LONG-ACTING FATTY ACID-CONJUGATED GnRH DERIVATIVES AND PHARMACEUTICAL COMPOSITIONS CONTAINING SAME
20220054593 · 2022-02-24
Assignee
Inventors
Cpc classification
A61K38/24
HUMAN NECESSITIES
A61K47/542
HUMAN NECESSITIES
A61P15/08
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61K38/24
HUMAN NECESSITIES
Abstract
An aspect of the present disclosure pertains to a novel long-acting fatty acid-conjugated gonadotrophin-releasing hormone (GnRH) derivative and a pharmaceutical composition containing the same. A GnRH derivative of the present invention is expected to greatly contribute, through excellent bioavailability, increased half-life in blood, and remarkably high therapeutic effects on sex hormone-dependent disease, to the reduction in drug dosing frequency and dosage and the like in the treatment of sex hormone-dependent diseases. Particularly, the GnRH derivative can overcome the disadvantages of existing GnRH sustained-release preparations, which have the side effects of residual feeling and pain at the injection site.
Claims
1. A pharmaceutical composition for preventing or treating a sex hormone-dependent disease, the pharmaceutical composition comprising: a long-acting fatty acid-conjugated gonadotropin-releasing hormone (GnRH) derivative in which the gonadotropin-releasing hormone (GnRH) derivative is conjugated with a fatty acid, or a pharmaceutically acceptable salt thereof as an active ingredient; and cyclodextrin, wherein the GnRH derivative comprises an amino acid sequence selected form the group consisting of SEQ ID NO: 2, and SEQ ID NOS: 4 to 11, wherein the fatty acid is a lauric acid, palmitic acid or arachidic acid, wherein a carboxyl group of the fatty acid of the GnRH derivative is conjugated through a peptide-bond to an amino terminus of the peptide portion of the GnRH derivative.
2. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of inorganic acids, organic acids, ammonium salts, alkali metal salts, and alkaline earth metal salts.
3. The pharmaceutical composition of claim 2, wherein the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, hydrobromide, phosphate, metaphosphate, nitrate, sulfate, acetate, sulfonate, benzoate, citrate, ethanesulfonate, furmarate, lactate, maleate, malate, succinate, tartrate, sodium salt, calcium salt, potassium salt, and magnesium salt.
4. The pharmaceutical composition of claim 1, wherein the sex hormone-dependent disease is selected from the group consisting of prostate cancer, breast cancer, ovarian cancer, endometriosis, uterine fibroid, polycystic ovary disease, central precocious puberty, hypertrichosis, gonadotroph pituitary adenoma, sleep apnea, irritable bowel syndrome, premenstrual syndrome, benign prostatic hyperplasia, and contraception.
5. The pharmaceutical composition of claim 1, wherein the cyclodextrin is methyl-β-cyclodextrin.
6. The pharmaceutical composition of claim 1, wherein the fatty acid-conjugated GnRH derivative and the cyclodextrin exist together as an inclusion complex.
7. The pharmaceutical composition of claim 1, wherein the cyclodextrin and the fatty acid-conjugated GnRH derivative are used at a molar ratio of 7:1 to 1:1.
8. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises a biodegradable polymer.
9. A method for preventing or treating a sex hormone-dependent disease, comprising administering a pharmaceutical composition to a patient in need thereof, wherein the pharmaceutical composition comprising: a long-acting fatty acid-conjugated gonadotropin-releasing hormone (GnRH) derivative in which the gonadotropin-releasing hormone (GnRH) derivative is conjugated with a fatty acid, or a pharmaceutically acceptable salt thereof as an active ingredient; and cyclodextrin, wherein the GnRH derivative comprises an amino acid sequence selected form the group consisting of SEQ ID NO: 2, and SEQ ID NOS: 4 to 11, wherein the fatty acid is a lauric acid, palmitic acid or arachidic acid, wherein a carboxyl group of the fatty acid of the GnRH derivative is conjugated through a peptide-bond to an amino terminus of the peptide portion of the GnRH derivative.
