RU2589258C1 - Agent of peptide structure, inhibiting dipeptidyl peptidase-4, and pharmaceutical composition based thereon - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to an agent for preventing, delaying progression and/or treating conditions pathogenesis of which involves DPP-4 enzyme, containing an active substance and a pharmaceutically acceptable carrier or carriers. As an active substance, agent comprises myristoyl tripeptide Myr-Trp-Arg-Glu-OH or hydrates, solvates or salt thereof.

EFFECT: invention provides a wider range of biologically active substances, used for preventing, delaying progression and/or treating diseases, disorders or conditions, which can be treated or prevented by inhibition of dipeptidyl peptidase-4 enzyme.

5 cl, 4 ex, 3 dwg, 3 tbl

Description

The invention relates to medicine and veterinary medicine, in particular to drugs used to prevent, slow the progression and / or treatment of conditions mediated by the enzyme dipeptidyl peptidase-4, i.e. diseases, disorders or conditions that can be treated or prevented by inhibiting this enzyme.

Dipeptidyl peptidase-4 (according to the IUBMB EC.3.4.14.5 enzyme nomenclature, hereinafter referred to as DPP-4) is a membrane-bound serine peptidase that is localized in a number of tissues (intestines, liver, lungs, kidneys), as well as on blood T-lymphocytes, where the enzyme is known as CD-26. In the literature, this enzyme has more names, including DPP4, DP4, DAP-IV, FAPS, protein 2, complexing with adenosine deaminase, protein binding of adenosine deaminase (ADAbp), dipeptidyl aminopeptidase IV; Xaa-Pro-dipeptidyl aminopeptidase; Gly-Pro-naphthylamidase; postprolindipeptidyl aminopeptidase IV; CD26 lymphocyte antigen; glycoprotein GP110; glycylproline-peptidase; glycyl proline aminopeptidase; X-prolyl dipeptidylamino peptidase; pepX; leukocyte antigen CD26; glycylprolyl dipeptidylamino peptidase; dipeptidyl peptide hydrolase; glycylprolylamino-peptidase; dipeptidyl aminopeptidase IV; aminoacylprolyl dipeptidylamino peptidase; T cell initiating Tp103 molecule; X-PDAP.

DPP-4 is a non-classical serine aminodipeptidase that cleaves Xaa-Pro dipeptides from the amino terminus (N-terminus) of polypeptides and proteins. It is also known that DPP-4-dependent slow release of X-Gly or X-Ser type dipeptides occurs in the case of some naturally occurring peptides.

DPP-4 inhibitors can be used as drugs to prevent, slow the progression and / or treatment of conditions mediated by DPP-4.

The studies revealed a number of compounds that inhibit DPP-4, which began to be used in medical practice.

For example, it is known to use a dipeptidyl peptidase 4 inhibitor (DPP-IV) for the treatment of preventing or reducing the risk of mortality due to cardiovascular diseases, including administering them to a suffering subject. An inhibitor is also administered to prevent or reduce the risk of non-fatal myocardial infarction and / or non-fatal stroke and / or diabetes mellitus. Saxagliptin, vildagliptin, sitagliptin (EA 201101231 A1, 09/30/2010) are used as such an inhibitor.

It is known that the use of DPP-4 inhibitors helps to restore impaired glucose-dependent secretion of insulin and to correct elevated glucagon levels - key disorders that are characteristic of diabetes mellitus.

In recent years, special attention has been paid all over the world to the role of the hormones of the gastrointestinal tract in the regulation of insulin secretion and, therefore, in the regulation of glucose homeostasis in the human body. Mainly, the above effects are due to the action of glucagon-like peptide-1 (GLP-1) (Green BD, Flatt PR Incretin hormone mimetics and analog in diabetes therapeutics // Best Practice & Research Clinical Endocrinology & Metabolism. - 2007. - T. 21. - No. 4. - C. 497-516). The disadvantage of GLP-1 is its short lifespan (4-5 minutes) due to the rapid inactivation by the enzyme DPP-4 (Hansen L., Deacon CF, Ørskov C. et al. Glucagon-Like Peptide-1- (7-36) Amide Is Transformed to Glucagon-Like Peptide-1- (9-36) Amide by Dipeptidyl Peptidase IV in the Capillaries Supplying the L Cells of the Porcine Intestine 1 // Endocrinology. - 1999.- T. 140.- No. 11. - C. 5356-5363.). Therefore, in clinical practice it is impossible to use GLP-1 as a drug.

