JP5099545B2 - Compound solubilizer and composition containing the same - Google Patents

Description

The present invention relates to a drug carrier based on an in-vivo product and a medicine using the same.
The present invention also relates to a solubilizing agent that improves the solubility when a compound is dissolved in water.

  In DDS (Drug Delivery System), development of a drug carrier is a key technology. Many drug carriers have been studied so far. One of the expected properties of drug carriers is the transportation of poorly water-soluble drugs. Even if the drug is effective, it is difficult to inject into the body due to poor water solubility. In addition, there are many cases where the efficacy is lowered when water-solubility is obtained by modification. If such a poorly water-soluble drug can be transported through the body by a drug transporter, the number of drugs that can be used is extremely large.

  As such a drug carrier, one having a configuration in which a poorly water-soluble drug is put therein by an outer layer having hydrophilic characteristics and an inner encapsulation having hydrophobic characteristics is considered (Patent Document 1).

  Examples of drug carriers that have been studied so far include liposomes, microparticles, nano-related substances, and drug / polymer conjugates. Among these, polymer micelles have attracted attention as an advantageous carrier for the transmission of poorly water-soluble drugs. For example, there is a micelle-forming composition composed of a hydrophobic core surrounded by a hydrophilic shell, wherein the hydrophilic shell is composed of PVP (N-vinyl-2-pyrrolidone) (Patent Document 2).

  By the way, not all the components in the body are water-soluble. For example, vitamins A, D, K, E and the like are poorly water-soluble. Nevertheless, it is circulated in the body because there is a protein called lipocalin family that carries them.

  For example, lipocalin-type prostaglandin D synthase (L-PGDS) is actively produced in various tissues and is involved in many physiological phenomena. Therefore, there are many proposals for its use, and it is possible to differentiate dementia diseases by measuring L-PGDS (Patent Document 3), to use for pathological management of renal diseases (Patent Document 4), or to detect water breakage (Patent Document 4). There is an invention used in Document 5).

However, these utilize the properties of L-PGDS as an enzyme. Note that the structure of L-PGDS was recently released (Patent Document 6).
Japanese National Patent Publication No. 10-502376 Special table 2004-501180 gazette JP 2000-212248 A JP 2000-246980 A Japanese Unexamined Patent Publication No. 2000-22929 Japanese Patent Laid-Open No. 2004-194581

  Drug carriers such as the virus type have anxiety in removing toxicity even though they have the ability to transport into cells. Therefore, many drug carriers are artificially synthesized as described above. However, there has been a problem that the artificially synthesized product is absorbed in the body or conversely remains without being discharged out of the body. Moreover, since the compound, especially the chemical | medical agent said to be sparingly water-soluble is insoluble in water, there existed a subject that utilization was restrict | limited.

The present gun invention, a lipocalin-type prostaglandin D synthase is a protein selected from lipocalin family, the compound employed, especially a poorly water-soluble drug as a solubilizing agent that allows dissolved in a polar solvent (especially water) Applications are also provided. The present invention also provides a composition comprising such a solubilizer, a compound, and a polar solvent.

  Since the lipocalin family is a substance produced in the first place, there is no worry about safety. In particular, L-PGDS wraps a polypeptide of a certain length and transports it in the body regardless of the structure of the drug. Therefore, it is possible to provide a safe and highly effective drug carrier and a drug with the drug carrier added thereto.

  In addition, proteins belonging to the lipocalin family can dissolve compounds that have limited molecular weight in water because they can be taken into pocket-shaped parts and dissolved in water. .

  In addition, with regard to drugs that have been developed against conventional epidemics, water solubility must be imparted in order to be ingested by the body, and there are cases in which the medicinal effect is somewhat sacrificed. The use eliminates the need to trade off medicinal properties and water solubility.

Lipocalin family The lipocalin protein belonging to the lipocalin family known as a group of lipophilic low-molecular transport proteins is a protein having 160 to 200 amino acid residues and a molecular weight of about 20,000. Moreover, it is known that the structural similarity is very high even though the homology of amino acid sequences is about 20%.

  FIG. 1 shows a part of the lipocalin family. (A) retinol binding protein, (b) major urine protein, (c) villin binding protein, (d) β-lactoglobulin, (e) lobster crustocyanin, (f) odorant binding protein are members of the lipocalin family is there.