10. The method of claim 9, wherein the pharmaceutically acceptable salt is selected from the group consisting of inorganic acids, organic acids, ammonium salts, alkali metal salts, and alkaline earth metal salts.
11. The method of claim 10, wherein the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, hydrobromide, phosphate, metaphosphate, nitrate, sulfate, acetate, sulfonate, benzoate, citrate, ethanesulfonate, furmarate, lactate, maleate, malate, succinate, tartrate, sodium salt, calcium salt, potassium salt, and magnesium salt.
12. The method of claim 9, wherein the sex hormone-dependent disease is selected from the group consisting of prostate cancer, breast cancer, ovarian cancer, endometriosis, uterine fibroid, polycystic ovary disease, central precocious puberty, hypertrichosis, gonadotroph pituitary adenoma, sleep apnea, irritable bowel syndrome, premenstrual syndrome, benign prostatic hyperplasia, and contraception.
13. The method of claim 9, wherein the cyclodextrin is methyl-β-cyclodextrin.
14. The method of claim 9, wherein the fatty acid-conjugated GnRH derivative and the cyclodextrin exist together as an inclusion complex.
15. The method of claim 9, wherein the cyclodextrin and the fatty acid-conjugated GnRH derivative are used at a molar ratio of 7:1 to 1:1.
16. The method of claim 9, wherein the pharmaceutical composition further comprises a biodegradable polymer.
Description
BRIEF DESCRIPTION OF DRAWINGS
[0064]
[0065]
[0066]
[0067]
[0068]
[0069]
[0070]
[0071]
[0072]
[0073]
[0074]
[0075]
DETAILED DESCRIPTION
[0076] Hereinafter, the embodiments of the present disclosure will be described by referring to the Preparation Examples and Examples, which are set forth to illustrate the present disclosure, but not construed to limit the present invention.
Preparation Example 1: Preparation of Gonadotrophin-Releasing Hormone (GnRH) Derivatives
[0077] Natural mammalian GnRH has the following sequence.
TABLE-US-00002 [Mammalian GnRH Sequence] (SEQ ID NO: 1) pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly
[0078] Leuprolide® having the mammalian GnRH sequence with the substituted D-Leu instead of Gly at position 6 and the substituted des-Gly instead of Gly at position 10 was used as a backbone for the derivative and the fatty acid-conjugate derivative according to an embodiment of the present disclosure.
TABLE-US-00003 [Leuprolide Sequence] (SEQ ID NO: 2) pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt
[0079] Derivatives where glutamate at position 1 on the Leuprolide sequence remains unsubstituted or was substituted with glutamine were prepared as follows.
[0080] For reference, the sequence of triptorelin is provided as follows:
TABLE-US-00004 [Triptorelin Seqeunce] (SEQ ID NO: 3) pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2
[0081] (1) Preparation Method for GnRH Derivative Peptides
[0082] The derivatives are synthesized using a general Fmoc/tBu solid-phase peptide synthesis (SPPS) method, where the α-amino groups of amino acid residues are protected by the base-labile group of Fmoc (fluorenylmethyloxycarbonyl chloride) while the side groups are protected by an acid-labile group. In the solid phase peptide synthesis method comprising the following steps, a peptide chain is sequentially extended by repetitive Fmoc cleavage and amino acid coupling.
[0083] {circle around (1)} Load Fmoc amino acid onto resin (Fmoc-Pro-trityl resin);
[0084] {circle around (2)} Remove Fmoc protecting group from Fmoc-AA-resin (20% piperidine/DMF);
[0085] {circle around (3)} Wash with DMF;
[0086] {circle around (4)} Bind amino acid after activation (DIC/HOBt used);
[0087] {circle around (5)} Wash with DMF;
[0088] {circle around (6)} Repeat steps {circle around (2)} to {circle around (5)} to bind amino acids sequentially;
[0089] {circle around (7)} Remove resin only from synthesized peptide (1.5% TFA/DCM);
[0090] {circle around (8)} Attach ethylamine to the amino terminus of the resulting peptide (using EDC.HCl/HOAt); and
[0091] {circle around (9)} Make overall cleavage of protected side chains from the resulting peptide (92.5% TFA/2.5% TIS/2.5% EDT/2.5% H2O).
