LONG-ACTING PALMITIC ACID-CONJUGATED GnRH DERIVATIVE, AND PHARMACEUTICAL COMPOSITION CONTAINING SAME

Abstract

The present invention relates to a novel long-acting palmitic 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 and increased half-life in blood, 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 long-acting palmitic acid-conjugated gonadotropin-releasing hormone (GnRH) derivative, comprising: palmitic acid conjugated to GnRH derivative comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2 to 6, wherein a carboxyl group of palmitic acid conjugated to an amino terminal of the GnRH derivative through a peptide-bond; and a pharmaceutically acceptable salt.

2.-5. (canceled)

6. The long-acting palmitic acid-conjugated GnRH derivative 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.

7. The long-acting palmitic acid-conjugated GnRH derivative of claim 6, 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.

8. The long-acting palmitic acid-conjugated GnRH derivative of claim 1, wherein the long-acting palmitic acid-conjugated GnRH derivative comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 to 6.

9. The long-acting palmitic acid-conjugated GnRH derivative of claim 8, wherein the long-acting palmitic acid-conjugated GnRH derivative comprises the amino acid sequence of SEQ ID NO: 4.

10. The long-acting palmitic acid-conjugated GnRH derivative of claim 8, wherein the long-acting palmitic acid-conjugated GnRH derivative comprises the amino acid sequence of SEQ ID NO: 5.

11. A pharmaceutical composition for prevention or treatment of sex hormone-dependent disease, comprising the long-acting palmitic acid-conjugated GnRH derivative of claim 1, wherein the sex hormone-dependent disease is selected from the group consisting of prostate cancer, breast cancer, endometriosis, and central precocious puberty.

12. (canceled)

13. The pharmaceutical composition of claim 11, further comprising a biodegradable polymer selected from PLA (poly-lactic acid), linear or branched PLGA (poly(lactic-co-glycolic acid)), PGA (poly-glycolic acid), and hydrogel.

14.-15. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0049] FIG. 1 is a graph showing the cell viability of the prostate cancer cell line DU-145 after treatment with various concentrations of GnRH or GnRH derivatives and the control drug. 1% DMSO was used as a negative control and 0.1% SDS as a positive control.

[0050] FIG. 2 is a graph showing the cell viability of the prostate cancer cell line DU-145 after treatment with sodium or acetate salt of GnRH derivatives and the control drug. 1% DMSO was used as a negative control and 0.1% SDS as a positive control.

[0051] FIG. 3 shows the results of measuring the solubility of the palmitic acid-conjugated GnRH derivatives having different first amino acid residues and salt types.

[0052] FIG. 4 is a graph showing the increased in vivo half-life of the palmitic acid-conjugated GnRH derivatives, where the blood concentration is plotted over time after the control drugs and the GnRH derivatives of the present disclosure are each subcutaneously administered once at a dose of 12.5 mg/kg to animals (rats).

[0053] FIG. 5 is a graph showing the increased in vivo half-life of the palmitic acid-conjugated GnRH derivatives, where the blood concentration is plotted over time after the control drug of Leuprolide and the GnRH derivatives of the present disclosure are each subcutaneously administered once at a dose of 12.5 mg/kg to animals (rats).

[0054] FIG. 6 is a graph showing the increased in vivo half-life of the palmitic acid-conjugated GnRH derivatives, where the blood concentration is plotted over time after the control drug of Leuprolide acetate depot one-month formulation and the GnRH derivatives of the present disclosure are each subcutaneously administered once at a dose of 12.5 mg/kg to animals (rats).

DETAILED DESCRIPTION

[0055] Hereinafter, the embodiments of the present disclosure will be described by referring to 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 Method for Gonadotrophin-Releasing Hormone (GnRH) Derivative

[0056] Natural mammalian GnRH has the following sequence.

TABLE-US-00002 [MammalianGnRHsequence] (SEQIDNO:1) pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly

[0057] 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 GnRH derivative and the palmitic acid-conjugate GnRH derivative of the present disclosure.

