Patent Application
20090221496
The present invention relates to an anti-thrombotic reagent, and a method of treating an ischemic disease such as myocardial infarction, cerebral infarction or occlusive arterial disease.
Until now, a variety of anti-thrombotic reagents have been available. These reagents are subdivided into two categories. That is, those acting on platelets and those modulating function of circulating coagulants. Reagents acting on platelets include those inhibiting cyclooxygenase (aspirin), those inhibiting phosphodiesterase (cilostazol), and those suppressing purinergic receptors on the cell membrane (thiazolidine derivatives). On the other hand, reagents controlling the action of coagulant factors include those inhibiting thrombin (anti-thrombin III), those inhibiting other coagulant factors (warfarin), and those activating fibrinolytic activities (tissue plasminogen activator; tPA and urokinase). These reagents have been used widely in the clinical field to treat patients with acute coronary insufficiency, stroke and cerebral ischemia.
However, these anti-thrombotic reagents have many problems, and thus an anti-thrombotic reagent having a novel action mechanism is required to treat such ischemic disease.
Under such circumstances, a novel pharmaceutical composition that can be used in an anti-thrombotic treatment, the treatment of an ischemic disease or the like is desired.
As a result of intensive studies, the present inventors have found that an albumin conjugated with polyethylene glycol (PEG) has a function of controlling blood coagulation and/or platelet aggregation in a vascular injury area, and that the albumin conjugated with polyethylene glycol exhibits an anti-thrombotic effect, thereby completing the present invention.
That is to say, the present invention is as follows.
As described above, the present invention provides a novel pharmaceutical composition. In a preferred embodiment of the present invention, the pharmaceutical composition can be used as an anti-thrombotic reagent or a reagent for treating an ischemic disease, and it has a function of controlling blood coagulation and/or platelet aggregation in a vascular injury area and the like and exhibits an anti-thrombotic effect and the like.
Hereinafter, the present invention will be described in detail. It should be noted that the scope of the present invention is not limited to the following description and that modifications to the embodiments disclosed herein may be made appropriately without departure of the spirit of the invention. This specification includes all of the contents as disclosed in the specification and/or drawings of U.S. Provisional Patent Application No. 60/778,489, which is a priority document of the present application. All documents and publications cited herein are incorporated herein by reference in their entirety.
As a result of intensive studies, the present inventors have developed a novel pharmaceutical composition used in an anti-thrombotic treatment, the treatment of an ischemic disease, or the like. In a preferred embodiment of the present invention, the pharmaceutical composition can be a novel anti-thrombotic reagent that differs from those belonging to the aforementioned category.
The pharmaceutical composition of the present invention comprises an albumin conjugated with polyethylene glycol (PEG) (PEG-conjugated albumin). An example of the PEG-conjugated albumin is a “PEG-conjugated human serum albumin” (PEG-HSA), and a particular example thereof is a “PEG-conjugated recombinant human serum albumin (rHSA)” (PEG−rHSA).
When a PEG-conjugated albumin such as PEG-HSA or PEG−rHSA is administered into blood, it increases the viscosity of the blood. It has been known that such increase in the viscosity of the blood increases a wall shear rate, thereby secondarily inducing nitrogen monoxide (NO) in vivo. Thus, in a preferred embodiment of the present invention, the pharmaceutical composition can be used as an anti-thrombotic reagent or a reagent for treating an ischemic disease, and it has a function of controlling blood coagulation and/or platelet aggregation in a vascular injury area and the like and exhibits an anti-thrombotic effect and the like.
In previous studies, an attempt has been made to apply PEG−rHSA to the control of hemorrhagic shock (References 1 and 2). However, it has not been known that a PEG-conjugated albumin such as PEG−rHSA has a function of controlling blood coagulation and/or platelet aggregation in a vascular injury area and the like because of a mechanism regarding redistribution and accumulation of circulating platelets under in vivo flow condition.
Moreover, the present invention also encompasses: a method of treating a thrombosis or an ischemic disease, comprising intravascularly administering an albumin conjugated with polyethylene glycol to a patient; use of an albumin-conjugated with polyethylene glycol for production of a pharmaceutical composition used in the treatment of an ischemic disease; and use of an albumin conjugated with polyethylene glycol for production of an anti-thrombotic pharmaceutical composition.
The “polyethylene glycol-conjugated albumin” used in the present invention is formed by allowing polyethylene glycol to bind to an albumin. The number of molecules of polyethylene glycol allowed to bind to a single molecule of albumin is, for example, 1 to 20, and preferably 8 to 15.