[0092] According to the preparation method above, derivatives having the sequences described in Table 2 were prepared. Lauric acid, palmitic acid, or arachidic acid was conjugated to the amino terminus of the obtained GnRH derivative, as instructed in Table 2. Conjugating a fatty acid to the amino terminus of the derivative was carried out in the same manner as the conjugation of general amino acids. The synthesis of the GnRH derivatives of the Comparative Examples and Examples was entrusted to Anygen Co. Ltd.
[0093] (2) Purification of GnRH Derivative Peptides
[0094] Following the TFA cleavage, the peptide was purified using a C18 column in the Shimadzu HPLC 10AVP system under HPLC conditions (A buffer 0.05% TFA/H.sub.2O, B buffer 0.05% TFA/acetonitrile, flow rate 1 mL/min, wavelength 230 nm). Purification results in each Example were entrusted to and obtained from Anygen Co. Ltd. and are shown in Table 1. In Table 1, the GnRH derivatives of Example 1 (L1), Example 3 (P1), Example 5 (P3), and Example 7 (A1) have glutamate as the amino acid residue at position 1, while the GnRH derivatives of Example 2 (L2), Example 4 (P2), Example 6 (P4), and Example 8 (A2) have glutamine as the amino acid residue at position 1 (see Table 2).
TABLE-US-00005 TABLE 1 MS GnRH Derivative Purity (%) Calc. (Da) Found (Da) Example 1(L1) 98.4 1409.7 1409.6 Example 2(L2) 99.0 1408.7 1409.0 Example 3(P1) 98.2 1465.8 1465.7 Example 4(P2) 98.2 1464.8 1464.9 Example 5(P3) 98.1 1465.8 1465.5 Example 6(P4) 98.3 1464.8 1464.0 Example 7(A1) 98.9 1521.7 1521.1 Example 8(A2) 98.8 1520.7 1520.9
[0095] Lauric acid, palmitic acid, and arachidic acid are sparingly soluble in water and exist as solid phases at room temperature with respective melting points of about 43.8° C., about 60° C., and about 75.5° C. Hence, as the fatty acid-conjugated GnRH derivatives might be poorly soluble in water, salting was further carried out. The fatty acid-conjugated GnRH derivatives were subjected to salting with sodium salt or acetate to prepare the fatty acid-conjugated GnRH derivative salts of Comparative Examples 1 to 3 and Examples 1 to 8, as shown in Table 2 below. These processes were entrusted to Anygen Co. Ltd.
TABLE-US-00006 TABLE 2 Derivative Backbone/Derivative Sequence and Salt Comparative pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH.sub.2 acetate Example 1 salt (SEQ ID NO: 1) (GnRH) Comparative pyroGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt acetate Example 2 salt (SEQ ID NO: 2) (Leuprolide) Comparative pyroGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH.sub.2 acetate Example 3 salt (SEQ ID NO: 3) (Triptorelin) Example 1 Lauric acid-Glu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt acetate salt (SEQ ID NO: 4) Example 2 Lauric acid-Gln-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt acetate salt (SEQ ID NO: 5) Example 3 Palmitic acid-Glu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt sodium salt (SEQ ID NO: 6) Example 4 Palmitic acid-Gln-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt sodium salt (SEQ ID NO: 7) Example 5 Palmitic acid-Glu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt acetate salt (SEQ ID NO: 8) Example 6 Palmitic acid-Gln-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt acetate salt (SEQ ID NO: 9) Example 7 Arachidic acid-Glu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt acetate salt (SEQ ID NO: 10) Example 8 Arachidic acid-Gln-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt acetate salt (SEQ ID NO: 11)
Preparation Example 2: Preparation of Pharmaceutical Composition Comprising Inclusion Complex of GnRH Derivative and Cyclodextrin
[0096] Methyl-β-cyclodextrin powder (SigmaAldrich; Lot no.: C4555; Mw.: 1303.311 g/mol) was dissolved in the amounts given in Table 3 in 1 mL of sterilized tertiary distilled water.
