Sustained-release formulation of human chorionic gonadotropin (hCG)

Abstract

It relates to sustained release formulations of human chorionic gonadotropin (hCG). The present disclosure relates to sustained release dosage forms of human chorionic gonadotropin (hCG) or derivatives or isoforms thereof. The sustained release hCG dosage forms include biodegradable polymer microspheres that provide sustained release of hCG over a desired period of time.

Description

[Background technology]

[0001] I. Background Human chorionic gonadotropin (hCG) is a hormone produced by the syncytiotrophoblast of the human placenta and gonads. hCG interacts with the luteinizing hormone / choriogonadotropin receptor (LHCGR), also known as the lutropin / choriogonadotropin receptor (LCGR) or luteinizing hormone receptor (LHR), found in the gonads of both sexes. The action of hCG is similar to that of pituitary luteinizing hormone (LH): both hormones stimulate the production of testosterone and other steroid hormones by the Leydig cells of the testes, and both hormones stimulate the production of progesterone by the corpus luteum of the ovaries.

[0002] During fetal development, hCG produced by the placenta stimulates the fetal testes to produce androgens, which are important for normal male development. In adults, administration of exogenous hCG stimulates testicular Leydig cells to produce testosterone. For men with hypogonadotropic hypogonadism, exogenously administered hCG can stimulate testicular Leydig cells and restore normal testosterone production. Administration of hCG can also stimulate testicular descent in boys with cryptorchidism if there is no anatomical obstruction to descent.

[0003] In women, hCG, produced by the placenta, stimulates the ovaries and promotes the maintenance of the corpus luteum during the onset of pregnancy. This allows the corpus luteum to secrete the hormone progesterone during the first trimester. Progesterone causes the uterus to develop a thick layer of blood vessels and capillaries so that it can support the growing fetus.

[0004] During the normal menstrual cycle, LH participates with FSH in normal follicle development and maturation, and the mid-cycle LH surge induces ovulation. In adult women, clinical administration of exogenous hCG can substitute for the LH surge. For women undergoing in vitro fertilization, hCG is widely used parenterally to induce final maturation. In the presence of one or more mature follicles, ovulation can be induced by administration of hCG. Furthermore, hCG may be used to enhance progesterone production for clinical purposes during infertility treatment.

[0005] Because the most abundant biological source is currently pregnant women, some organizations collect urine from pregnant women to extract hCG for pharmaceutical use in dosage forms marketed under the trade names Novarel® and Pregnyl®. Recombinant hCG is produced in Chinese hamster ovary (CHO) cells and is commercially available in a dosage form marketed under the trade name Ovidrel®.

[0006] Currently available commercial dosage forms of hCG are limited to intramuscular (IM) or subcutaneous (SC) injection forms that raise serum hCG levels to therapeutic levels for a short period of time. Several clinical applications requiring sustained dosing of hCG require frequent injections. These applications include, but are not limited to, the treatment of hypogonadotropic hypogonadism and the stimulation of progesterone production for female pregnancy. There is a need in the art for sustained-release dosage forms of hCG. The present invention fulfills this need. Summary of the Invention [Means for solving the problem]

[0007] II. Overview The present disclosure relates to a long-felt need in the art for sustained-release human chorionic gonadotropin (hCG) formulations. In particular, the present disclosure is directed to hCG dosage forms having sustained-release profiles. In some embodiments, the hCG dosage forms exhibit a release profile of about 1 week to about 2 months. In other embodiments, the hCG dosage forms described herein may have a sustained-release profile of about 1 week to about 6 months.

[0008] In some embodiments, the sustained-release hCG dosage form comprises hCG encapsulated in microspheres. In further embodiments, the microspheres are formed from copolymers. In even further embodiments, the copolymers are block copolymers or multiblock copolymers. In such embodiments, the block copolymers can comprise, or consist essentially of, polyethylene glycol (PEG) or PEG-containing polymer blocks and one or more other polymer blocks.

[0009] The hCG sustained-release formulations described herein may be useful in various treatments related to hormone therapy, including, but not limited to, the treatment of infertility and pituitary disorders. Accordingly, further aspects of the present disclosure relate to methods of administering the sustained-release hCG formulations described herein and methods of treating with the sustained-release hCG formulations described herein. Such methods include the treatment of pregnancy and pituitary disorders. Further methods disclosed herein include the treatment of breast cancer. In some embodiments, the treatment is for existing breast cancer in nulliparous women. In some embodiments, the woman is about 25 years of age or younger.

[0010] In another embodiment, methods are encompassed in which the described sustained release hCG dosage forms are administered according to a regimen that achieves or approximates the theoretical sustained release profile of FIG.

