hemorrhage, there are cardiovascular events such as neurogenic pulmonary edema and heart failure associated with a rapid increase in intracranial pressure in the ultra-early stage, re-rupture, primary brain injury associated with the hemorrhage itself, early brain injury due to increased intracranial pressure and subarachnoid hematoma, and delayed cerebral ischemia disorders represented by cerebral vasospasm that subsequently occurs. In addition, from the subacute stage to the chronic stage, there is dysfunction due to hydrocephalus after subarachnoid hemorrhage.

[0004] As acute treatment for ruptured cerebral aneurysms that cause subarachnoid hemorrhage, first, there is surgical clipping or endovascular coil embolization to prevent re-rupture, and then intensive perioperative management is performed medically to prevent delayed cerebral ischemia disorder.

[0005] The incidence of delayed cerebral ischemia disorder has been reported to be 5.5 - 20% even limited to symptomatic cases presenting some neurological symptoms, and it occurs in the majority including those detected only by image diagnosis and asymptomatic ones (Non-Patent Documents 1 - 4). It has been shown by multiple animal experiments that the cause of cerebral vasospasm is due to the hematoma leaked into the subarachnoid space.

[0006] Based on this, there were reports in the 1980s and 1990s of hematoma removal following craniotomy clipping, but they did not show a remarkable improvement in prognosis (Non-Patent Literature 5). Furthermore, the results of the large-scale ISAT study (Non-Patent Literature 6) also showed no superiority of craniotomy clipping, which removes the hematoma, compared to coil embolization, which does not remove the hematoma. As a result, direct hematoma removal in the acute phase is no longer recognized as a valid treatment strategy, and currently, the prevention of cerebral vasospasm through drug therapy is the main approach. The following are possible reasons why conventional techniques for hematoma removal were ineffective: The cisterns are not a single space, but are divided into several compartments by the arachnoid membrane, while the hematoma in subarachnoid hemorrhage is widely distributed throughout these compartments. Furthermore, since subarachnoid hematoma is a blood clot and does not circulate as well as normal cerebrospinal fluid, the problem with direct hematoma removal is that removing the hematoma, which is firmly adhered to the brain surface, simultaneously damages the pia mater on the brain surface.

[0007] Furthermore, in a nationwide survey conducted in Japan in 2022 comparing craniotomy clipping and coil embolization (Non-Patent Literature 7), univariate analysis showed that craniotomy clipping was significantly associated with a better prognosis than coil embolization. While multivariate analysis did not show a statistically significant difference, the adjusted odds ratio was 0.82 (95% CI 0.63-1.07), suggesting the superiority of craniotomy clipping.

[0008] On the other hand, tissue plasminogen activator (hereinafter sometimes referred to as "t-PA") is a bioactive substance in the body that converts plasminogen to plasmin on a thrombus. In 1981, Collen succeeded in purifying human t-PA, and the following year, in 1982, Genentech Corporation in the United States succeeded in mass-producing high-purity human recombinant t-PA using genetic recombination. In 1987, it was approved and marketed in the United States as a thrombolytic agent for acute myocardial infarction. In Japan, Kyowa Hakko Kogyo Co., Ltd. (now Kyowa Kirin Co., Ltd.) and Mitsubishi Chemical Corporation (now Tanabe Mitsubishi Pharma Corporation) began clinical trials in 1986 and obtained approval and began marketing in March 1991.

[0009] There have been some past reports on the use of t-PA or urokinase with similar thrombolytic effects for the removal of cisternary hematomas in patients with subarachnoid hemorrhage. However, the method of spraying t-PA into the cistern during surgery after clipping (Non-Patent Documents 8-10) is basically a method that relies on spraying to dissolve the hematoma afterward, and does not involve situationally releasing the hematoma from the surrounding tissue through the action of t-PA and then washing and aspirating it. Furthermore, there are reports that washing and aspirating hematomas with urokinase dissolved in physiological saline during surgery is useful in preventing delayed cerebral vasospasm (Non-Patent Document 11), but to date there have been no reports of washing and aspirating target hematomas with a washing solution in which t-PA is dissolved in physiological saline or artificial cerebrospinal fluid (ArtCeleb®). [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Brami J, Chousterman B, Boulouis G, Dorze ML, Majlath M, Saint-Maurice JP, Civelli V, Froelich S, Houdart E, Labeyrie MA. Delayed Cerebral Infarction is Systematically Associated with a Cerebral Vasospasm of Large Intracranial Arteries. Neurosurgery. 2020 Feb 1;86(2):E175-E183. doi: 10.1093 / neuros / nyz340. PMID: 31501886. [Non-Patent Document 2] Inagawa T, Yahara K, Ohbayashi N. Risk factors associated with cerebral vasospasm following aneurysmal subarachnoid hemorrhage. Neurol Med Chir (Tokyo). 2014 Jun 17;54(6):465-73. doi: 10.2176 / nmc.oa.2013-0169. Epub 2014 Mar 27. PMID: 24670311; PMCID: PMC4533446.