[0097] The GnRH derivatives were added in the amounts given in Table 3 to respective 15-mL conical tubes to which 1 mL of the aqueous β-cyclodextrin solution corresponding to the mole ratios of Table 3 was then added before mixing overnight at room temperature with the aid of a low-speed rotator. Subsequently, the solutions contained in the 15-mL tubes were collected by a brief spin down and then transferred to 1.5 mL tubes, followed by secondary mixing overnight at room temperature by a low-speed rotator. Finally, pharmaceutical compositions containing each the active ingredients at a concentration of about 10 mM were obtained. The compositions were used in the following Experimental Examples optionally after being diluted. For Comparative Examples 1 to 3, the compositions were diluted to 20 mM in tertiary distilled water.
TABLE-US-00007 TABLE 3 Pharmaceutical Composition (mg/1 mL) EXAMPLE 9 10 11 12 13 14 15 16 C. Example Example 1 2 3 1 2 3 4 5 6 7 8 GnRH 24 mg 24 mg 24 mg 14 mg 14 mg 15 mg 15 mg 15 mg 15 mg 15 mg 15 mg Derivative Methyl-β-cyclodextrin — — — 13 mg 13 mg 13 mg 65 mg 13 mg 65 mg 13 mg 13 mg Mole ratio — — — 1:1 1:1 1:1 5:1 1:1 5:1 1:1 1:1 (cyclodextrin:GnRH Derivative)
[0098] The following Experimental Examples were carried out with the pharmaceutical compositions.
[Experimental Example 1] Evaluation of Inclusion Complex of Fatty Acid-Conjugated GnRH Derivative and Cyclodextrin
[0099] For use as a control, a solution of 13 mg of methyl-β-cyclodextrin powder in 1 mL of sterile, tertiary distilled water was prepared in the same manner as in Preparation Example 2 in a 1.5-mL tube (concentration 10 mM).
[0100] The prepared control, and the compositions of Examples 9 to 16 finally prepared by mixing in 1.5-mL tubes were observed with the naked eye. They were spun down at 10,000 rpm for 40 seconds to 50 seconds (less than 1 minute) and again observed with the naked eye to evaluate solubility. The results of Examples 9, 10, 15, and 16 are depicted in
[0101] As shown in
[0102] As implied by the results, the pharmaceutical compositions containing GnRH derivatives and cyclodextrin according to an embodiment of the present invention exist as suspensions in which the ingredients are well suspended, thus achieving the effects and purposes of long-acting or sustained released agents.
[Experimental Example 2] Assay for Viability of Prostate Cancer Cell Lines
[0103] GnRH derivatives are clinically applied to the therapy of diseases including breast cancer, prostate cancer, endometriosis, central precocious puberty, and the like. Hence, different prostate cancer cell lines (DU-145, PC3, and LNCaP cell lines) were each cultured in an RPMI 1640 medium (containing 10% FBS, penicillin/streptomycin, 1% non-essential amino acids) in a T75 flask and cultured at 37° C. under the atmosphere of 5% CO2/95% air in a sterile incubator. An assay for cell viability was performed using the Cell Counting Kit-8 (CCK-8, manufactured by DOJINDO). Each of the cell lines was separated from the T75 flask by trypsinization and transferred to 96-well plates at a density of 1×104 cells/mL for DU-145 and at a density of 1×105 cells/mL for PC3 and LNCaP, followed by incubation for one hour for attachment.