DPP-4 inhibitors prevent the rapid inactivation of endogenous GLP-1, thereby enhancing and prolonging its action. Known DPP-4 inhibitors - gliptins (sitagliptin (Januvia ^® ), vildagliptin (Galvus ^® ), saxagliptin (Onglisa ^® ) and linagliptin (Trazhenta ^® )) are small molecules that inhibit the catalytic site of DPP-4.

Glyptins, inhibiting DPP-4, increase, in particular, the concentration of 2 known hormones of the incretin family: GLP-1 and glucose-dependent insulinotropic polypeptide. The use of DPP-4 inhibitors helps restore impaired glucose-dependent secretion of insulin and corrects elevated glucagon levels, key disorders that are characteristic of diabetes mellitus.

Gliptins lead to more than 90% inhibition of the enzyme (Fukuda-Tsuru S. et al. 2012) and, as a result, significantly reduce the formation of the metabolite GLP-1 (9-36) amide, which potentiates the action of the main hormone GLP-1 ( 7-36) amide.

Peptide inhibitors of DPP-4 are promising from the point of view of the relationship between safety and efficacy. Thus, for tripeptides containing Trp-Arg-Xaa, the IC50 for inhibiting DPP-4 is less than 500 μM (Lan, Ito, Ito, & Kawarasaki. (2014). Trp-Arg-Xaa tripeptides act as uncompetitive-type inhibitors of human dipeptidyl peptidase IV. Peptides, 54, 166-170). Peptides containing Trp-Arg-Xaa exhibit a competitive type of inhibition of DPP-4. In competitive inhibitors, whose effect is associated with interaction with the substrate, specific inhibition of DPP-4 occurs, which reduces potential toxic effects (Eckhardt, Langkopf, Mark et al. (2007) .8- (3- (R) -Aminopiperidin-1 -yl) -7-but-2-ynyl-3-methyl-1- (4-methyl-quinazolin-2-ylmethyl) -3, 7-dihydropurine-2, 6-dione (BI 1356), a highly potent, selective, long-acting, and orally bioavailable dpp-4 inhibitor for the treatment of type 2 diabetes. Journal of medicinal chemistry, 50 (26), 6450-6453).

Thus, there is a high demand for new DPP-4 inhibitors.

The objective of the present invention is to develop means with inhibitory DPP-4 action and the creation on its basis of a means to prevent, slow the progression and / or treatment of conditions in the pathogenesis of which the enzyme DPP-4 is involved.

The technical result provided by the invention is to expand the spectrum of biologically active substances used to prevent, slow the progression and / or treatment of conditions mediated by the enzyme dipeptidyl peptidase-4, i.e. diseases, disorders or conditions that can be treated or prevented by inhibiting this enzyme.

To solve this problem, a tool is proposed for preventing, slowing the progression and / or treatment of conditions in the pathogenesis of which the DPP-4 enzyme is involved, containing the active substance and a pharmaceutically acceptable carrier or carriers, which as an active principle includes myristoyl tripeptide or its hydrates, solvates or salts of the following structure:

Myr-Trp-Arg-Glu-OH

For structural analogues of the claimed compounds, the presence of specific inhibitory activity against DPP-4 was shown (Lan et al. 2014). The formation of peptide conjugates with fatty acids improves the pharmacokinetic profile and can lead to improved oral bioavailability (Novakovic, Anderson, Grasso. (2014). Myristic acid conjugation of [D-Leu-4] -OB3, a biologically active leptin-related synthetic peptide amide, significantly improves its pharmacokinetic profile and efficacy. Peptides. 62. 176-182). Natural objects, such as salmon, cattle meat, etc., can also serve as a source of isolation of peptides that have an inhibitory effect on DPP-4 (Li-Chan, Hunag, Jao, Ho, & Hsu. (2012). Peptides derived from Atlantic salmon skin gelatin as dipeptidyl-peptidase IV inhibitors. Journal of Agricultural and Food Chemistry, 60, 973-978; WO 2006068480 A3, Priority date 12/23/2004). It was previously shown that a complex of biologically active substances isolated from the gonads of sea urchins Strongylocentrotus droebachiensis, including peptides, amino acids and fatty acids, has an antidiabetic effect (Pat RU No. 2520695, C1, priority 04.02.2013). Further separation and purification revealed that the lipophilic gonad peptide of sea urchins S.droebachiensis has inhibitory activity against DPP-4. However, during storage, its destruction was observed. To ensure stability, an attempt was made to synthesize an analog of a natural peptide.