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 10

Non-Patent Document 11

[0011] In a nationwide survey comparing craniotomy clipping and coil embolization (Non-Patent Literature 7), the inventors believe that the superiority of craniotomy clipping was due to hematoma removal, which is only possible during craniotomy, improving the prognosis. Given this background, while it is expected that simple hematoma removal will not have a significant or minimal effect on improving the prognosis of subarachnoid hemorrhage cases by preventing delayed cerebral ischemic injury, the inventors believe that if an aggressive hematoma removal therapy within the cisternae can be established, using tissue plasminogen activator as a washing agent to release hematomas adhering to the cisternae, and then aspirating and removing the released hematomas, an improvement in prognosis can be expected.

[0012] The present invention aims to provide a pharmaceutical composition that can prevent the occurrence of delayed cerebral ischemic injury and improve prognosis by performing aggressive hematoma removal using a tissue plasminogen activator. [Means for solving the problem]

[0013] The gist of this invention is as follows: (1) A pharmaceutical composition containing tissue plasminogen activator as an active ingredient, which is used for the hematoma removal therapy in the cerebral cavity. During the acute stage of subarachnoid hemorrhage associated with ruptured cerebral aneurysm, at the same time as treating the cerebral aneurysm by craniotomy clipping, the tissue plasminogen activator is used in the state of an aqueous solution as a cleaning agent to aspirate and remove the free hematoma. (2) The pharmaceutical composition according to (1) above, wherein the tissue plasminogen activator is dissolved in physiological saline or artificial cerebrospinal fluid and used as a cleaning solution. (3) The pharmaceutical composition according to (1) above, which is used within 72 hours after the onset of subarachnoid hemorrhage. (4) The pharmaceutical composition according to (1) above, wherein the hematoma removal therapy includes pressurized washing in the cerebral cavity. (5) The pharmaceutical composition according to (1) above, wherein the aqueous solution contains tissue plasminogen activator at a concentration in the range of 5,000 - 20,000 IU / ml. (6) The pharmaceutical composition according to (2) above, which contains a tissue plasminogen activator preparation diluted 10 - 200 times with physiological saline or artificial cerebrospinal fluid. This specification incorporates the disclosure content of Japanese Patent Application No. 2024 - 053288, which is the basis of the priority of this application.

Effects of the Invention

[0014] According to the present invention, by performing active hematoma removal using a tissue plasminogen activator preparation, the occurrence of delayed cerebral ischemic disorder can be prevented, and a prognostic improvement effect can be obtained.

Brief Description of the Drawings

[0015] [Figure 1] Figure 1 shows the state of the blood clot at 24 hours after blood sampling of specimens A, B, and C. [Figure 2] Figure 2 shows the state of the blood clot at 48 hours after blood sampling of specimens A, B, and C. [Figure 3] Figure 3 shows the color tone of the blood clot in the storage container at 48 hours after blood sampling of specimens A, B, and C.

Modes for Carrying Out the Invention

[0016] The present invention provides a pharmaceutical composition containing tissue plasminogen activator as an active ingredient, for use in cisternectomy therapy, in which tissue plasminogen activator is used as a washing agent in aqueous solution to remove the free hematoma simultaneously with the treatment of the cerebral aneurysm by craniotomy clipping during the acute phase of subarachnoid hemorrhage associated with a ruptured cerebral aneurysm, and the freed hematoma is aspirated and removed.

[0017] The pharmaceutical composition of the present invention is a washing solution in the form of an aqueous solution obtained by dissolving tissue plasminogen activator in physiological saline or artificial cerebrospinal fluid (e.g., ArtCeleb®), and can be used for washing and aspirating hematomas to be treated.

[0018] Tissue plasminogen activators have been identified as natural proteins in various animals. For example, in humans, a protein consisting of 562 amino acids is listed in databases such as the National Center for Biotechnology Information (NCBI) as Accession: NP_000921.1.