[0011] The invention described and claimed herein has many features and embodiments, including, by way of example and not limitation, those described or illustrated or referenced in this Summary. It is not exhaustive, and the invention described and claimed herein is not limited to or by the features or embodiments identified in this Summary, which is included for illustrative and not limiting purposes only. Additional embodiments may be disclosed in the following drawing descriptions and detailed description. III. BRIEF DESCRIPTION OF THE DRAWINGS [Brief explanation of the drawings]

[0012] [Figure 1] 1 is an exemplary theoretical release profile for an hCG sustained release formulation. [Figure 2] Cumulative release profiles (μg released) of hCG sustained release formulations containing hCG microspheres showing the total amount of hCG released over a 14 day period; Panel A shows the cumulative release of each individual formulation, and Panel B shows the average cumulative release of each formulation. [Figure 3] Figure 1 shows the release profiles of hCG sustained-release formulations containing hCG microspheres normalized to the total hCG content (% release) of the hCG microspheres. The release profiles show the total amount of hCG released over a 14-day period; Panel A shows the cumulative release of each individual formulation, and Panel B shows the average cumulative release of each formulation. [Figure 4] Calculated observed in vitro release profile (% released) over a 14 day period for Formulation 11, an hCG sustained release formulation containing hCG microspheres. [Figure 5] Predicted release profile of Formulation 11 based on regimented dosing of 3 mg every 7 days with a Cmin of 18 ng / ml. [Figure 6] Predicted release profile of Formulation 11 based on regimented dosing of 3 mg every 14 days with a Cmin of 1.7 ng / ml. [Figure 7]Predicted release profile of Formulation 11 based on regimented dosing of 3 mg every 28 days with a Cmin of 0.07 ng / ml. [Figure 8] Predicted release profile of Formulation 11 based on regimented dosing of 250 μg every 7 days with t½ of 36 hours and Cmin of 3 ng / ml. [Figure 9] Predicted release profile of Formulation 11 based on regimented dosing of 250 μg every 14 days with t½ of 36 hours and Cmin of 0.3 ng / ml. [Figure 10] Cumulative release profiles (μg released) of hCG sustained release formulations containing hCG microspheres demonstrating the total amount of hCG released over a 50 day period; Panel A shows the average cumulative release of each individual formulation, and Panel B shows the normalized release profile (% released) of each formulation. [Figure 11] HPLC chromatogram demonstrating the purity of hCG upon concentration. [Figure 12] Gel analysis of the molecular weight of Ovidrel® compared to Dong-A hCG. [Figure 13] Purity analysis of hCG from round 1 in vitro release. [Figure 14] Cumulative release profiles of sustained release formulations containing hCG microspheres demonstrating the total amount of hCG released over a 40-day period (three replicates run for each formulation): Panel A shows the cumulative release profile (μg released) of each replicate, Panel B shows the normalized profile (% based on high performance size exclusion chromatography), and Panel C shows the normalized release profile (% released) of each formulation. [Figure 15] The average release profiles of the replicates shown in Figure 14 . [Figure 16]Cumulative release profiles for three replicate runs of sustained release formulation 695-01-0034 containing hCG microspheres demonstrating the total amount of hCG released over a 40-day period: Panel A shows the cumulative release profile (μg released) of each replicate, Panel B shows the normalized profile (% based on high performance size exclusion chromatography), and Panel C shows the normalized release profile (% released) of each formulation. [Figure 17] RP-UPLC chromatogram of concentrated hCG solution. [Figure 18] SEM image of hCG sustained-release microspheres composed of a blend of SynBiosys 50CP10C20-LL40 and 30CP30C40-LL40. [Figure 19] Cumulative release profiles of hCG sustained-release formulations containing hCG microspheres composed of a blend of SynBiosys 50CP10C20-LL40 and 30CP30C40-LL40. Release profiles (average of three replicates for each formulation) were normalized for the total hCG content (% released) of each formulation and represent the total amount of hCG released over a 60-day period. [Figure 20] Cumulative release profile of hCG sustained-release formulation (JA16043) containing hCG microspheres composed of a 33 / 66% w / w blend of SynBiosys 50CP10C20-LL40 and 30CP30C40-LL40. The release profiles (average of three replicates for each formulation) were normalized for the total hCG content (% released) of the formulation and show the total amount of hCG and % intact hCG (relative to total) released over a 35-day period. [Figure 21] 20 SEM photograph of hCG sustained-release microspheres composed of [PCL-PEG3000-PCL]-b-[PDO] (batch number JA16101). [Figure 22]Cumulative release profiles of hCG sustained-release formulations (JA16101 and JA16102) composed of SynBiosys20[PCL-PEG3000-PCL]-b-[PDO]. Release profiles (average of three replicates for each formulation) were normalized for the total hCG content (% released) of each formulation and show the total amount of hCG and % intact hCG (relative to total) released over a 70-day period. [Figure 23] Serum hCG levels (Panel A) and serum testosterone levels (Panel B) in control monkeys receiving daily hCG injections. [Figure 24] Serum r-hCG and testosterone levels in monkeys treated with hCG sustained-release microspheres representing total doses of 200, 600, and 1200 μg of hCG: Panel A shows serum r-hCG levels for the first 48 hours, Panel B shows serum r-hCG levels for the full 28-day duration of the study, and Panel C shows serum testosterone levels for the full 28-day duration of the study. [Figure 25] Serum r-hCG and testosterone levels in monkeys treated with hCG sustained-release microspheres representing total doses of 200 μg (Panel A), 600 μg (Panel B), and 1200 μg hCG (Panel C). [Figure 26] SEC-UPLC chromatogram of rhCG and its potential degradation products. [Figure 27] Cumulative release profiles of Ovidrel hCG sustained-release formulations composed of SynBiosys20[PCL-PEG3000-PCL]-b-[PDO]. Release profiles (average of three replicates for each formulation) are normalized for the total hCG content (% released) of each formulation and represent the total amount of hCG released over a 49-day period as measured using an optimized in vitro dissolution method. [Figure 28]Percentage of hCG-induced progesterone produced relative to progesterone produced by Ovidrel. A) hCG released from microsphere lots at 2 hours, 23, and 37 days; B) hCG extracted from microspheres in extraction buffer (AMD-18-022-1) or added hCG (AMD-18-022-4) and C) mean percent of progesterone produced by hCG released from lots MS18-037, MS18-038, and SR18-031 as a function of time relative to Ovidrel. [Figure 29] Results of 2-AB glycan mapping of hCG extracted from hCG sustained-release microspheres. DETAILED DESCRIPTION OF THE INVENTION