The type of the polyethylene glycol used in the present invention is not particularly limited. It is preferably an aqueous, biocompatible liquid. The molecular weight of the polyethylene glycol used in the present invention is preferably within the range of 100 to 20,000 Daltons (Da), and more preferably within the range of 1,000 to 10,000 Daltons (Da). Such polyethylene glycol can be easily produced according to known techniques, or commercially available products can be purchased.
Examples of the albumin used in the present invention include: animal albumins such as an egg albumin, a serum albumin, a lactalbumin and a myoalbumin; and vegetable albumins such as leucosin, legumelin and ricin. In particular, it is preferable to use a human serum albumin in the present invention. In addition, a recombinant human serum albumin may also be used. Such a recombinant human serum albumin may have an amino acid sequence identical to that of a human serum albumin, or it may also be a recombinant albumin comprising a deletion, substitution or addition of one or several amino acids. Such an albumin may be easily produced according to known techniques, or commercially available products may be purchased.
The amount of the polyethylene glycol-conjugated albumin contained in the pharmaceutical composition of the present invention is not particularly limited. It can be appropriately determined by persons skilled in the art.
The present invention includes use of the polyethylene glycol-conjugated albumin for production of an anti-thrombotic pharmaceutical composition.
Moreover, the composition of the present invention may comprise pharmaceutically acceptable additives for the purposes of: (i) the improvement of the stability of the drug in a polymeric composition; (ii) the control of the concentration of hydrogen ion in the composition; (iii) the control of the release rate of the drug and the decomposition rate of polymer; etc. Such additives are preferably selected from the group consisting of surfactants, inorganic salts, sugars, natural polymers and mixtures thereof. Either ionic or non-ionic surfactants may be used in the composition of the present invention. Typical ionic surfactants include sodium dodecylsulfate and typical non-ionic surfactants include polysorbate, polyvinylpyrrolidone, poloxamers, glyceryl monooleate, glyceryl monostearate, polyoxyethylene alkyl ether and mixtures thereof.
The sugars which can be used as additives in the present invention include mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose and mixtures thereof.
Examples of inorganic salts which can be used as additives in the present composition include sodium chloride, calcium chloride, zinc chloride, magnesium chloride, calcium carbonate, zinc carbonate, zinc acetate, zinc lactate, magnesium hydroxide, aluminum chloride, aluminum hydroxide, zinc oxide and mixtures thereof.
Examples of natural polymer which can be used as additives in the present composition include cyclodextrin, gelatin, hyaluronic acid, chitosan, sodium carboxymethylcellulose and mixtures thereof.
The content of additives in the composition of the present invention is preferably within the range of 0.01 to 10 wt %, and more preferably within the range of 0.1 to wt %.
In a preferred embodiment of the present invention, by administering the pharmaceutical composition into a living body, blood coagulation and/or platelet aggregation can be controlled in a vascular injury area, so that formation of thrombus can be effectively prevented. Thus, the pharmaceutical composition of the present invention is useful for an anti-thrombotic treatment or the treatment of an ischemic disease.
The administration route of the pharmaceutical composition of the present invention is not particularly limited. For example, a parenteral administration is preferable. In the case of such a parenteral administration, the pharmaceutical composition is preferably administered in the form of a liquid preparation such as an injection. In the present invention, such an injection is particularly preferably a drip injection. When the pharmaceutical composition is administered in the form of an injection, it is administered as an intravenous injection, a subcutaneous injection, an intracutaneous injection, an intramuscular injection, etc. The dose of the pharmaceutical composition of the present invention is different depending on the age, sex and symptoms of a patient, an administration route, the number of doses, and a dosage form. Thus, the dose of the present pharmaceutical composition is not particularly limited, and it can be selected, as appropriate. For example, the dose is appropriately selected by a clinician.
A method of producing the composition of the present invention is not particularly limited, and a known production method can be applied. For example, when the pharmaceutical composition of the present invention is produced in the form of an injection, it is prepared by dissolving, suspending or emulsifying an albumin conjugated with polyethylene glycol in a sterile aqueous or oily liquid. Such an aqueous liquid used for injection includes a normal saline solution, an isotonic solution containing glucose or other auxiliary agents (e.g. D-sorbitol, D-mannitol, sodium chloride, etc.), and the like. Suitable solubilizing agents such as alcohol (e.g. ethanol, etc.), polyalcohol (e.g. propylene glycol, polyethylene glycol, etc.), and a nonionic surfactant (e.g. polysorbate 80, HCO-50, etc.) may be used together with such an aqueous liquid. On the other hand, an oily liquid includes sesame oil and soybean oil. Solubilizing agents such as benzyl benzoate and benzyl alcohol may be used together with such an oily liquid. In addition, such an oily liquid may also be mixed with a buffer (e.g. a phosphate buffer, a sodium acetate buffer, etc.), a soothing agent (e.g. benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (e.g. polyethylene glycol, etc.), a preservative (e.g. benzyl alcohol, phenol, etc.), and the like. The prepared injection is generally filled into an ampule.