[0019] The tissue plasminogen activator used in the present invention may be the natural one described above. In this case, since it is contained in the washing solution that will ultimately be aspirated and removed, it can be derived from humans, or from non-human animals such as mammals like cattle, pigs, mice, and rats, or birds like chickens, and is not particularly limited. Furthermore, the tissue plasminogen activator used in the present invention may be synthetic, and those obtained by genetic recombination are particularly preferred. Examples of genetically modified products include alteplase (ACTIVASE®, etc.), reteplase (RETEVASE®, RAPILYSIN®, etc.), and tenecteplase (TNKase®, etc.), and these can be used as appropriate.

[0020] Alteplase, leteplase, and tenecteplase are all known as thrombolytic agents in intravenous thrombolytic therapy. For example, alteplase is known to be administered intravenously to adult humans at a dose of 0.6 mg / kg to improve functional impairment associated with the acute phase of ischemic cerebrovascular disease, and to adult humans at a dose of 0.5-0.75 mg / kg in the form of an aqueous solution of 600,000 international units / ml (IU / ml) to dissolve coronary artery thrombi in acute myocardial infarction. Here, "IU" is a standardized unit defined by t-PA activity. Alteplase is contraindicated in patients suspected of having subarachnoid hemorrhage.

[0021] Compared to alteplase, leteplase has a smaller molecular weight, a longer half-life, and is considered to have a lower probability of causing bleeding. Tenecteplase is a modified form of alteplase in which some amino acids are substituted, and it is also believed to have a longer half-life than alteplase. Clinical trials of tenecteplase are being conducted with intravenous administration at a dose of 0.25 mg / kg.

[0022] The tissue plasminogen activator in the pharmaceutical composition of the present invention is used as a washing agent in aqueous solution. The aqueous solution is not limited, but examples include physiological saline or artificial cerebrospinal fluid in which the tissue plasminogen activator is dissolved. The concentration of the tissue plasminogen activator in the aqueous solution is not particularly limited, but is preferably in the range of 5,000 to 20,000 international units / ml (IU / ml), 6,000 to 15,000 IU / ml, or 6,000 to 12,000 IU / ml.

[0023] Clinically, as described below, it is preferable to dissolve and dilute existing tissue plasminogen activator preparations (e.g., alteplase with 24 million IU / V) when aspirating and removing intracisional hematomas, and the pharmaceutical composition of the present invention is intended to encompass both the high-concentration composition before dilution (tissue plasminogen activator preparation) and the diluted composition (aqueous solution).

[0024] Accordingly, in one embodiment, the pharmaceutical composition of the present invention is a composition obtained by diluting a tissue plasminogen activator preparation, which is conventionally used at a concentration of 600,000 IU / ml, with physiological saline or artificial cerebrospinal fluid to a concentration of 10 to 200 times, for example, 20 to 150 times, 30 to 120 times, 40 to 100 times, or 50 to 100 times.

[0025] The pharmaceutical composition of the present invention is used simultaneously with the treatment of cerebral aneurysms by craniotomy clipping during the acute phase of subarachnoid hemorrhage. The acute phase of subarachnoid hemorrhage is generally considered to be about 14 days from the onset, but in the present invention, it is preferable to use it as early as possible after the onset, for example, within 72 hours, within 48 hours, or within 24 hours.

[0026] The pharmaceutical composition of the present invention is used for hematoma removal therapy within the cerebral cisterns. Hematoma removal therapy is not limited to, but preferably includes pressurized irrigation of the cerebral cisterns. The pressure required for pressurized irrigation is not particularly limited, as long as it is within a range that allows the hematoma to be released from the brain surface and surrounding tissues without damaging the pia mater of the brain surface. For example, pressurized irrigation of the cerebral cisterns can be performed using irrigation and suction devices that are already in clinical use, such as the Super Bypass Suction Irrigation System (Takayama Medical Instruments Co., Ltd.) or the Strike Flow 2 Suction / Irrigation System (Stryker Japan Ltd.). As the device used for pressurized irrigation, a combination of a device for pressurized irrigation and a device for aspirating and removing the cleaning agent (including the hematoma) after irrigation can be used. Alternatively, it is preferable to use an irrigation and suction device that can perform both pressurized irrigation and suction operations, as this simplifies the operation. In this case, the cleaning agent can be used as the irrigation perfusion fluid within the cerebral cisterns for pressurized irrigation.

[0027] Hematoma aspiration and removal should be continued until the hematoma is visually removed or until the irrigation fluid is depleted. The amount of irrigation fluid used may vary depending on the size of the hematoma, but is typically in the range of approximately 2.5L to 4L.