[0013] IV. Detailed Description The present invention is directed to a sustained-release hCG dosage form. Such a dosage form has long been needed in the art, as currently all commercially available hCG dosage forms are liquids for intramuscular or subcutaneous injection. Furthermore, currently available hCG dosage forms all have an immediate release profile, resulting in unsustained serum levels and limited by the half-life of hCG, approximately 36 hours. This means that patients need to receive frequent injections to achieve the desired hCG therapeutic level over a period of time, which can be inconvenient and therefore can lead to poor patient compliance or lead patients and providers to choose less effective treatments.

[0014] In some embodiments, the sustained-release hCG dosage form comprises hCG or a derivative or isoform thereof encapsulated in microspheres. In further embodiments, the microspheres are formed from copolymers. In even further embodiments, the copolymers are block copolymers or multiblock copolymers. In such embodiments, the block copolymers can comprise, or consist essentially of, polyethylene glycol (PEG) or PEG-containing polymer blocks and one or more other polymers.

[0015] Embodiments according to the present disclosure are described more fully below. However, aspects of the present disclosure may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The terminology used in the detailed description herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0016] A. Human chorionic gonadotropin As used herein, the term "human chorionic gonadotropin" or "hCG" refers to the defined glycoprotein associated with this name and any other molecule with similar biological function that shares at least about 80% amino acid sequence identity with naturally occurring hCG or its isoforms. hCG derivatives and isoforms can also be utilized in the compositions and methods described herein. In other embodiments, hCG variants having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% amino acid sequence identity with naturally occurring hCG can be used in the compositions of the invention.

[0017] In fact, hCG is produced by the syncytiotrophoblast in the placenta after implantation and in small amounts by the gonads. It is an analog of LH, which is produced in the pituitary gland of men and women of all ages. hCG can also be produced synthetically and is commercially available. As used herein, hCG can refer to recombinant versions of glycoproteins, such as, but not limited to, Ovidrel®, Dong-A's recombinant hCG [product code DA-3803], other recombinant hCGs that demonstrate bioequivalence to any of these products, or generic versions thereof. See, e.g., Seo et al., BioDrugs, 1;25(2):115-27 (2011). In some embodiments, such recombinant hCG is produced by animal (e.g., human or other mammalian) cell lines or bacterial cell lines. In some embodiments, such recombinant hCG is about 40 to about 60 kDa, e.g., at least about 40 kDa or about 40 kDa, at least about 41 kDa or about 41 kDa, at least about 42 kDa or about 42 kDa, at least about 43 kDa or about 43 kDa, at least about 44 kDa or about 44 kDa, at least about 45 kDa or about 45 kDa, at least about 46 kDa or about 46 kDa, at least about 47 kDa or about 47 kDa, at least about 48 kDa or about 48 kDa, or at least about 48 kDa. The hCG may be at or about 49 kDa, at least at or about 50 kDa, at least at or about 51 kDa, at least at or about 52 kDa, at least at or about 53 kDa, at least at or about 54 kDa, at least at or about 55 kDa, at least at or about 56 kDa, at least at or about 57 kDa, at least at or about 58 kDa, at least at or about 59 kDa, or about 60 kDa. As used herein, hCG may also refer to isolated naturally occurring hCG, such as, but not limited to, Novarel®, Pregnyl® urinary hCG, another recombinant hCG that demonstrates bioequivalence to any of these products, or generic versions thereof.

[0018] hCG is a heterodimer of approximately 38 kDa containing alpha and beta subunits. The alpha subunit has 92 amino acids and two N-linked carbohydrate chains. The beta subunit has 145 amino acids and two N-linked and four O-linked carbohydrate chains. While the individual subunits are held together in the heterodimer by noncovalent interactions, the heterodimer and the alpha and beta subunits have little or no hydrophobic core. Five disulfide bonds in the alpha subunit and six in the beta subunit stabilize the structure. There is a homologous arrangement of 10 half-cystines in the alpha subunit, while the beta subunit contains 12 conserved half-cystines. Banerjee et al., Indian J. of Exp. Biol., 40:434-447 (2002). Both subunits consist of a cystine knot motif formed by three disulfide bridges and four polypeptide chains. In each subunit, three hairpin loops emerge from a central cystine knot. Glycoprotein hormones, such as hCG, are the most complex molecules with hormonal activity. Cahoreau et al., Front Endocrinol. (Lausanne), 6:26 (2015).