[0104] Subsequently, each cell line was treated with 100 μM and 200 μM of each of the derivatives of Comparative Examples 1 to 3 and Examples 9 to 16 and the control. In brief, 1% methyl β-cyclodextrin was used as a negative control for cell viability while 0.1% sodium dodecyl sulfate (SDS) served as a positive control. After 48 hours of incubation, the existing culture medium was removed, and 100 μL of fresh culture medium and 10 μL of CCK-8 solution were applied to each cell line. Again, after 48 hours of incubation, the medium solution was replaced by 100 μL of fresh culture medium and 10 μL of CCK-8 solution. The cells were incubated for 4 hours, and then the absorbance was measured at 450 nm to assess cell viability. The measurement results are provided in Tables 4 to 6 and
TABLE-US-00008 TABLE 4 Viability of DU-145 relative to negative control Treatment 1% methyl-β-cyclodextrin Statistical Derivative conc. (μM) (%) significance Example 9 100 50.00 ± 0.63 <0.01 200 30.80 ± 0.66 <0.01 Example 10 100 36.40 ± 6.64 <0.01 200 25.40 ± 3.96 <0.01 Example 11 100 47.25 ± 2.50 <0.02 200 35.25 ± 0.25 <0.01 Example 12 100 36.75 ± 1.11 <0.01 200 33.50 ± 0.50 <0.01 Example 13 100 81.50 ± 1.44 <0.01 200 41.75 ± 1.18 <0.01 Example 14 100 38.50 ± 0.29 <0.01 200 32.00 ± 0.71 <0.01 Example 15 100 68.25 ± 0.95 <0.01 200 51.25 ± 2.95 <0.01 Example 16 100 50.25 ± 3.35 <0.01 200 39.00 ± 2.27 <0.01 C. Example 1 100 107.50 ± 2.56 not significant (N.S) 200 101.08 ± 3.26 N.S C. Example 2 100 99.42 ± 2.06 N.S 200 100.17 ± 3.07 N.S C. Example 3 100 100.92 ± 2.98 N.S 200 94.27 ± 3.47 N.S Positive 18.18 ± 2.32 <0.01 Control (0.1% SDS)
TABLE-US-00009 TABLE 5 Viability of PC3 relative to negative control Treatment 1% methyl-β-cyclodextrin Statistical Derivative conc. (μM) (%) significance Example 9 100 41.00 ± 4.36 <0.02 200 39.00 ± 4.73 <0.02 Example 10 100 41.00 ± 1.53 <0.02 200 23.00 ± 5.51 <0.02 Example 11 100 65.60 ± 1.69 <0.01 200 29.80 ± 2.54 <0.01 Example 12 100 29.20 ± 3.04 <0.01 200 19.20 ± 1.93 <0.01 Example 13 100 99.60 ± 5.84 N.S 200 104.60 ± 3.52 N.S Example 14 100 90.80 ± 4.04 N.S 200 54.80 ± 3.93 <0.01 Example 15 100 72.50 ± 5.33 <0.02 200 63.75 ± 5.56 <0.02 Example 16 100 45.75 ± 5.41 <0.02 200 41.33 ± 3.38 <0.02 C. Example 1 100 105.44 ± 1.76 N.S 200 100.11 ± 1.82 N.S C. Example 2 100 104.22 ± 3.15 N.S 200 104.11 ± 2.66 N.S C. Example 3 100 99.67 ± 3.17 N.S 200 95.44 ± 4.03 N.S Positive 15.11 ± 1.72 <0.01 Control (0.1% SDS)
TABLE-US-00010 TABLE 6 Viability of LNCaP relative to negative control Treatment 1% methyl-β-cyclodextrin Statistical Derivative conc. (μM) (%) significance Example 9 100 91.33 ± 10.73 N.S 200 45.33 ± 5.61 <0.02 Example 10 100 28.33 ± 3.18 <0.02 200 22.33 ± 2.33 <0.02 Example 11 100 37.00 ± 0.58 <0.02 200 30.67 ± 0.67 <0.02 Example 12 100 29.67 ± 1.20 <0.02 200 30.33 ± 0.88 <0.02 Example 13 100 72.33 ± 5.84 <0.02 200 66.33 ± 5.24 <0.02 Example 14 100 33.00 ± 1.53 <0.02 200 30.33 ± 0.67 <0.02 Example 15 100 64.25 ± 3.52 <0.02 200 36.50 ± 1.94 <0.02 Example 16 100 18.25 ± 0.63 <0.02 200 18.50 ± 0.50 <0.02 C. Example 1 100 105.64 ± 4.03 N.S 200 93.64 ± 4.20 N.S C. Example 2 100 99.22 ± 6.11 N.S 200 84.78 ± 6.16 N.S C. Example 3 100 92.89 ± 4.33 N.S 200 90.22 ± 4.03 N.S Positive 20.45 ± 1.58 <0.01 Control (0.1% SDS)
[0105] With reference to Tables 4 to 6 and
[0106] In detail, the prostate cell line DU-145 survived the compositions of Comparative Examples 1 to 3 at substantially the same rates as the negative control. Only the composition of Comparative Example 3 allowed a viability of about 94% for the cells. In contrast, the compositions according to an embodiment of the present disclosures reduced the cell viability to at least about 80% and to down to about 25%, compared to the negative control. The remarkably reduced cell viabilities by the compositions according to an embodiment of the present disclosure were statistically significant.