  The folding structure of these lipocalin proteins consists of eight antiparallel β strands that are continuously hydrogen bonded, and has a barrel shape surrounding the hydrophobic low molecular (ligand) binding portion. Seven loops connecting eight β-strands are six short hairpins and one omega loop, and this omega loop is considered to serve as a lid for the ligand binding part. It also has one α helix. That is, the structural similarity of these lipocalin families is that they have eight β-strands, one omega loop, and one α-helix. In the present invention, the lipocalin family includes proteins having 8 β strands, 1 omega loop, 1 α helix, and about 160 to 200 amino acid residues. In FIG. 1, α helix and omega loop of each member are indicated by reference numerals 9 and 10.

  In this structure, the inside of the barrel structure is hydrophobic and the outside of the barrel structure is hydrophilic. Thus, the hydrophobic ligand can be taken up and transported into the barrel. Here, the ligand means a low molecular substance having a molecular weight of about several hundreds or less.

  FIG. 2 shows the amino acid sequences of these proteins. Although the homology of the amino acid sequences of each protein is low, there are cysteine residues at the 89th and 186th positions, and a disulfide bond is formed between them. This is also one of the features common to the lipocalin family.

  In the present invention, these proteins belonging to the lipocalin family are used as a drug carrier for DDS. It is also used as a solubilizing agent for substances.

FIG. 3 shows a schematic diagram of the crystal structure of a recently published lipocalin-type prostaglandin D synthase (L-PGDS). L-PGDS is an enzyme that synthesizes PGD ₂ using PGH ₂ synthesized from arachidonic acid by the action of cyclooxygenase as a substrate. In the central nervous system, PGD ₂ exhibits actions such as sleep induction, hypothermia, suppression of lutein hormone secretion, and regulation of pain and odor responses. In peripheral nerves, PGD ₂ is released from mast cells as an allergic mediator, It is a substance that exhibits actions such as bronchoconstriction and inhibition of platelet aggregation.

  On the other hand, L-PGDS belongs to the lipocalin family, and has 8 β-strands, 1 α-helix, and 1 omega loop, like the lipocalin family shown in FIG. In FIG. 3, the barrel structure is numbered from 1 to 8, and the α helix and the omega loop are denoted by reference numerals 9 and 10, respectively. FIG. 2 also shows the amino acid sequence of L-PGDS. Like other lipocalin families, there are cysteine residues at positions 89 and 186, and a disulfide bond is formed between them. Below, the example which utilized this L-PGDS as a drug carrier of DDS and a solubilizing agent is demonstrated.

Example 1
<Preparation of recombinant L-PGDS>
Since L-PGDS is an enzyme produced in vivo, when it is used as a drug carrier, it must be artificially synthesized and, if necessary, artificially remade. Therefore, L-PGDS produced by the living body was first prepared as a recombinant.

1. Cloning of cDNA encoding L-PGDS RNA was extracted from whole mouse brain by a standard method, and cDNA was synthesized by reverse transcription using this as a template. This cDNA was amplified by polymerase chain reaction (PCR) using primers designed based on the base sequence of L-PGDS derived from mouse (EMBL / GenBank / DDBJ ^™ accession number D83329). The primers are shown in Table 1 as SEQ ID NOS: 1 and 2.

  PCR was carried out by heat denaturation at 94 ° C. for 90 seconds, followed by 30 reaction cycles of 94 ° C. for 30 seconds, aligning at 55 ° C. for 30 seconds, and 73 ° C. for 1 minute, followed by 72 An extension reaction was carried out at 7 ° C. for 7 minutes.

The amplified cDNA was ligated to the Srf I site in the cloning vector pCR-Scrpit (Stratagene), and a part of the reaction solution was transformed into E. coli to obtain a clone. The obtained clone was determined by the sequence Mo using the dideoxy method (determined using the Thermo Sequence Cy5 Dye Terminator Cycle Sequencing Kit (Amersham) and the Long-Read Tower DNA sequencer (Amersham) according to the manual). It was confirmed that the PGDS protein was encoded (SEQ ID NO: 3). Table 1 shows it as SEQ ID NO: 3. This vector was named “L-PGDS / pCR-script”.