This agent with an inhibitory effect of DPP-4 contains Myr-Trp-Arg-Glu-OH and pharmaceutically acceptable excipients. Preferably, it is intended to normalize the level of incretins in the blood and / or normalize the function of endothelial cells.

The proposed tool is performed in the form of various dosage forms, preferably solid dosage forms — tablets, sustained-release tablets, capsules, granules, and powders. As auxiliary substances, substances commonly used in the pharmaceutical industry for the production of solid dosage forms can be used, for example, starch, sugar, cellulose and its derivatives, gelatin, polyvinylpyrrolidone, polyethylene oxide, calcium phosphate, a lubricant, a wetting agent such as sodium lauryl sulfate, polyoxyethylene sorbitan esters and fatty acids (twins), esters of sorbitan and fatty acids (spana), preferably starch, including modified starch, lactose, microcrystalline cellulose lulose, croscarboxymethyl cellulose sodium, polyvinylpyrrolidone, excipients - release inhibitors, lubricant. Examples of the latter are stearic acid and / or its salts — calcium stearate, magnesium stearate, zinc stearate, talc, colloidal silicon dioxide, polyethylene glycol, hydrogenated vegetable oil, liquid paraffin. The new composition may also contain flavors, colorants and / or flavors.

Preferably, the preparation is made in the form of a tablet, which may be coated. The presence of the latter improves the appearance, organoleptic properties of the dosage form, protects it from mechanical damage.

A preferred amount of active principle (Myr-Trp-Arg-Glu-OH) in a single dose is from 1 mg to 50 mg, more preferably 5 mg. As used herein, the term Myr-Trp-Arg-Glu-OH also includes hydrates, solvates and salts of Myr-Trp-Arg-Glu-OH, for example, hydrochloride.

Obtaining the claimed dosage form can be carried out in accordance with known methods for the manufacture of solid dosage forms. All methods include the step of combining the active ingredient with a carrier, which consists of one or more accessory ingredients. In the General case, the claimed pharmaceutical composition is obtained by unifying and uniformly combining Myr-Trp-Arg-Glu-OH with a liquid or finely divided solid carrier, or both, and then, if necessary, give the product the desired shape, for example, by the wet method granulation, followed by the addition of a lubricant to the dry granules, molding the final mixture of ingredients to form a dosage form of a given configuration and size, and, if necessary, by coating, or by direct compression followed by the application shell.

The basis of the invention lies unexpectedly discovered by the authors of the property of the previously not described compound Myr-Trp-Arg-Glu-OH in relation to the key enzyme of DPP-4 metabolism. As a result of studies, Myr-Trp-Arg-Glu-OH was identified as a tool for inhibiting the enzyme DPP-4.

The results of mass spectrometric analysis and chromatographic purity are attached to the description as illustrative materials (Figures 1 and 2).

Figure 1 shows a mass spectrometric analysis confirming the structure of myristoyl tripeptide (Myr-Trp-Arg-Glu-OH).

Figure 2 - HPLC chromatogram of the inventive myristoyl tripeptide. Analysis conditions: a Shimadzu chromatograph (Japan) with a Luna C ₁₈ (2) column of 4.6 × 150 mm (particle size of the sorbent 5 μm) and a pre-column (3 mm) filled with the same sorbent (Phenomenex, USA) in the gradient mode of elution with the mixture 0.03% solution of trifluoroacetic acid (phase A) and acetonitrile (phase B), eluent feed rate 1.0 ml / min, sample volume 20 μl, detection wavelength 210 nm).

Figure 3 - inhibitory activity of the studied drugs.

Pharmacological properties and the method of obtaining claimed in the present invention myristoyl tripeptide (Myr-Trp-Arg-Glu-OH) are not described in the literature.

The synthesis of the claimed myristoyl tripeptide was carried out according to the following procedure. The list of abbreviations used is presented in table 1.