[0028] The following is an example of a procedure for aspirating and removing an intracerebral hematoma using the pharmaceutical composition of the present invention. 1. After the craniotomy and clipping procedure is completed, prepare saline solution or artificial cerebrospinal fluid (Artcele) containing tissue plasminogen activator and set it in the high-pressure irrigation device (recommended: 4L). Also, prepare a suction device for aspirating and removing free hematomas. 2. In a compartment of one cistern, a solution is sprayed between the brain surface and the hematoma, and this process is repeated to detach the hematoma from the brain surface and surrounding tissues. The detached hematoma is then removed by suction using a suction device. 3. Repeat the procedure in step 2 above for the accessible compartments of the brain cisterns.

[0029] The aforementioned method of aspiration and removal of intracisional hematomas is thought to allow for the efficient removal of more hematomas in a shorter time and without damaging the brain, compared to conventional methods that only remove hematomas from the surface. Furthermore, the combined use of a lavage and aspiration device allows for the active dislodgement and removal of hematomas in deeper areas near the base of the brain, which are difficult to reach with simple cisternous drainage, although this depends on the craniotomy method (anterior or lateral approach).

[0030] The present invention also provides a method for removing an intracranial hematoma in a patient, comprising washing and / or aspirating the hematoma using a washing agent containing tissue plasminogen activator in aqueous solution. In one embodiment, the present invention is a method for treating subarachnoid hemorrhage, and is performed on patients in the acute phase of subarachnoid hemorrhage in combination with, preferably after, treatment of a cerebral aneurysm by craniotomy clipping or the like. As the cleaning agent, the pharmaceutical composition of the present invention described above can be used.

[0031] The present invention also provides a tissue plasminogen activator for use in the treatment of subarachnoid hemorrhage. The tissue plasminogen activator is used in the form of an aqueous solution. In one embodiment, the tissue plasminogen activator for use of the present invention can be used in combination with, preferably after, treatment of a cerebral aneurysm by craniotomy clipping during craniotomy surgery for patients in the acute phase of subarachnoid hemorrhage, to wash and / or aspirate the hematoma at the site with a washing solution in the form of an aqueous solution. As the cleaning agent, the pharmaceutical composition of the present invention described above can be used. [Examples]

[0032] The present invention will now be described in more detail with reference to examples, but the present invention is not limited thereto.

[0033] [Example 1] In the following examples, alteplase, a recombinant tissue plasminogen activator preparation for intravenous injection, was used as the tissue plasminogen activator. (1) Equipment and chemicals used (a) Surgical microscope (b) Super Bypass Suction Irrigation System (Takayama Medical Equipment Manufacturing Co., Ltd.) (c) Medical gauze (d) Alteplase 6 million international units / vial, 2 vials (e) Alteplase 12 million international units / vial, 2 vials (f) Artificial cerebrospinal fluid (ArtCeleb) 500ml / bottle, 18 bottles (g) Human blood 20ml / person, enough for 3 people (used for samples A, B, and C respectively)

[0034] (2) Method Alteplase was diluted in cerebrospinal fluid, and concentrations of 6,000 international units / ml (IU / ml), 12,000 IU / ml, and a control group consisting of cerebrospinal fluid alone were used. Human blood (3 ml) was immersed in medical gauze (four-fold gauze) and left for approximately two hours to allow coagulation. By allowing the blood clot to become entangled in the mesh of the gauze, a hematoma that had entered between the arachnoid columns, arachnoid membrane, and normal blood vessels within the cisternus was simulated and designated as specimens A, B, and C. Human blood was collected from healthy adult volunteers after obtaining their informed consent.

[0035] The effectiveness of washing away blood clots from medical gauze was evaluated 24 and 48 hours after immersion in human blood. Furthermore, the blood clots that replicated the hematoma as described above were left for 2 hours, and once coagulation was confirmed, they were immersed in artificial cerebrospinal fluid to prevent drying and stored in a 37°C incubator until use for evaluation. Figures 1 and 2 show the state of the blood clots before washing 24 hours and 48 hours after blood collection for samples A, B, and C, respectively, and Figure 3 shows the color of the blood clots immersed in artificial cerebrospinal fluid in their storage containers 48 hours after blood collection for samples A, B, and C.

[0036] Under a neurosurgical microscope, a super-bypass suction irrigation system (set to 200 mmHg pressure) was used to spray the blood clots with artificial cerebrospinal fluid containing alteplase at various concentrations, and immediately aspirate them. Washing and aspiration were repeated until all clots were removed, or until the entire 250 mL of washing solution was used for each specimen.