[0019] hCG Heterogeneity: Multiple molecular forms of hCG exist in pregnancy serum, including dissociated or degraded molecules that have no biological activity. Examples of known isoforms of hCG include intact hCG ("hCG"), nicked hCG ("hCGn"), hCG beta subunit ("hCGβ"), nicked hCG beta subunit ("hCGβn"), hCG beta core fragment ("hCGβcf"), and hCG alpha subunit ("hCGα").

[0020] B. Copolymer microspheres As used herein, the term "microsphere" refers to a spherical or spheroidal particle of about 999 μm or less in diameter that encapsulates an internal void that can be loaded with one or more therapeutic agents for drug delivery.

[0021] Aspects of the present disclosure relate to microspheres formed by copolymers. In some embodiments, these copolymers can be selected based on copolymers or portions thereof having one or more of the following characteristics: (i) the polymer forms a lattice to allow free flow of acid or release of acid, (ii) the polymer protects the hCG active ingredient from the environment in which it is stored and / or from which the microspheres are released (e.g., temperature stability), (iii) the polymer is hydrophilic, (iv) the polymer degrades without affecting the purity of the hCG, (v) the polymer is biodegradable, (vi) the polymer allows diffusion of the active ingredient (hCG), and / or (vii) the polymer accommodates the hydrodynamic radius of hCG (e.g., that of Ovidrel® or Dong-A hCG).

[0022] The microspheres may release from less than about 3% to about 40% of the hCG in the microspheres, based on the total weight of the microspheres, within about 24 hours.

[0023] It was unexpected that stable, sustained-release formulations of hCG could be made utilizing the polymers described herein, since hCG is known to decompose at elevated temperatures, e.g., body temperature. It is also known to decompose in response to pH changes. Therefore, prior to the present invention, it was believed that the only successful way to administer hCG to a patient or subject was via injection. The present invention details the surprising discovery that polymers can be used to form stable, sustained-release formulations of hCG that are not degraded by either the temperature or pH present in vivo, where the component hCG exists.

[0024] Microspheres containing hCG or its derivatives or isoforms can be prepared by techniques known to those skilled in the art, including, but not limited to, solvent evaporation and spray drying. In some embodiments, the microspheres are formed using a water-polymer ratio of about 0.1 to about 1.0, including, but not limited to, about 0.5 to about 1.0, about 0.55 to about 1.0, about 0.6 to about 1.0, about 0.65 to about 1.0, about 0.7 to about 1.0, about 0.75 to about 1.0, about 0.8 to about 1.0, about 0.85 to about 1.0, about 0.9 to about 1.0, or about 0.95 to about 1.0. In some embodiments, the water-polymer ratio of the microspheres is about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95, or about 1.0. These water-polymer ratios can be adjusted to achieve a particular release profile based on the concentration of hCG or its derivative or isoform and / or polymer used to form the microspheres. See Bos et al., Pharmaceutical Technology, October 2011:110-120.

[0025] In some embodiments, the microspheres have a diameter of at least about 1 μm, at least about 2 μm, at least about 5 μm, at least about 10 μm, at least about 20 μm, at least about 30 μm, at least about 40 μm, at least about 50 μm, at most about 50 μm, at most about 60 μm, at most about 70 μm, at most about 80 μm, at most about 90 μm, or at most about 100 μm. In some embodiments, the microspheres have a diameter of about 20 μm to about 100 μm, about 30 μm to about 100 μm, about 30 μm to about 50 μm, or about 50 μm to about 100 μm.

[0026] In some embodiments, the concentration of hCG used to prepare the microspheres is at least about 5 mg / ml, at least about 10 mg / ml, at least about 15 mg / ml, at least about 20 mg / ml, at least about 25 mg / ml, at least about 30 mg / ml, at least about 35 mg / ml, at least about 40 mg / ml, at least about 45 mg / ml, at least about 50 mg / ml, at least about 55 mg / ml, at least about 60 mg / ml, at least about 65 mg / ml, at least about 70 mg / ml, at least about 75 mg / ml, at least about 80 mg / ml, at least about 85 mg / ml, at least about 90 mg / ml, at least about 95 mg / ml, or at least about 100 mg / ml. The concentration of hCG used to prepare the microspheres can be about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, about 50 mg / ml, about 55 mg / ml, about 60 mg / ml, about 65 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 85 mg / ml, about 90 mg / ml, about 95 mg / ml, about 100 mg / ml or more of protein.