TABLE-US-00003 [LeuprolideSequence] (SEQIDNO:2) pGlu-His-Trp-Ser-Tyr[D-Leu6]-Leu-Arg-Pro-NHEt

[0058] Derivatives where glutamate at position 1 on the Leuprolide sequence remains unsubstituted or was substituted with glutamine were prepared as follows.

[0059] (1) Preparation Method for GnRH Derivative Peptides

[0060] The derivatives are synthesized using a general Fmoc/tBu solid-phase peptide synthesis (SPPS) method, where the -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.

[0061] {circle around (1)} Load Fmoc amino acid onto resin (Fmoc-Pro-trityl resin);

[0062] {circle around (2)} Remove Fmoc protecting group from Fmoc-AA-resin (20% piperidine/DMF);

[0063] {circle around (3)} Wash with DMF;

[0064] {circle around (4)} Bind amino acid after activation (DIC/HOBt used);

[0065] {circle around (5)} Wash with DMF;

[0066] {circle around (6)} Repeat steps {circle around (2)} to {circle around (5)} to bind amino acids sequentially;

[0067] {circle around (7)} Remove resin only from synthesized peptide (1.5 TFA/DCM);

[0068] {circle around (8)} Attach ethylamine to the amino terminus of the resulting peptide (using EDC.HCl/HOAt); and

[0069] {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).

[0070] Palmitic acid was conjugated to the amino terminus of the obtained GnRH derivative. Conjugating palmitic acid to the amino terminus of the derivative was carried out in the same manner as the conjugation of general amino acids.

[0071] (2) Purification of GnRH Derivative Peptides

[0072] 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). In Table 1, P1 and P3 GnRH derivatives have glutamate as the amino acid residue at position 1, while P2 and P4 GnRH derivatives have glutamine as the amino acid residue at position 1.

TABLE-US-00004 TABLE 1 MS GnRH derivative Purity (%) Calculated (Da) Measured (Da) P1 98.2 1465.8 1465.7 P2 98.2 1464.8 1464.9 P3 98.1 1465.8 1465.5 P4 98.3 1464.8 1464.0

[0073] Palmitic acid is sparingly soluble in water with a solubility of 0.04 mg/ml and exists as a solid phase at room temperature with a melting point of 60 C. Hence, as the palmitic acid-conjugated GnRH derivative might be poorly soluble in water, salting was further carried out. The palmitic acid-conjugated GnRH derivatives were subjected to salting with sodium salt or acetate to prepare P1 to P4 palmitic acid-conjugated GnRH derivative salts as shown in Table 2 below.

TABLE-US-00005 TABLE2 Deri- vative Backbone/Derivativesequenceandsalt GnRH pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg- Pro-Gly-NH.sub.2 Leupro- pyroGlu-His-Trp-Ser-Tyr-DLeu-Leu-Arg- lide Pro-NHEt P1 Palmitate-Glu-His-Trp-Ser-Tyr-DLeu-Leu- Arg-Pro-NHEtsodiumsalt(SEQIDNO:3) P2 Palmitate-Gln-His-Trp-Ser-Tyr-DLeu-Leu- Arg-Pro-NHEtsodiumsalt(SEQIDNO:4) P3 Palmitate-Glu-His-Trp-Ser-Tyr-DLeu-Leu- Arg-Pro-NHEtacetatesalt(SEQIDNO:5) P4 Palmitate-Gln-His-Trp-Ser-Tyr-DLeu-Leu- Arg-Pro-NHEtacetatesalt(SEQIDNO:6)

[0074] Subsequent experiments were performed using the prepared GnRH derivatives and palmitic acid-conjugated GnRH derivative salts.