Moreover, the polyethylene glycol-conjugated albumin used in the present invention can be obtained by allowing polyethylene glycol (PEG) to react with an albumin (e.g. a human serum albumin (HSA) or a recombinant human serum albumin (rHSA)). Specifically, the polyethylene glycol-conjugated albumin solution of the present invention can be produced by a method which comprises a step of mixing and dissolving a water-soluble biocompatible liquid polyethylene glycol and an albumin (e.g. HSA, rHSA, etc.) as a physiologically active substance in an organic solvent or in a mixture of an organic solvent and water to obtain a polymeric solution. Furthermore, such a polymeric solution is filtrated by passing it through a membrane for sterilization, and the thus filtrated polymeric solution is then freeze-dried or evaporated under reduced pressure, so that the organic solvent may be eliminated.
More specifically, the polyethylene glycol-conjugated albumin solution of the present invention can be produced by dissolving a liquid polyethylene glycol, an albumin (e.g. HSA, rHSA, etc.), and any given additives in an organic solvent or in a mixture of an organic solvent and water. Examples of an organic solvent suitable for preparing the solution include acetonitrile, acetone, acetic acid, dimethyl acetamide, ethanol, isopropanol, and dioxane.
The present invention also provides a method of treating a thrombosis or an ischemic disease, which comprises intravascularly administering a polyethylene glycol-conjugated albumin. Targets to be treated are not particularly limited. Examples of such a therapeutic target include a human, and organisms other than a human including nonhuman mammals such as a bovine, a simian, an Aves, a feline, a mouse, a rat, a guinea pig, a hamster, a swine, a canine, a rabbit, a sheep or a horse.
Herein, it is considered that the therapeutic method of the present invention functions by taking advantage of the action of the albumin molecule during an ischemic (“oxygen-depleted”) event. It is to be noted that the term “ischemic event” is used in the present specification to mean that the patient has experienced a local and/or temporary ischemia due to insufficient blood flow (partial or total obstruction of the blood circulation) to an organ. Moreover, the term “anti-thrombotic treatment” is used herein to mean a treatment for suppressing thrombus formation.
Furthermore, the term “ischemic disease” is used to mean a disease that causes such an ischemic event. Examples of such an ischemic disease include external injuries, immunological rejection occurring during transplantation, ischemic cerebral vascular accident (e.g. apoplectic stroke, cerebral infarction, etc.), ischemic renal disease, ischemic pulmonary disease, ischemic disease associated with infectious disease, ischemic disease of limbs (e.g. arteriosclerosis obliterans), and ischemic heart disease (e.g. ischemic cardiomyopathy, myocardial infarction, ischemic heart failure, etc.). Other examples also include diseases that cause ischemic events, such as cerebral edema and acute coronary failure.
Preferably, a polyethylene glycol-conjugated albumin is intravascularly administered in the form of a liquid preparation such as an injection. In addition, in a preferred embodiment of the present invention, such a polyethylene glycol-conjugated albumin is intravenously administered.
In addition, the present invention also includes use of a polyethylene glycol-conjugated albumin for production of a pharmaceutical composition used in the treatment of a human ischemic disease.
Hereinafter, the present invention will be described in more detail with reference to the following Example. However, the present invention is not limited to this Example.
Male Wistar rats weighing 230-290 g were overnight fasted under free access of water and anesthetized with isoflurane inhalation according to the previous methods (reference 3). Bleeding times were determined using a method previously described (reference 4). A 2.5 mm long 1 mm deep template-guided incision was made in the tails of anesthetized rats, 1 cm from the tip of the tail; 45 degrees from the dorsal vein. Tails were immersed in 45 ml of normal saline, and the time until bleeding stopped was measured with a stopwatch.