2. Preparation of Insert In order to prepare an insert for L-PGDS expression, the above-mentioned L-PGDS / pCR-script was treated with a restriction enzyme to obtain a fragment. Briefly, 10 μL of Bam HI (Takara), 10 U of Eco RI (Takara), 10 × K Buffer (Takara) was added to 10 μg of L-PGD / pCR-script to prepare a 20 μl reaction solution at 37 ° C. For 1 hour. Next, this reaction solution was electrophoresed in a 2.0% nucleic acid electrophoresis agarose gel (nacalai tesque) to confirm that it was approximately 500 bp including the added restriction enzyme site, and using PCR DNA and Gel Band Purification Kit. According to the manual, an expression insert was collected from the gel and used as a polypeptide expression insert.

3. Preparation of vector pGEX-2T was used as an expression vector. In order to insert the above cDNA downstream of the Glutathione S-transferase (GST) gene in the pGEX-2T vector, the vector pGEX-2T was treated with a restriction enzyme. Briefly, 10 μg of Bam HI (Takara), 10 U of Eco RI (Takara), 10 × K Buffer (Takara) was added to 10 μg of pGEX-2T vector to prepare a 20 μl reaction solution at 37 ° C. for 1 hour. Reacted.

  Next, this reaction solution was electrophoresed in a 1.0% nucleic acid electrophoresis agarose gel (nacalai test), confirmed to be 5000 bp, and expressed from the gel according to the manual using PCR DNA and Gel Band Purification Kit. The insert was recovered and used as a polypeptide expression vector. The collected vector was used for ligation with each of the above inserts.

4). Ligation and transformation In the ligation reaction, ligation was performed between 500 ng of the insert and 50 ng of the vector according to the manual using Ligation-High (TOYOBO).

  After ligation, a part of the reaction solution was transformed into E. coli to obtain a clone. The obtained clone was treated with a restriction enzyme to obtain an expression vector having an insert for polypeptide expression. Briefly, 1 μg of the obtained clone was added with 1 U of Bam HI (Takara), 1 U of Eco RI (Takara), and 10 × K Buffer (Takara) to prepare a 20 μl reaction solution at 37 ° C. for 1 hour. Reacted. Subsequently, this reaction solution was electrophoresed in a 2.0% nucleic acid electrophoresis agarose gel, and it was confirmed that the insert was about 500 bp and the vector was about 5000 bp.

  Subsequently, E. coli BL21 (DE3) strain was transformed with the vector into which the insert was inserted. Thereafter, transformed clones were selected by culturing overnight at 37 ° C. in LB agar medium (containing 100 μg / ml ampicillin). These clones (hereinafter “L-PGDS / pGEX”) were stored at −80 ° C. as glycerol stocks.

  L-PGDS / pGEX was cultured overnight at 37 ° C. in 5 ml of LB liquid medium (containing ampicillin). Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.6 mM to induce the expression of the insert DNA-derived protein, and the culture was further continued for 6 hours. The polypeptide is expressed as a fusion protein with a GST tag.

  The culture solution was centrifuged at 8,000 × g for 10 minutes at 4 ° C. to recover the cells. Ten times the amount of Lysis buffer (10 mM disodium hydrogen phosphate, 1.8 mM dipotassium hydrogen phosphate, 140 mM sodium chloride, 2.7 mM potassium chloride, pH 7) to which the collected bacterial cells were added with 0.5 mM Phenylmethylsulfide Fluoride (WAKO). .5) was crushed by sonication (ULTRASONIC DISCURPTOR, TOMY SEIKO). The crushed material was centrifuged at 10,000 × g for 20 minutes at 4 ° C., and the supernatant was recovered as a protein extraction fraction.

5. Purification of polypeptide A GST-tagged polypeptide was purified from the protein extract fraction using Glutathione Sepharose 4B (GE Healthcare) as follows. The GST tag fusion polypeptide was adsorbed on a Glutathione gel according to the manual. Subsequently, in order to elute L-PGDS, 100 units of Thrombin Protease (SIGMA) was added to GST-L-PGDS adsorbed on Glutathione gel (1 ml) and reacted at room temperature for 16 hours.