Example 1: Effect of Palmitic Acid Conjugation on Prostate Cancer Cell Death

[0075] GnRH derivatives are clinically applied to the treatment of diseases including breast cancer, prostate cancer, endometriosis, central precocious puberty, and the like. The DU-145 cell line, which is a type of prostate cancer cells, was cultured in T75 flasks containing an adequate amount of RPIM 1640 culture medium (containing 10% FBS, penicillin/streptomycin, and 1% non-essential amino acids) in a sterile incubator having 5% CO.sub.2/95% air at 37 C. Cell death assay was performed using the Cell Counting Kit-8 (CCK-8, manufactured by DOJINDO). The DU-154 cells were separated from the T75 flasks by trypsinization treatment and transferred to 96-well plates at a density of 1104 cells/well, followed by incubation for one hour for attachment.

[0076] Subsequently, the cells were treated with various concentrations of GnRH or GnRH derivatives, and control reagents. Specifically, 1% DMSO was used as a negative control for cell death while 0.1% SDS served as a positive control. After 48 hours of incubation, the existing culture medium was removed, and 100 L of fresh culture medium and 10 L of CCK-8 solution were applied to each well. Again, after 48 hours of incubation, the medium solution was replaced by 100 L of fresh culture medium and 10 L 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 Table 3 and FIGS. 1 and 2.

TABLE-US-00006 TABLE 3 Statistical significance Treatment conc. Viability relative (compared to 1% Derivative (M) to control (%) DMSO control) P1 200 99.7 P2 200 30.7 <0.0001 P3 200 25.8 <0.0001 P4 200 98.3 Leuprolide 50 99.1 100 93.6 200 89.7 GnRH 50 90.9 100 88.7 200 93.4 1% DMSO 91.0 0.1% SDS 21.7 <0.0001

[0077] The P2 and P3 derivatives were observed to exhibit about 7- to 7.5-fold greater cytotoxic effects, compared to Leuprolide or GnRH, which exhibits a cell death rate of about 10% on prostate cancer cells. The results suggest that combinations of amino acid substitution at position 1 and salting bring about unpredicted excellent cytotoxic effects on prostate cancer cells.

[0078] GnRH derivatives significantly differ from each other in terms of the cytotoxic effect on prostate cancer cell lines, depending on the type of the first amino acids and salting.

Example 2: Differences in Solubility of Palmitic Acid-Conjugated GnRH Derivative by Saltine Type

[0079] The solubility of GnRH in water is estimated to be about 1 mg/mL under experimental conditions and about 0.0588 mg/mL under real conditions. In contrast, acetate is known to increase the solubility of GnRH about 10 times or more to 10 mg/mL (https://www.drugbank.ca/drugs/DB00644).

[0080] The salt is understood to increase the solubility by lowering the pH of the solvent, along with ionization thereof. In order to increase the solubility of commercially available GnRH and GnRH derivatives, various linkages were tried at the carboxy terminal (C terminal) of GnRH and GnRH derivatives, as summarized in Table 4 below.

TABLE-US-00007 TABLE 4 Treatment Dose Salt type at Product Name concentration (Strength) C terminal Factrel Inj. 0.5 mg/vial Powder, for 0.5 mg GnRH solution hydrochloride Factrel Pws 100 mcg/vial Powder, for 100 mcg solution Factrel Pws 500 mcg/vial Powder, for 500 mcg solution Lutrepulse Powder, for 3.2 mg GnRH acetate solution Lutrepulse Pws 0.8 mg/vial Powder, for 0.8 mg solution

[0081] The water solubilities of commercially available GnRH derivative salts are provided as shown in Table 5 below.

TABLE-US-00008 TABLE 5 Ingredient CAS No. Avg. Weight (Da) Water solubility GnRH 33515-09-2 1182.2901 1 mg/mL GnRH acetate 52699-48-6 1260.378 10 mg/mL GnRH hydrochloride 51952-41-1 1218.77 0.0498 mg/mL

[0082] Additionally, the four types of palmitic acid-conjugated GnRH derivatives were subjected to a solubility test. First, the peptides in the form of powder at room temperature were weighed with a microbalance and dissolved in the organic solvent 100% DMSO with a final concentration of 20 mM. They were all observed with the naked eye to dissolve very quickly and completely. The obtained solutions were used as stocks and diluted in various concentrations in PBS buffer solutions or cell culture medium to examine the solubility thereof.