[0107] For PC3 prostate cancer cells, the compositions of Comparative Examples 1 to 3 showed slight reducing effects on cell viability. Only the triptorelin of Comparative Example 3 allowed about 95% for the cell viability. In contrast, the compositions according to an embodiment of the present disclosure showed a very significant reduction in the cell viability. Inter alia, the composition of Example 12 exhibited very highly reduced cell viability, compared to those of the Comparative Examples. Particularly, the composition of Example 12 reduced the cell viability to a similar degree to that which the positive control achieved.
[0108] When applied at a concentration of 200 μM to LNCaP cells, the compositions of Comparative Examples 1, 2, and 3 allowed cell viabilities of about 93%, about 85%, and about 90%, respectively. However, the compositions according to an embodiment of the present disclosure showed very high cell death effects, compared to the compositions of the Comparative Examples. Particularly, the cell death effect was found to be similar between the composition of Example 10 and the positive control and higher in the composition of Example 16 than the positive control.
[0109] According to the experimental data on the three different prostate cancer cell lines, the compositions of Examples 10, 12, 14, and 16 comprising the derivatives of Examples 2, 4, 6, and 8 in which the amino acid at position 1 is substituted from glutamic acid to glutamine were superior in cell death effect to those of Examples 9, 11, 13, and 15 comprising the derivatives of Examples 1, 3, 5, and 7 in which the amino acid at position 1 remains intact. These data indicate that the GnRH derivatives characterized by amino acid substitution at position 1, fatty acid conjugation, and conversion to salt according to an embodiment of the present disclosure exhibit an unpredictably excellent prostate cancer cell death effect.
[Experimental Example 3] Assay for Ovarian Morphological Change
[0110] Experiments were carried out to examine whether or not the fatty acid-conjugated GnRH derivatives maintain the functional characteristics and incur a morphological change in the ovary.
[0111] The pharmaceutical compositions of Examples 9, 10, 13, 14, 15, and 16 were each subcutaneously injected at a single dose of 12.5 mg/kg once at the back of the neck to female rats 9 weeks old. Rats to which no drugs were administered were used as a non-treated control while an aqueous solution of 3 mg of methyl-β-cyclodextrin was injected to a negative control. Each group consisted of 3 rats.
[0112] On day 28 after injection, the rats were subjected to an autopsy to excise the ovaries which were then stained with hematoxylin-eosin (H&E stain). The stained ovaries were observed for histological change and pathogenic aberration.
[0113] Briefly, ovaries were stained with H&E and observed as follows.
[0114] 1 Immediately after autopsy, the excised tissue (ovary) was fixed for 12 hours in a fixation solution.
[0115] 2. The sufficiently fixed tissue was washed with water to remove the fixation reagent.