[0037] (3) Evaluation method Washing was performed by moving from the upper left to the upper right of the gauze-soaked blood clot, and as the clots were washed away, washing was continued downwards until the lower right. If all clots were removed, the total volume of solution and the total amount of alteplase units were measured and recorded. If any clots remained, this was noted. The washing solution used was blinded to the experimenter and later verified.

[0038] The dissolving and washing effect was determined when the blood clots were completely removed in all three samples. The blood clots included those attached to the surface of the gauze and those entangled in the mesh of the gauze. The amount of washing solution used when the blood clots on the surface of the gauze were removed was recorded, and it was also evaluated whether the blood clots in the mesh of the gauze were dissolved and removed. The Japanese Standard Commodity Classification number and lot number of the alteplase used were recorded. The experimental results were recorded in a digitally signed PDF and in a laboratory notebook with the date. Experimental videos were also recorded and saved.

[0039] (4) Disposal All consumables used in the study, including residual and recovered washing solution or saline solution, gauze, and residual blood, were disposed of as infectious waste.

[0040] (5) Results The results for samples A, B, and C are shown in Tables 1-3, respectively.

[0041] [Table 1]

[0042] [Table 2]

[0043] [Table 3]

[0044] (6) Discussion As shown in Tables 1-3, when the pharmaceutical composition of the present invention was used (alteplase 6,000 IU / ml or 12,000 IU / ml group), blood clots entangled in the mesh of the gauze were removed by pressurized washing particularly well in a dose-dependent manner at 48 hours compared to the control group (artificial cerebrospinal fluid only), and blood clots were removed in all cases at 48 hours. Furthermore, at 48 hours, the blood clots in samples A and B had begun to dissolve and become softer compared to 24 hours later, but the blood clots in sample C had hardly dissolved at all (see Figure 3).

[0045] [Example 2] Except as described below, separate tests were conducted and evaluated using the same procedure as in Example 1. Human blood was immersed in medical gauze, and left for 3 hours to artificially recreate a hematoma, allowing the blood clot to become entangled in the gauze's mesh. The blood clot was then washed away 24 and 72 hours after immersion in human blood, and its effectiveness was evaluated. The recreated hematoma was then immersed in artificial cerebrospinal fluid after 3 hours and stored in a 37°C incubator until use for evaluation. The washing solution used was blinded to the experimenters and later verified.

[0046] Table 4 summarizes the results for the three samples, A, B, and C. Each evaluation was performed three times under each condition, and the solution volume shown in the table represents the amount (ml) required to wash away the surface blood clots.

[0047] [Table 4]

[0048] As shown in Table 4, after 24 hours, blood clots could not be washed away in 2 / 3 of the samples in the control group (artificial cerebrospinal fluid only). However, when the pharmaceutical composition of the present invention was used (alteplase 6,000 IU / ml or 12,000 IU / ml group), the clots were washed away without any problems, confirming the usefulness of the pharmaceutical composition of the present invention.

[0049] On the other hand, after 72 hours, blood clots adhering to the surface were washed away in all groups. However, blood clots entangled in the gauze mesh remained in two-thirds of the control group and two-thirds of the alteplase 6,000 IU / ml group, even after using the entire washing solution. In contrast, alteplase 12,000 IU / ml could be used to wash them away, and good results were obtained by increasing the dose. Furthermore, at 72 hours, the blood clots had softened during storage in artificial cerebrospinal fluid. They were softer before washing compared to the blood clots at 24 hours, and the surface clots were easily blown off by water pressure alone. Therefore, in all groups, surface clots could be removed with less solution at 72 hours than at 24 hours. All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A pharmaceutical composition containing tissue plasminogen activator as an active ingredient, for use in hematoma removal therapy, which involves pressurized lavage of the cerebral cisternae using an aqueous solution containing tissue plasminogen activator at a concentration in the range of 5,000 to 20,000 IU / ml as a lavage agent, simultaneously with treatment of the cerebral aneurysm by craniotomy clipping in the acute phase of subarachnoid hemorrhage associated with a ruptured cerebral aneurysm, and using 2.5 L to 4 L of the lavage agent to aspirate and remove the released hematoma.

2. The pharmaceutical composition according to claim 1, wherein tissue plasminogen activator is dissolved in physiological saline or artificial cerebrospinal fluid and used as a washing solution.

3. The pharmaceutical composition according to claim 1, to be used within 72 hours after the onset of subarachnoid hemorrhage.

4. The pharmaceutical composition according to claim 2, wherein the washing solution contains a tissue plasminogen activator preparation diluted 10 to 200 times with physiological saline or artificial cerebrospinal fluid.

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