[0027] B1. hCG Microspheres Composed of PolyActive™ Multiblock Copolymers In some embodiments, the copolymer is a block copolymer and can optionally comprise or consist essentially of polyethylene glycol (PEG) and one or more other polymers. In some embodiments, the one or more other polymers can optionally be selected from polyethylene terephthalate (PET), polypropylene terephthalate, and polybutylene terephthalate (PBT). Non-limiting exemplary polymers include those having the chemical structure of the repeating polymer unit, e.g., [ka] and OctoPlus and / or PolyActive™ polymers produced by Dr. Reddy's Laboratories, having the formula:

[0028] PolyActive™ polymers represent a series of poly(ether ester) multiblock copolymers based on poly(ethylene glycol), PEG, and poly(butylene terephthalate), PBT. The main advantage of this system is the ability to vary the amount and length of each of the two building blocks to create a diverse family of customized polymers. Polymer matrix characteristics, such as the rate of controlled release, degradation, swelling, and strength, can be precisely controlled by the appropriate combination of the two copolymer segments.

[0029] While PolyActive™ polymers have been used in drug delivery systems prior to the present invention, prior to the present invention, the polymers have not been well formulated for highly complex glycoproteins, such as hCG. In particular, prior publications on PolyActive™ polymers have described the polymers as useful for the controlled release of "biopharmaceuticals and small lipophilic molecules," P. Mansell, "OctoPlus broadens rights to PolyActive delivery technology," In-Pharma Technologist.com, dated April 26, 2007 (see http: / / www.in-pharmatechnologist.com / Ingredients / OctoPlus-broadens-rights-to-PolyActive-delivery-technology, downloaded on March 9, 2016). Examples of biopharmaceuticals include proteins.

[0030] When a PEG polymer is present in an hCG microsphere formulation prepared from a PolyActivePEG / PBT multiblock copolymer, the length of the PEG can vary from about 1000 to about 2500 g / mol. Non-limiting examples include PEG lengths of at least about 1000 g / mol, at least about 1100 g / mol, at least about 1200 g / mol, at least about 1300 g / mol, at least about 1400 g / mol, at least about 1500 g / mol, at least about 1600 g / mol, at least about 1700 g / mol, at least about 1800 g / mol, at least about 1900 g / mol, at most about 2000 g / mol, at most about 2100 g / mol, at most about 2200 g / mol, at most about 2300 g / mol, at most about 2400 g / mol, or at most about 2500 g / mol.

[0031] In some embodiments, the PEG and one or more other polymers present in the hCG microsphere dosage form prepared from PolyActivePEG / PBT multiblock copolymers are present in a ratio (by weight) of about 80 / 20 to about 60 / 40, for example, but not limited to, about 79 / 21 to about 60 / 40, about 78 / 22 to about 60 / 40, about 77 / 23 to about 60 / 40, about 76 / 24 to about 60 / 40, about 75 / 25 to about 60 / 40, about 74 / 26 to about 60 / 40, about 73 / 27 to about 60 / 40, about 72 / 28 to about 60 / 40, about 71 / 29 to about 60 / 40, about 70 / 30 to about 60 / 40, about 69 / 31 to about 60 / 40, about 68 / 32 to about 60 / 40, and about 67 / 33 to about 60 / 40. Examples of weight ratios include about 80 / 20, about 79 / 21, about 78 / 22, about 77 / 23, about 76 / 24, about 75 / 25, about 74 / 26, about 73 / 27, about 72 / 28, about 71 / 29, about 70 / 30, about 69 / 31, about 68 / 32, or about 67 / 33.

[0032] B2. hCG microspheres composed of multiblock copolymers containing crystalline poly(L-lactide) building blocks Other exemplary polymers that can be utilized in the sustained-release hCG compositions of the present invention include SynBiosys® polymers produced by Innocore Pharmaceuticals. InnoCore's SynBiosys® technology provides a novel platform of bioabsorbable polymers specifically designed to function as drug delivery systems. The polymers are composed of D,L-lactide, glycolide, ε-caprolactone, and polyethylene glycol, which are approved for human use. While SynBiosys® polymers have been used in drug delivery systems, prior to the present invention, polymers have not been well formulated for highly complex glycoproteins, such as hCG.

[0033] SynBiosys® polymers are described in WO-A2013 / 015685, the entire contents of which are incorporated herein by reference, which describes biodegradable, semi-crystalline, phase-separated, thermoplastic multi-block copolymers comprising at least one hydrolyzable prepolymer (A) segment and at least one hydrolyzable prepolymer (B) segment, the multi-block copolymers having a T of 37° C. or less under physiological conditions. g and T of 110 to 250°C m wherein the segments are linked by a polyfunctional chain extender, the segments are randomly distributed on the polymer chain, and the prepolymer (A) segment comprises polyethylene glycol.

[0034] The form and properties under physiological conditions (i.e., in the body) may differ from the form and properties under ambient (dry, room) temperatures. g and T m refers to the corresponding value of the material when applied in vivo, i.e., at equilibrium with an atmosphere saturated with water vapor and at body temperature, which can be simulated in vitro by carrying out DSC measurements after equilibrating the material with a water-saturated atmosphere.