[0116] 3. For use in paraffin embedding, the ovarian tissue was dehydrated with graded alcohols. The dehydration was conducted in a series of gradually graded alcohols beginning from a low concentration to higher concentrations and finally with 100% pure alcohol and benzene.
[0117] 4. A solution of paraffin in benzene was allowed to infiltrate into the dehydrated ovarian tissue which was then treated with pure paraffin in a liquid state at a high temperature (60° C.) to completely infiltrate paraffin into the tissue.
[0118] 5. The paraffin-embedded tissue was cut into blocks of suitable sizes and then sectioned at a 4 μm thickness on a microtome.
[0119] 6. The sectioned tissue was mounted on a glass slide and deparaffinized with an organic solvent such as xylene.
[0120] 7. The slide having the tissue attached thereto was hydrated by treatment with a series of alcohols from high to low concentrations.
[0121] 8. An aqueous hematoxylin solution was first applied to stain the nucleus and other acidic structures (RNA-rich structures, etc.) blue.
[0122] 9. Various substances and organelles in the cytoplasm and extracellular matrix were secondarily stained red with eosin.
[0123] 10. After completion of the staining, the specimen was dehydrated with graded alcohols and then cover-slipped using an adhesive such as a resin (balsam or a synthetic resin). This process is called mounting.
[0124] 11. After finishing the above procedures, the specimen slides were scanned using a ScanScope® AT slider scanner (Aperio) and the images thus stored were analyzed using the ImageScope program (Aperio). The scanned images are depicted in
[0125] Results
[0126] In female rats, an estrous cycle of diestrus, proestrus, estrus, and metestrus lasts about 4 to 5 days, with the ovary morphologically changing depending on the estrous stages.
[0127] It is known that when administered to rats, a GnRH agonist reduces the formation of secondary follicles and Graafian follicles (see: Mohammadbeigi et al., Short-term Administration of Gonadotropin-releasing Hormone Agonist (Buserelin) Induces Apoptosis in Rat Ovarian Developmental Follicles, July 2016). In this experiment, secondary and Graafian follicles on ovary images in each treatment group were indicted by arrows to examine whether the number of the follicles is reduced or not.
[0128] Referring to
[0129] From the data, it is understood that the fatty acid-conjugated GnRH derivatives according to an embodiment of the present disclosure have an advantageous effect, specifically, an effect of deterring sexual maturation in rats.
[0130] In addition, the observation of a reduction in the number of secondary or Graafian follicles even 28 days after the single dose indicates that the fatty acid-conjugated GnRH derivatives according to an embodiment of the present disclosure or the inclusion complexes of the derivatives and cyclodextrin show sustained release and as such, can be used as long-acting formulations.
[Experimental Example 4] Measurement of Rate of Increase of In Vivo Half-Life
[0131] The present inventor carried out animal experiments (female SD rats, nine weeks old) in order to examine the increased in vivo half-lives of the prepared fatty acid-conjugated GnRH derivatives. In brief, Leuprolide formulation for one-day administration (n=6), Leuprolide acetate depot formulation for one-month administration (3.75 mg/month; n=7), and the derivative of Example 4 (n=6) or the derivative of Example 6 (n=6) were subcutaneously administered once at a dose of 12.5 mg/kg to rats of each group, followed by monitoring blood concentrations over time. DMSO (dimethyl sulfoxide) was used as a solvent as needed. Before administration and at 0.5, 1, 2, and 6 hours and on days 1, 3, 7, 10, 14, 21, and 28 after administration, blood samples were taken from the tail vein of the rats and measured for the blood concentrations of Leuprolide and the derivatives of the Examples, using LC/MSMS. If the concentration reached about 4 ng/mL at a specific time point, no measurements were further made for the next time point.
[0132] The experimental results are summarized as follows. In the following table, the numerical unit is ng/mL.