1.2 Preparation of PEG−rHSA
Recombinant human serum albumin (rHSA) is a generous gift from NIPRO Inc (Shiga, Japan). Polyethylene glycol (PEG) with a molecular weight of 10,000 was commercially available. PEG-bound rHSA was prepared by reacting rHSA with succinimidyl-PEG precursor which has the ability to react with lysine residue of the protein, according to previous methods (reference 5). The number of PEG bound per a molecule of rHSA was calculated by determining free PEG in the reaction mixture for the PEG−rHSA preparation using high-performance liquid chromatography (HPLC, TSKgel column with Waters 2695 separation module). In order to quantify the free unbound PEG in the reaction mixture, known concentrations of PEG were prepared for establishment of the calibration line. The amounts of PEG were determined by calculating the areas of the peak of HPLC charts. As seen in
The effect of PEG−rHSA on the blood viscosity was examined in vitro using a rotation fluid viscometer. Parameters for characterization of the samples were shown in Table 1. As shown in
Rats were anesthetized with diethyl ether followed by isoflurane. An arterial catheter was implanted in the left femoral artery, connected to a pressure transducer, and mean arterial pressure (MAP) was amplified and monitored continuously, and the data was collected on a computer. The right femoral vein was cannulated for infusion of solutions and the right femoral artery was cannulated for withdrawing blood and sampling arterial blood for the blood gas analysis, hematocrit and platelet count. Samples were taken in EDTA tubes twice before and after the hemodilution (at the cannulation and the end of the observation period). Rectal temperature was monitored and kept stably from the start of monitoring.
After three catheter implantations, the stability of MAP was observed in 5 minutes. Each solution (rHSA, PEG−rHSA conjugate, PEG+rHSA mixture) was infused with a dual syringe pump into the right femoral vein catheter in 5 minutes, and blood was simultaneously withdrawn from the right femoral artery catheter at the same rate. The volume of exchange transfusion was calculated as a 30% of the rat's blood volume, estimated as 56 ml/kg of body weight for rats. The rate of exchange transfusion was calculated as: (The volume of exchange transfusion)/5 [ml/min]
At 10 min after initiation of the blood exchange, the position of the rat was changed and prepared for the tail cut. At 15 min after initiation of the blood exchange, in five minutes after the position-change, a template-guided incision (Quikheel, Becton-Dickinson, San Jose, Calif.) 2.5 mm in length and 1.0 mm in depth was made 1 cm from the tip of the tail. The tail was immersed in a 50 ml cylinder of saline, and the time taken for bleeding to stop was measured. The monitoring of MAP was continued until 50 min, when the second sample was collected.
1.4 Intravital Observation of Platelet Adhesion and Aggregation in vivo Using Laser Ablation
In separate sets of experiments, differences in platelet adhesion and aggregation at the site of vascular injury between the group treated with PEG−rHSA and that treated with free rHSA were compared using an in vivo model of microvascular laser ablation according to a known method (reference 6). In brief, rats were injected with carboxy fluorescein diacetate succinimidyl ester (CSFE), a fluorescent precursor that can stain circulating platelets and partly leukocytes when injected into circulation (reference 7). After the CFSE injection, 200 ml of PEG-coated liposome labeled with rhodamine G was injected. This liposomal particle does not accumulate nor adhere to the intact vascular wall, but accumulate quickly to the subendothelial space of the injured vascular endothelial cells.
To elicit microvascular thrombosis, the inventors used a multi-pinhole confocal laser microscope (Yokokawa Medical Inc., Tokyo) equipped with a nitrogen-dye pulse laser system that allows the inventors to carry out pin-point laser ablation of the microvascular endothelial cells located at the area of interests. The energy of the laser ablation was set constant among different experiments. When the pulse laser was applied to a microvessel chosen for the analysis, the damaged endothelial cells became noticeable through accumulation of the rhodamine-labeled liposome which can be visualized as red color (or magenta) under excitation with 530 nm. On the other hand, alterations in platelet adhesion and aggregation were visualized under epi-illumination at 488 nm as green color. Using a wide-range band-pass filter, both green and red fluorescent signals at the site of the vascular injury were visualized simultaneously through intensified CCD camera (Hamamatsu Photonics Inc., Hamamatsu City) and a digital video recording system.
Values are presented as means±S.E. unless otherwise indicated. Statistical significance is reported at the P<0.05.
2.1 Improving Effects of PEG−rHSA on MAP and Heart Rates During 30% Isovolemic Hemodilution Procedure
2.2 Administration of PEG−rHSA Elongates Bleeding Time in vivo
2.3 Administration of PEG−rHSA Attenuates Laser-Induced Microvascular Thrombogenesis in vivo
Based on results shown in
Since the systemic blood pressure was slightly higher in the PEG−rHSA-treated group than in rHSA-treated group, the inventors chose post-capillary venules where the local perfusion pressure would be comparable between the groups. As seen in
PEG−rHSA has the remarkable effect to maintain retention of water into an intravascular space and helps improvement of microvascular hemodynamics with anti-platelet properties. Thus, the PEG−rHSA administration is likely to be beneficial to treat patients with acute ischemic stroke of cerebral microcirculation and brain edema or with acute coronary insufficiency.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2007/054489 | 3/1/2007 | WO | 00 | 10/6/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60778489 | Mar 2006 | US |