  On the next day, PBS (-) was added thereto to elute L-PGDS and Thrombin Protease. The eluted L-PGDS was concentrated using a concentration filter (apollo), and after gel filtration with 5 mM Tris / Cl, pH 8.0 (superdex 75, GE healthcare), the L-PGDS fraction was recovered and 1 mg / ml Freeze-incubated at -20 ° C in concentration. The collected L-PGDS is called rL-PGDS. PBS (phosphate-buffered saline) is phosphate buffered saline.

<Diazepam experiment>
In order to confirm that L-PGDS transports a drug in vivo and the transported drug shows an effect, the pentobarbital anesthetic time extending action of the diazepam complex was examined. The anxiolytic diazepam is a hydrophobic compound that is extremely insoluble in aqueous solvents and is suspended in a cellulose solvent or the like when applied to animals.

  As an index that can detect the pharmacological activity of diazepam, there is a pentobarbital anesthetic time extension action. When diazepam works, the anesthesia time becomes longer. Therefore, the presence or absence of pharmacological activity is determined by examining the length of anesthesia time with and without L-PGDS.

  Specifically, a diazepam and L-PGDS diazepam complex solution solubilized in an aqueous solvent was administered into the cerebral ventricles of mice, and the effect on pentobarbital anesthesia time was examined.

<Preparation of drug solution>
Diazepam (3 mg / mL) was sufficiently suspended in an aqueous solvent (5 mM Tris-HCl buffer pH 8.0) and passed through a 0.2 μm filter to remove insoluble particles to obtain a diazepam solution. Moreover, what was prepared similarly in presence of rL-PGDS (0.5, 2 and 5 mg / mL) was used as the rL-PGDS-diazepam complex solution. A solution in which 2 mg / mL of rL-PGDS was dissolved was used as an L-PGDS solution.

<Intraventricular administration of chemicals and measurement of pentobarbital anesthesia>
In the examples, ddY male mice (6 weeks old) were used. The scalp was incised under light ether anesthesia, and intraventricular administration (5 μL / mouse) was performed by inserting a two-stage needle (3 mm) with a microsyringe from a position 1 mm to the right of the cranial bregma. Each administration group was set as follows. The amount of diazepam actually dissolved is shown together with the amount of rL-PGDS and the amount of diazepam in this order.
(1) Solvent administration group (5 mM Tris-HCl buffer pH 8.0)
(2) rL-PGDS (2 mg / mL) only (3) Diazepam solution administration group (2.6 mg / mL)
(4) rL-PGDS-diazepam complex solution administration group (0.5 mg / mL: 3.5 mg / mL)
(5) rL-PGDS-diazepam complex solution administration group (2 mg / mL: 8.0 mg / mL)
(6) rL-PGDS-diazepam complex solution administration group (5 mg / mL: 13.6 mg / mL)

  Ten minutes after intracerebroventricular administration, pentobarbital (35 mg / kg) was intraperitoneally administered, and the mouse was held in the supine position to observe its direct reflex. The time when the specular reflex disappeared was recorded as the start time of anesthesia induction. Even after the induction of anesthesia, the forward reflex was observed as needed, and the time when the reflex was completely recovered was recorded as the awakening time. The anesthesia time was determined by dividing the anesthesia induction start time from the awakening time, and the average value of the solvent administration group was defined as 100% and expressed as an average value ± standard error.

<Result>
The results are shown in FIG. The vertical axis represents the relative anesthesia time, and the solvent administration group (1) is shown as 100%. The horizontal axis represents the administration groups (1) to (6) above.

  There was no difference in pentobarbital anesthesia time between the solvent-administered group (indicated as “control” in the figure) and the (3) diazepam solution-administered group (indicated as “DZP / PBS” in the figure). From these results, it was considered that the diazepam solution prepared in this study did not reach the effective concentration showing an anesthetic extension action.

  On the other hand, the rL-PGDS-diazepam complex solution administration group of (4) to (6) above (the numbers in parentheses and “Complex” and the concentrations of diazepam and rL-PGDS are shown) are rL-PGDS- A pentobarbital anesthetic time-extending effect was observed depending on the concentration of the diazepam complex solution, and a significant effect was exhibited when the rL-PGDS-diazepam complex solution concentration was 400 μM or more. In FIG. 4, (5) and (6) indicated by “*” are judgments at a significance level of 0.05.