TABLE-US-00011 TABLE 7 C. Example 4 C. Example 1 (Leuprolide acetate Example 4 Example 6 (Leuprolide acetate 1-month formulation (P2; Pal_[Q1] (P4; Pal_[Q1] 1-day formulation) (3.75 mg)) GnRH) GnRH_AcOH) Time Mean SD Mean SD Mean SD Mean SD 0 — — — — — — — — 0.5 hr 1020 177 133 54.1 11.8 4.362 6.51 2.30 1 hr 769 572 164 89.1 16.9 3.16 9.22 2.57 2 hr 228 267 93.0 51.5 29.1 4.97 18.6 2.83 6 hr 2.88 4.09 19.6 7.04 52.2 14.1 45.7 12.0 1 Day — — 19.0 7.49 45.0 6.07 23.2 6.75 3 Days — — 7.47 3.45 21.4 2.73 9.60 1.58 7 Days — — 5.24 1.72 11.9 2.86 7.20 1.79 10 Days — — 10.8 2.89 8.18 2.83 8.20 3.80 14 Days — — 15.1 5.01 4.35 1.67 4.58 1.88 21 Days — — 4.73 4.15 — — 1.26 0.72 28 Days — — 1.47 1.77 — — — —
[0133] The measurement results are graphically depicted in
TABLE-US-00012 TABLE 8 C. Example 1 C. Example 4 Example 4 Example 6 t.sub.1/2 0.03 4.17 4.80 4.03 [Day] CL 0.180 0.049 0.051 0.075 [(mg/kg)/(ng/mL)/Day] Vd 0.007 0.296 0.351 0.436 [(mg/kg)/(ng/mL)] Tmax 0.02 0.04 0.25 0.25 [Day] Cmax 1020.0 164.0 52.2 45.7 [ng/mL] AUCt 69.21 245.22 216.24 159.51 [ng/mL*d]
[0134] As can be understood from the data, the fatty acid-conjugated GnRH derivatives according to an embodiment of the present disclosure are significantly superior to Leuprolide (Comparative Example 1) in terms of the in vivo half-life, clearance rate, volume of distribution, and systemic exposure (AUCt). Furthermore, the fatty acid-conjugated GnRH derivatives according to an embodiment of the present disclosure were found to have similar levels of half-life, clearance rate, and systemic exposure (AUCt) to those of the existing Leuprolide formulation for one-month administration containing a physically mixed biodegradable polymer and particularly to exhibit a superior volume of distribution, a delayed time to reach the maximum concentration following drug administration, and a reduced maximum concentration for a prolonged period of time, compared to the commercially available product. Taken together, the data demonstrate that the fatty acid-conjugated GnRH derivatives according to an embodiment of the present disclosure allow GnRH to maintain a suitable concentration for a prolonged period of time in vivo.
[0135] In light of the excellent properties thereof, the fatty acid-conjugated GnRH derivatives according to an embodiment of the present disclosure can be used at a remarkably reduced volume, compared to the existing products in which biodegradable polymers are mixed with GnRH derivatives to achieve a sustained release, and thus can overcome the disadvantage of pain and exclude the side effect that the biodegradable polymer remains in vivo for a long period of time. These properties are advantageous particularly to children. In addition, having excellent release sustainability, the fatty acid-conjugated GnRH derivatives according to an embodiment of the present disclosure can exhibit excellent effects of killing prostate cancer cells as well as deterring sexual maturation by reducing the numbers of secondary or graafian follicles in the ovary, as identified in Experimental Examples 2 and 3.
[0136] Meanwhile, when the fatty acid-conjugated GnRH derivatives according to an embodiment of the present disclosure were used in combination with the biodegradable polymer used in the conventional products, the half-life is remarkably increased, compared to conventional drugs such as Leuprolide, to become as long as drugs used in invasive methods (surgery), such as for implants (several months to one year).
[0137] Although the technical idea of the present disclosure has been described by the examples described in some embodiments and illustrated in the accompanying drawings, it should be noted that various substitutions, modifications, and changes can be made without departing from the scope of the present disclosure which can be understood by those skilled in the art to which the present disclosure pertains. In addition, it should be noted that such substitutions, modifications and changes are intended to fall within the scope of the appended claims.