6.0 g of 2-chlorotrityl resin (1.5 mmol / g capacity) in 45 ml of DCM was suspended in a solid-phase synthesis reactor, kept for 5 min, the resin was filtered off, washed with 2 × 30 ml of DCM. A solution of 4.25 g (10.0 mmol) of Fmoc-Glu (OtBu) -OH and 6 ml (36 mmol) of DIPEA in 30 ml of DCM was added to the resin, stirred for 60 min at room temperature. The resin was filtered off, washed with 2 × 30 ml of DCM, treated with 2 × 30 ml of a mixture of DCM / methanol / DIPEA (17: 2: 1) for 10 min, washed with 2 × 30 ml of DCM and 3 × 30 ml of DMF. 30 ml of a 20% solution of diethylamine in DMF were loaded into the reactor, stirred for 5 min, filtered, washed with 3 × 30 ml of DMF, 30 ml of a 20% solution of diethylamine in DMF was added, kept for 20 min, filtered, washed with 5 × 30 ml DMF

A cooled (4 ° C) solution of 12.98 g (20.0 mmol) of Fmoc-Arg (Pbf) -OH, 2.98 g (22.0 mmol) of HOBt and 3.42 ml (22.0 mmol) of DIC in 30 ml of DMF was loaded into the reactor, stirred for 2 hours at room temperature. The resin was filtered, washed with 6 × 30 ml of DMF, 30 ml of a 20% diethylamine in DMF solution was added, stirred for 5 min, filtered, washed with 3 × 30 ml of DMF, 30 ml of a 20% diethylamine in DMF solution was added, kept for 20 min , filtered, washed with 5 × 30 ml of DMF.

A cooled (4 ° C) solution of 10.53 g (20.0 mmol) of Nα-Fmoc-N (in) -Boc-L-Trp-OH, 2.98 g (22.0 mmol) of HOBt and 3.42 ml (22.0 mmol) of DIC in 30 were charged into the reactor ml of DMF was stirred for 2 hours at room temperature. The resin was filtered, washed with 6 × 30 ml of DMF, 30 ml of a 20% diethylamine in DMF solution was added, stirred for 5 min, filtered, washed with 3 × 30 ml of DMF, 30 ml of a 20% diethylamine in DMF solution was added, kept for 20 min , filtered, washed with 5 × 30 ml of DMF.

A cooled (4 ° C) solution of 4.57 g (20.0 mmol) of myristic acid (MA), 2.98 g (22.0 mmol) of HOBt and 3.42 ml (22.0 mmol) of DIC in 30 ml of DMF was loaded into the reactor, stirred for 2 hours at room temperature . The resin was filtered off, washed with 6 × 30 ml of DMF, 4 × 30 ml of DCM, dried, 30 ml of a mixture of TFA / TIS / EDT / H ₂ O (37: 1: 1: 1) was added, kept for 4 hours at room temperature, filtered off, washed with 3 × 20 ml trifluoroacetic acid, the combined filtrates were evaporated to a volume of ~ 20 ml, 60 ml of dry ether was added to the residue. The precipitate was filtered off, washed on the filter with ether, and dried. The resulting product was dissolved in 50 ml of water, the solution was frozen and lyophilized. The lyophilisate was dissolved in 30 ml of water and applied to an Amberlite IRA-400 ion exchange resin column (Cl ^- form). The column was washed with water, the fractions containing the product were combined, evaporated to a volume of ~ 40 ml and applied to a Waters X-Bridge C18 reverse phase column, 10 μm, 127 Å, 50 × 250 mm. Elution was carried out at an eluent flow rate of 50 ml / min. Phase A: 0.1% HCl / H ₂ O, B: / acetonitrile. Gradient: 0% (B) -70% (B) in 70 minutes. Fractions containing the main product were combined, evaporated to a volume of ~ 50 ml, frozen and lyophilized. Received 5.2 g (74%) of product with a purity (HPLC) of 95.3%. Mass spectrum: calculated M ⁺ 700.88, obtained M ⁺ 700.44. Amino acid analysis: arginine 1.00 (1), glutamic acid 0.95 (1), tryptophan 1.00 (1).

The following examples of pharmacological studies confirm the possibility of using the claimed Myr-Trp-Arg-Glu-OH as an agent with an inhibitory effect of DPP-4.