[0083] When the peptide stocks were diluted in PBS buffer solutions or cell culture medium, the solutions became milky, with the generation of aggregates. The degree of the aggregation differed in each GnRH derivative types. The degree of aggregation was examined by centrifuging the aggregates. As a result, P2 and P3 derivatives, which were identified to have excellent cytotoxic effects on prostate cancer cell lines, were observed to be very high in solubility (FIG. 3).

[0084] In the two derivatives, sodium salt and acetate, respectively, were conjugated to the carboxyl terminal of GnRH, which indicates that the solubility of the palmitic acid-conjugated GnRH derivatives of the present disclosure is influenced by the type of the first amino acid residues and salting.

Example 3: Measurement of Increase Rate of In Vivo Half-Life

[0085] 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 palmitic acid-conjugated GnRH derivatives. In brief, Leuprolide (n=6), Leuprolide acetate depot formulation for one-month administration (3.75 mg/month; n=7), and GnRH derivative P2 (n=6) or P4 (n=6) were subcutaneously administered once at a dose of 12.5 mg/kg to rats of each group, followed by a monitoring of blood concentrations over time. 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 GnRH derivatives, 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.

[0086] The experimental results are summarized as follows.

TABLE-US-00009 TABLE 6 Comparative Example 2 Comparative Example 1 (sustained release) P4 (natural) Leuprolide acetate P2 Pal_[Q1]GnRH Leuprolide (3.75 mg) Pal_[Q1]GnRH AcOH Time Mean S.D. Mean S.D. Mean S.D. Mean S.D. 0 0.5 hr. 1020 177 133 54.1 11.8 4362 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

[0087] The measurement results are graphically depicted in FIGS. 4 to 6. Based on the results, pharmacokinetic analysis was carried out by calculating the half-life (t.sub.1/2), clearance rate (CL), volume of distribution (Vd), time to reach the maximum concentration following drug administration (T.sub.max), maximum concentration following drug administration (C.sub.max), and systemic exposure to drug (AUC.sub.t). The analysis results are as follows.

TABLE-US-00010 TABLE 7 Leuprolide P2 P4 acetate Pal_ Pal_[Q1]GnRH Leuprolide (3.75 mg) [Q1]GnRH AcOH t.sub.1/2 [day] 0.03 4.17 4.80 4.03 CL[(mg/kg)/(ng/ 0.180 0.049 0.051 0.075 ml)/day] Vd[(mg/kg)/ 0.007 0.296 0.351 0.436 (ng/ml)] T.sub.max [day] 0.02 0.04 0.25 0.25 C.sub.max [ng/ml] 1020.0 164.0 52.2 45.7 AUC.sub.t [ng/ml*d] 69.21 245.22 216.24 159.51

[0088] As can be understood from the data, the palmitic acid-conjugated GnRH derivatives of the present disclosure are significantly superior to Leuprolide in terms of the in vivo half-life, clearance rate, volume of distribution, and systemic exposure (AUC.sub.t). Further, the palmitic acid-conjugated GnRH derivatives of the present disclosure were found to have similar levels of half-life, clearance rate, and systemic exposure (AUC.sub.t) to those of the commercially available Leuprolide formulation for one-month administration containing a 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 palmitic acid-conjugated GnRH derivatives of the present disclosure allow GnRH to maintain its suitable concentrations for a prolonged period of time in vivo.

[0089] In light of the excellent properties thereof, the palmitic acid-conjugated GnRH derivatives of the present disclosure can be used at a remarkably reduced volume, compared to the existing products comprising a biodegradable polymer for 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. Meanwhile, when the palmitic acid-conjugated GnRH derivatives 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).

[0090] 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 that such substitutions, modifications and changes are intended to fall within the scope of the appended claims.