  From (4) to (6), it can be seen that the concentration of diazepam increases as the concentration of rL-PGDS increases.

  This result is considered that the solubility of diazepam was increased in the rL-PGDS-diazepam complex solution, and the effective concentration was reached. From the above results, it was shown that L-PGDS can improve the solubility of poorly water-soluble compounds and that the complex can exert the action of the drug in vivo.

(Example 2)
Male and female sand mice (body weight 70-80 g) were anesthetized with 1-2% Frocene after light ether anesthesia. A sand mouse was fixed in the dorsal position, the midline neck was incised, and the bilateral common carotid artery and right common jugular vein were carefully detached. A PE10 catheter filled with heparinized saline was placed in the right common jugular vein.

  Forebrain ischemia was performed by clipping the bilateral common carotid artery. The clip was removed 5 minutes after the start of ischemia, and blood flow to the forebrain was resumed. Immediately after recirculation, the solvent and specimen (NBQX (2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo [f] quinoxaline-7-sulfonamide) / rL-PGDS complex) were simply supplied from the catheter. Immediately after intravenous administration, continuous intravenous administration (flow rate of 0.2 mL / h) was started in conjunction with an infusion pump (Harvar apparator model 11). Continuous intravenous administration was performed for 2 hours.

  After the administration, the catheter tip was tied to prevent backflow of blood and specimen, and the catheter was buried subcutaneously and sutured. In addition, in order to prevent the nonspecific body temperature fall resulting from anesthesia or a surgical operation, all the processes were implemented under the incandescent lamp, and the rectal temperature was maintained at 36-38 degreeC.

  One week after the forebrain ischemia surgery, formalin reflux fixation was performed transcardially under pentobarbital deep anesthesia, and after removing the whole brain, a coronal brain section (2 mm) containing the hippocampus was prepared. The number of hippocampal CA1 pyramidal neurons was evaluated by hematoxylin and eosin (HE) staining.

  5 to 8 show photos of hippocampal staining. In FIG. 5 to FIG. 6, the arrows indicate the hippocampal CA1 pyramidal neurons. Since the sand mouse is in the forebrain ischemia state as in the above experiment, the CA1 pyramidal neurons are damaged. NBQX is known as a drug that recovers this forebrain ischemia. However, it is also known as a drug that is difficult to dissolve and difficult to give in such an amount that a medicinal effect appears.

  FIG. 5 (a) shows normal CA1 pyramidal neurons that have not undergone forebrain ischemia. CA1 pyramidal neurons were stained and could be confirmed as dark curves. FIG.5 (b) shows the case where only PBS is administered to the forebrain ischemia state. CA1 pyramidal neurons remained damaged and were observed as a thin curve compared to the case of FIG. 5 (a). FIG. 5C shows the result when only rL-PGDS is given. It can be seen that there was not much difference from the case of FIG. In FIG. 5 and FIG. 6, the thick black line at the lower right of the photograph is 200 μm.

  In FIG. 6, the same photograph as FIG. 5A is shown in FIG. 5A for easy comparison. FIG. 6D shows the result when NBQX is given. NBQX was administered by dissolving it in a solvent (PBS) as much as possible. NBQX has a medicinal effect to recover forebrain ischemia, but almost no difference from the cases of FIGS. 5B and 5C, and almost no recovery of CA1 pyramidal neurons was observed.

  FIG. 6 (e) shows the result of administration of NBQX and rL-PGDS dissolved together. The CA1 pyramidal neurons were found to have recovered to the same extent as in FIG. 6 (a) without forebrain ischemia.

  FIG. 7 shows a result obtained by increasing the magnification. 7 (b), (c), and FIG. 8 (d) showing only PBS, only rL-PGDS, and only NBQX at this magnification, the CA1 pyramidal neurons remain damaged. In the case of the mixture of NBQX and rL-PGDS shown by 8 (e), it was found that almost all CA1 pyramidal neurons were alive. 7 and 8, the thick black line represents 50 μm.