Example 1. Determination of the activity of DPP-4 in vitro using a recombinant human enzyme and an enzyme contained in rat plasma

The activity of the DPP-4 enzyme was determined using glycyl-L-proline-p-nitroanilide as a chromogenic substrate, from which glycyl-L-proline dipeptide (Gly-Pro) and p-nitroaniline are formed under the action of DPP-4. In the presence of DPP-4 inhibitors, the concentration of reaction products decreases. For determination, we used a substrate solution with a concentration of 1 mmol / L (glycyl-L-proline-p-nitroanilide), in 0.1 M Tris-buffer solution, pH = 8.0 (working buffer).

The analytical part of the study was carried out in 96-well plates. 20 μl of rat plasma or enzyme solution (0.025 IU / ml) and 40 μl of working buffer solution were added to the rows of wells. Then, 40 μl of substrate solution was added to all wells to start the reaction, after which the plate was incubated for 30 min at 37 ° C. During this time, the optical density of the reaction mixture at 405 nm every 5 minutes was measured on an xMark ™ Microplate Spectrophotometer (Bio-Rad, USA).

After incubation, the dependence of the change in optical density at 405 nm on the time of the enzyme reaction D = f (t, min) for each sample was built. The linear region of variation of the optical density was determined and the linear equation y = a × x + b was constructed with a regression coefficient of at least 0.99. For further calculations, we used the value of the slope of the linear dependence (tanα, equal to the value of coefficient “a”).

The% inhibitory activity was calculated by the formula:

IA (%) = (tgα _control -tgα _test ) ∗ 100 / tgα _control ,

where tgα _control is the slope of the linear dependence obtained for the positive control (100% enzyme activity); tgα _sample - the slope of the linear dependence obtained for the tested samples (samples with tested drugs and a specific inhibitor).

According to the analysis of a series of solutions of the studied drugs with different concentrations, for which the IA values (%) are in the range from 10 to 90%, the dependence of IA on the drug concentration (mg / ml) was built. By the obtained regression equation determined depending IC ₅₀ - the concentration of the substance (mg / ml) causing 50% inhibition of DPP-4.

For determination, we used blood plasma obtained from outbred male rats aged 10-12 weeks with a body weight of 200-250 g. Blood was taken postmortally. Euthanasia was performed using a CO ₂ camera. The animals were there until complete loss of consciousness, then the animal was removed, the chest cavity was opened, and complete sterile bleeding with a syringe from the heart cavities was carried out.

Blood was collected in test tubes, heparin was used as an anticoagulant. Blood samples were centrifuged at 1800 g for 20 min to obtain plasma at a temperature of + 4 ° C. Frozen plasma was stored until analysis at -20 ° C.

Before the quantitative determination of the activity of DPP-4, the samples were thawed. Sitagliptin and a specific inhibitor of DPP-4, S-EPA, were used as reference preparations. Inhibitory activity depending on the dose of the studied drugs are presented in table 2.

The inhibitory activity of the test substance against DPP-4 of human blood plasma and laboratory animals is presented in table 3.

To determine the type of inhibition and Michaelis constants for calculating K _m was used graphical method using double reverse coordinates Lineweaver-Burk, i.e. built dependencies 1 / v from 1 / [S]. By the nature of the dependencies, a conclusion was drawn about the type of inhibition.

It was found that Myr-Trp-Arg-Glu-OH exhibits the properties of a competitive inhibitor of DPP-4, while the calculated value of K _m against the background of the use of Myr-Trp-Arg-Glu-OH was 0.842, against 0.486 in the control experiment.

Example 2. Determination of the inhibitory activity of Myr-Trp-Arg-Glu-OH in vivo

The study of inhibitory activity was carried out with intragastric administration to outbred male rats. 7 groups of animals were formed, 10 pieces each. Myr-Trp-Arg-Glu-OH was administered in three doses: 0.1 mg / kg, 1.0 mg / kg and 10 mg / kg. Comparison drug sitagliptin was administered at a dose of 100 mg / kg (Fukuda-Tsuru, Anabuki, Abe et al. (2012). A novel, potent, and long-lasting dipeptidyl peptidase-4 inhibitor, teneligliptin, improves postprandial hyperglycemia and dyslipidemia after single and repeated administrations. European journal of pharmacology, 696 (1), 194-202) and additionally at doses of 1 mg / kg and 10 mg / kg. The activity of DPP-4 was evaluated before administration, 1, 6, and 24 hours after administration of the objects. Blood for analysis was taken from the tail vein in a volume of 0.5 ml in heparinized tubes. Then centrifuged for 15 minutes (1800 g, 4 ° C). The obtained blood plasma was stored at -20 ° C until analysis. The analytical part of the study and the calculation of inhibitory activity was carried out analogously to example 1. The results of the determination are presented in Figure 3.