[0035] The prepolymer (A) segment may comprise the reaction product of an ester-forming monomer selected from a diol, a dicarboxylic acid, and a hydroxycarboxylic acid. Preferably, the prepolymer (A) segment comprises the reaction product of glycolide, lactide (D and / or L), ε-caprolactone, and / or δ-valerolactone.

[0036] The prepolymer (A) segment has an M of about 500 g / mol or more, e.g., about 700 g / mol or more, about 1000 g / mol or more, about 2000 g / mol or more, about 3000 g / mol or more, or about 4000 g / mol or more. n Typically, the prepolymer (A) segment has an M of about 80,000 g / mol or less. n It has.

[0037] The prepolymer (B) segment is preferably poly(L-lactide), more preferably having an M of about 1000 g / mol or more, e.g., about 2000 g / mol or more, about 3000 g / mol or more, or about 4000 g / mol or more. n Typically, the prepolymer (B) segment comprises a poly(L-lactide) having an M of about 80,000 g / mol or less. n It has.

[0038] The content of prepolymer (A) in the multi-block copolymer can be from about 10% to about 90%, for example, from about 30% to about 75%, or from about 50% to about 70%, based on the total weight of the multi-block copolymer.

[0039] The content of prepolymer (B) in the multi-block copolymer can be from about 10% to about 90%, for example, from about 25% to about 70%, or from about 30% to about 50%, based on the total weight of the multi-block copolymer.

[0040] The polyfunctional chain extender may be a difunctional aliphatic chain extender, preferably a diisocyanate, such as 1,4-butane diisocyanate or 1,6-hexane diisocyanate.

[0041] The polyethylene glycol in the poly(L-lactide)-based SynBiosys® multiblock copolymers has an M of about 150 to about 5000 g / mol, e.g., about 200 g / mol to about 1500 g / mol, about 600 to about 1000 g / mol, about 400 to about 3000 g / mol, about 600 to about 1500 g / mol, about 600 to about 5000 g / mol, or about 1000 to about 3000 g / mol. n may have:

[0042] Preferably, the biodegradable multi-block copolymer has a swelling ratio under physiological conditions of about 1 to about 4, preferably about 1 to about 2, more preferably about 1 to about 1.5.

[0043] In one embodiment, the biodegradable multi-block copolymer is a [poly(ε-caprolactone)-co-polyethylene glycol-co-poly(ε-caprolactone)]-b-[poly(L-lactide)] multi-block copolymer.

[0044] B2. hCG microspheres composed of multiblock copolymers containing crystalline poly(p-dioxanone) building blocks In some embodiments, the biodegradable multi-block copolymer comprises a biodegradable, phase-separated, thermoplastic multi-block copolymer comprising at least one amorphous hydrolyzable prepolymer (A) segment and at least one semi-crystalline hydrolyzable prepolymer (B) segment; said multi-block copolymer under physiological conditions has a T of about 37° C. or less g and T of about 50 to about 110°C m having; - the segments are linked by a multifunctional chain extender; - The segments are randomly distributed on the polymer chain; - the prepolymer (B) segment comprises an XYX triblock copolymer, where Y is a polymerization initiator and X is a poly(p-dioxanone) segment having a block length of about 7 or more p-dioxanone monomer units;

[0045] X is preferably a poly(p-dioxanone) segment having a block length of from about 7 to about 35, e.g., from about 8 to about 30, from about 9 to about 25, from about 10 to about 20, or from about 12 to about 15 p-dioxanone monomer units.

[0046] At least a portion of the prepolymer (A) segments, for example, about 30% or more, about 40 to about 95%, about 50 to about 90%, or about 60 to about 85%, based on the total weight of the prepolymer (A), can be derived from a water-soluble polymer.

[0047] Prepolymer (A) may comprise, for example, a reaction product of cyclic and / or acyclic monomers. Suitable acyclic monomers may be selected from the group consisting of succinic acid, glutaric acid, adipic acid, sebacic acid, lactic acid, glycolic acid, hydroxybutyric acid, ethylene glycol, diethylene glycol, 1,4-butanediol, and / or 1,6-hexanediol. Suitable cyclic monomers may be selected from the group consisting of glycolide, lactide, ε-caprolactone, δ-valerolactone, trimethylene carbonate, tetramethylene carbonate, 1,5-dioxepan-2-one, 1,4-dioxan-2-one (p-dioxanone), and / or cyclic anhydrides, such as oxepane-2,7-dione.

[0048] Preferably, the prepolymer (B) segment contains a relatively large poly(p-dioxanone) portion, for example, about 70% or more, preferably about 80% or more, and more preferably about 90% or more, based on the total weight of the prepolymer (B) segment, may be poly(p-dioxanone).