FIG. 9 shows the result of counting the number of cells photographed in FIG. The vertical axis represents the number of cells per square mm, and the horizontal axis represents the sample. (A) to (e) shown in the bar graph correspond to (a) to (e) shown in FIGS. When the forebrain ischemia indicated by the symbol (a) was not caused, the cells that were about 3300 cells / mm ² were 500 cells as indicated by (b) to (d) because the forebrain ischemia occurred. / Mm ² disappeared. This indicates that forebrain ischemia is hardly recovered by PBS, rL-PGDS, and NBQX alone. However, the mixture (e) in which NBQX was mixed with rL-PGDS (denoted as “complex” in FIG. 9) recovered about 80% or more.

  When the concentration of NBQX in the solution at this time was examined, it was 0.9 mM in the case of NBQX alone, but when rL-PGDS was mixed, the solubility was improved by about 4.6 times to 4.6 mM. .

  From this, rL-PGDS can improve the solubility of NBQX, and NBQX mixed with rL-PGDS crosses the blood-brain-barrier and enters the brain, and CA1 of the hippocampus It was concluded that the drug was effective in the area.

  Moreover, since the molecular weight of NBQX is 336 and rL-PGDS is tens of thousands, it is unlikely that rL-PGDS has passed through the blood-brain barrier, which is said to block substances having a molecular weight of about 500 or more. Therefore, it is considered that NBQX that was dissolved in a large amount by binding to rL-PGDS was separated from rL-PGDS at the blood-brain barrier and entered the brain.

  Thus, rL-PGDS not only improves the solubility of a substance, but can also separate and release only a substance in the body. That is, it can also be used as a DDS.

(Example 3)
In order to confirm that rL-PGDS improves the solubility of a substance, it was confirmed how the solubility changed in several substances.

  Substances examined were genistein, diazepam, retinoic acid, NBQX, bilirubin. In particular, diazepam and NBQX are artificially synthesized substances. The concentration when each substance was dissolved in PBS alone and the concentration when each substance was dissolved together with rL-PGDS were examined. If the amount of rL-PGDS is increased, the amount to be dissolved is increased. Therefore, here, rL-PGDS was set to be constant at 425 μM. Table 2 shows the molecular weight of the specimen and the results.

The specimens genistein, diazepam, retinoic acid, NBQX, and bilirubin dissolve only 58, 150, 22, 740, and 0.28 μM in PBS. However, when it was combined with rL-PGDS, it dissolved in a very large amount of 1.2, 1.1, 0.24, 1.7, and 0.03 mM. In order to make this easier to understand, the ratio of the concentration with PBS alone and the concentration in the presence of rL-PGDS was taken. The results are 21, 7.3, 11, 2.3, and 110 times the respective samples, and it can be seen that the solubility of each sample was improved.

  The present invention can be used for a DDS that supplies a drug into the body. Moreover, it can utilize as a solubilizing agent which improves the solubility of a ligand by mixing with the ligand containing a drug in polar solvents, such as water.

It is a figure which shows the structure of a lipocalin family. It is a figure which shows the characteristic of the amino acid sequence of a lipocalin family. It is a figure which shows the structure of a lipocalin type prostaglandin D synthase. It is a figure which shows the experimental result which confirms the pentobarbital anesthesia time extension effect of rL-PGDS diazepam complex. It is a figure which shows the photograph which dye | stained CA1 area | region of the hippocampus. It is a figure which shows the photograph which dye | stained CA1 area | region of the hippocampus. It is a figure which shows the photograph which dye | stained CA1 area | region of the hippocampus. It is a figure which shows the photograph which dye | stained CA1 area | region of the hippocampus. It is a graph which shows the cell number dye | stained by the CA1 area | region of the hippocampus for every sample.

Explanation of symbols

1 to 8 β strand 9 α helix 10 omega loop

Claims (7)

A solubilizing agent for improving the solubility of a compound having a molecular weight of 150 to 700 in a polar solvent ,
The solubilizing agent, wherein the solubilizing agent is a lipocalin type prostaglandin D synthase . Solubilizing agent according to claim 1, wherein said compound is an artificial synthetic. A polar solvent,
A compound having a molecular weight of 150 to 700;
Look contains a solubilizing agent for said compound,
A composition in which the solubilizing agent is lipocalin type prostaglandin D synthase . The composition according to claim 3 , wherein the polar substance is water. The composition according to claim 3 or 4 , wherein the compound is an artificial compound. The composition according to any one of claims 3 to 5 , wherein the compound is diazepam. The compounds, compositions according to any of claims 3 to 5 is NBQX.

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