The introduction of sitagliptin at a dose of 100 mg / kg led to 90% inhibition after 1 hour and 6 hours and decreased to 20% after 24 hours after administration, which is consistent with published data. A single injection of Myr-Trp-Arg-Glu-OH at a dose of 0.1 mg / kg did not affect the activity of the enzyme DPP-4. The introduction of Myr-Trp-Arg-Glu-OH at a dose of 1 mg / kg had an inhibitory effect (17%) after 6 hours. With the introduction of Myr-Trp-Arg-Glu-OH at a dose of 10 mg / kg, the enzyme activity was reduced on average 34% and was expressed during the day of observation.

Example 3 Preparation of Coated Tablets

The sifted powders of Myr-Trp-Arg-Glu-OH in an amount of 20 g, 162 g of microcrystalline cellulose and 6 g of crospovidone are mixed until homogeneous and moistened with a 40% low molecular weight polyvinylpyrrolidone solution, the mass is granulated, the granulate is dried at 45-55 ° C to a residual moisture of 2 , 0-3.0%. The dry substance was granulated again, 2 g of magnesium stearate was added to the granulate and tabletted on a tablet press to obtain tablet cores that contained 10 mg of Myr-Trp-Arg-Glu-OH.

The composition for the gastro-soluble coating is prepared as follows. 6.5 g of hydroxypropyl methylcellulose are added to 69.5 g of water with stirring with stirring and allowed to swell with stirring. Separately, a solution of polyethylene glycol is prepared from 3 g, 1 g of polysorbate and 12.5 g of water, 4.5 g of talc and 3 g of titanium dioxide are added to it, mixed, the resulting suspension is added to a solution of hydroxypropyl methylcellulose. The mixture is mixed until uniform and used to coat the core tablets until a uniform coating is obtained with an average tablet weight increase of about 2.5-3%.

Example 4 Softgels with enteric coating.

333 g of Myr-Trp-Arg-Glu-OH are dissolved with stirring in 1667 g of medium chain triglycerides. Separately, a solution of agar-agar polysaccharide is prepared and 0.3 g soft capsules with enteric coating are prepared in accordance with the application 2014142863 (dated 23.10.2014).

Thus, a decrease in the activity of DPP-4 leads to the stabilization of incretin received from the outside, i.e., by using Myr-Trp-Arg-Glu-OH, the splitting of incretin in the blood can be controlled, thereby normalizing the level of incretins. This implies the possibility of using Myr-Trp-Arg-Glu-OH to prevent, slow the progression and / or treatment of conditions / diseases mediated by the enzyme dipeptidyl peptidase IV, preferably to normalize the level of incretins in the blood and / or normalize the function of endothelial cells.

Claims (5)

1. Means for preventing, slowing the progression and / or treatment of conditions in the pathogenesis of which the DPP-4 enzyme is involved, containing the active substance and a pharmaceutically acceptable carrier or carriers, which as an active principle includes myristoyl tripeptide or its hydrates, solvates or salts of the following structure:
Myr-Trp-Arg-Glu-OH 2. The tool according to p. 1, characterized in that it is one of the following dosage forms suitable for oral or parenteral use: sublingual tablets, tablets and capsules, injection, nasal spray, nasal drops, subbuccal patch, rectal suppositories. 3. The tool according to p. 1 or 2, characterized in that the amount of active principle (Myr-Trp-Arg-Glu-OH) in a single dose of the drug is: from 1 mg to 50 mg, more preferably 5 mg. 4. The tool according to p. 1 or 2, characterized in that the active substance is in the form of its salt - hydrochloride. 5. The tool according to p. 1 or 2, characterized in that it further comprises pharmaceutically acceptable excipients.

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