[0049] The prepolymer (B) segment has a number average molecular weight M of about 1300 to about 7200 g / mol, preferably about 1300 to about 5000 g / mol, more preferably about 1500 to about 4500 g / mol, even more preferably about 2000 to about 4000 g / mol, for example, about 2200 to about 3000 g / mol. n may have:

[0050] The prepolymer (B) segment has a weight average molecular weight M of about 1800 to about 10800 g / mol, preferably about 1800 to about 7000 g / mol, more preferably about 2100 to about 6300 g / mol, even more preferably about 2600 to about 5600 g / mol, for example, about 3000 to about 4200 g / mol. w may have:

[0051] The prepolymer (B) segment has a T of less than about 0° C., for example, less than about −20° C., or less than about −40° C. g The prepolymer (B) segment may have a T in the range of about 60 to about 100°C, preferably about 75 to about 95°C. m may have:

[0052] The water-soluble polymer can be selected from or derived from the group of polymers consisting of polyethers, such as polyethylene glycol (PEG), polytetramethylene oxide (PTMO), polypropylene glycol (PPG), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyvinylcaprolactam, poly(hydroxyethyl methacrylate) (poly-HEMA), polyphosphazenes, or copolymers of these polymers. Preferably, the water-soluble polymer is derived from polyethylene glycol. More preferably, the water-soluble polymer has an M of about 150 to about 5000 g / mol. n It is derived from polyethylene glycol having the formula:

[0053] The chain extender may be a difunctional aliphatic chain extender. Preferably, the chain extender is a diisocyanate, such as 1,4-butane diisocyanate or hexamethylene diisocyanate.

[0054] In one embodiment, the biodegradable multi-block copolymer is a [poly(ε-caprolactone)-co-polyethylene glycol-co-poly(ε-caprolactone)]-b-[poly(p-dioxanone)] multi-block copolymer.

[0055] In some embodiments, the biodegradable multi-block copolymer comprises [(R 1 R 2 n R 3 ) q ] r [(R 4 p R 5 R 6 p )] s is represented by During the ceremony, R 1 , and R 3 are respectively [ka] and R 2 teeth, [ka] and R 4 and R 6 are respectively [ka] and n is the number of repetitions R 2 is the number of moieties, from about 20 to about 115, preferably from about 35 to about 100, more preferably from about 45 to about 85; p is the repetition R 4 and R6 is the number of moieties, which is about 7 or more, preferably about 7 to about 35, more preferably about 10 to about 20, and even more preferably about 10 to about 14; q is (R 1 R 2 n R 3 ) number average molecular weight of the block, which is from about 1000 to about 7000 g / mol, preferably from about 3000 to about 5000 g / mol, more preferably from about 3800 to about 4200 g / mol; r / s is the ratio of prepolymer (A) segments to prepolymer (B) segments and is from about 0.10 to about 1.0, for example, from about 0.15 to about 0.50, or from about 0.20 to about 0.30.

[0056] In one embodiment, the biodegradable multi-block copolymer comprises [(R 1 R 2 n R 3 ) q ] r [(R 4 p R 5 R 6 p )] s and R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently as defined above, n is from about 65 to about 71, p is from about 11 to about 13, q is from about 3800 to about 4200, r is from about 15 to about 25, and s is from about 75 to about 85.

[0057] When PEG polymers are present in hCG microsphere formulations prepared from poly(p-dioxanone)-based multiblock copolymers, the length of the PEG can vary from about 1000 to about 5000 g / mol. Non-limiting examples include PEG lengths of at least about 1000 g / mol, at least about 1200 g / mol, at least about 1400 g / mol, at least about 1600 g / mol, at least about 1800 g / mol, at least about 2000 g / mol, at least about 2200 g / mol, at least about 2400 g / mol, at least about 2600 g / mol, at least about 2800 g / mol, at least about 3000 g / mol, at least about 3200 g / mol, at least about 3400 g / mol, at least about 3600 g / mol, at least about 3800 g / mol, at most about 4000 g / mol, at most about 4200 g / mol, at most about 4400 g / mol, at most about 4600 g / mol, at most about 4800 g / mol, or at most about 5000 g / mol.

[0058] C. Formulations Aspects of the present disclosure relate to formulations comprising a plurality of hCG microspheres. Such formulations may comprise a homogeneous or heterogeneous mixture of microspheres according to any one of the parameters disclosed herein. Further such formulations may optionally further comprise pharmaceutically acceptable excipients and / or other components relevant to the specified indication being treated.

[0059] D. Methods of Administration and Release Profiles A feature of the microspheres disclosed herein is a release profile that allows for sustained release of hCG or its derivatives or isoforms. In some embodiments, the release of hCG or its derivatives or isoforms in the microspheres or microsphere formulations is less than or about 1 / 7, e.g., less than or about 1 / 14, less than or about 1 / 21, less than or about 1 / 28, less than or about 1 / 29, less than or about 1 / 30, less than or about 1 / 31, less than or about 1 / 33, or less than or about 1 / 34. or less than about 1 / 34, 1 / 35, 1 / 42, 1 / 49, 1 / 56, 1 / 57, 1 / 58, 1 / 59, 1 / 60, 1 / 61, or 1 / 62 is released in the first 24 hours after administration. In some embodiments, about 2% to about 40%, e.g., about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, about 12.5%, about 13%, about 13.5%, about 14%, about 14.5%, about 15%, about 15.5% of the hCG or derivative or isoform thereof in the microsphere or microsphere formulation. , about 16%, about 16.5%, about 17%, about 17.5%, about 18%, about 18.5%, about 19%, about 19.5%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40%, or any range including two of those values, for example, about 18.5% to about 26%, is released in the first 24 hours.

[0060] Without being bound by any theory, the above release profiles may be effective for a specified period of time, for example, but not limited to, about 1 week or more, about 2 weeks or more, about 3 weeks or more, about 4 weeks or more, about 5 weeks or more, about 6 weeks or more, about 7 weeks or more, about 8 weeks or more, about 1 month or more, about 2 months or more, or about 7 days or more, about 8 days or more, about 9 days or more, about 10 days or more, about 11 days or more, about 12 days or more, about 13 days or more, about 14 days or more, about 15 days or more, about 16 days or more, about 17 days or more, about 18 days or more, about 19 days or more, about 20 days or more, about 21 days or more, about 22 days or more, about 23 days or more, about 24 days or more, about 25 days or more, about 26 days or more, about 27 days or more, about 28 days or more, about 29 days or more, about 30 days or more, about 31 days or more, about It is envisioned that sustained release of hCG or a derivative or isoform thereof for 32 days or more, about 33 days or more, about 34 days or more, about 35 days or more, about 36 days or more, about 37 days or more, about 38 days or more, about 39 days or more, about 40 days or more, about 41 days or more, about 42 days or more, about 43 days or more, about 44 days or more, about 45 days or more, about 46 days or more, about 47 days or more, about 48 days or more, about 49 days or more, about 50 days or more, about 51 days or more, about 52 days or more, about 53 days or more, about 54 days or more, about 55 days or more, about 56 days or more, about 57 days or more, about 58 days or more, about 59 days or more, about 60 days or more, about 61 days or more, or about 62 days or more, about 1 month or more, about 2 months or more, about 3 months or more, about 4 months or more, about 5 months or more, or about 6 months or more is possible.

[0061] It is contemplated that the microspheres and formulations comprising these microspheres can be administered according to any mode of administration known in the art, including, but not limited to, topical, enteral, parenteral, oral, sublingual, via inhalation, nasal, via injection, intradermal, transdermal, intramuscular, subcutaneous, bolus administration, infusion, and / or any other suitable method.

[0062] In certain aspects, the present disclosure relates to methods of administration that achieve or approximate a theoretical release profile according to Figure 1. In some embodiments, the release profile exhibits minimal or no burst release.

[0063] Embodiments of the present disclosure relate to administration regimens that approximate, for example, the release profiles according to Figure 1. Such regimens include, but are not limited to, about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 1 month, 2 months, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about These include administration of a sustained release hCG formulation every 1, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, or about 62 days.

[0064] E. Treatment Methods The hCG microspheres and formulations thereof disclosed herein can be used to treat a variety of conditions and can be administered according to appropriate cycles and dosages based on the indication. The indication can be human or animal. Exemplary indications include hypogonadotropism, cryptorchidism, luteal phase maintenance (e.g., in assisted reproductive technology), contraception, weight loss, pituitary disorders, breast cancer, and / or any other disease or disorder associated with hCG deficiency.

[0065] Certain embodiments relate to the treatment of breast cancer with the hCG microspheres and formulations disclosed herein. Pregnancy is known to be protective against breast cancer. Without being bound by theory, it is predicted that administration of the hCG microspheres and formulations disclosed herein may achieve an approximately 30% to approximately 40% reduction in breast cancer risk. See Russo and Russo, Molecular Basis of Breast Cancer: Prevention and Treatment (Springer Science & Business Media, 2004). Furthermore, unlike methods disclosed in the art for administering hCG, the hCG microspheres and formulations disclosed herein require fewer administrations, e.g., less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, less than about 5, less than about 4, less than about 3, or about 1 or 2 administrations, compared to the 45 administrations of conventional hCG over a three-month period. In other embodiments, the described formulations may be effective in a once-weekly administration or a once-, twice-, or three-times-monthly administration schedule for the treatment and / or prevention of breast cancer. In some embodiments, the treatment is for existing breast cancer in nulliparous women. In some embodiments, the described formulations are in the form of a single injection that provides a therapeutic effect for more than one month.

[0066] F. General Definition As used in the detailed description of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0067] As used herein, the term "about," when referring to a measurable value, such as an amount or concentration, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the stated amount.

[0068] The terms "acceptable," "effective," or "sufficient," when used to describe the selection of any component, range, dosage form, etc. disclosed herein, are intended to mean that said component, range, dosage form, etc. is suitable for the purpose disclosed.

[0069] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, and does not include combinations when interpreted as alternatives ("or").

[0070] As used herein, the term "comprising" shall mean that the formulations and methods include the recited elements but do not exclude others. As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) shall be interpreted to encompass the recited materials or steps "and that do not materially affect the basic and novel characteristic(s)" of the recited embodiment. See In re Herz, 537 F.2d 549,551-52,190 USPQ 461,463 (CCPA 1976) (emphasis in original); see also MPEP § 2111.03. Thus, as used herein, the term "consisting essentially of" should not be interpreted as equivalent to "comprising." "Consisting of" shall mean excluding more than trace amounts of other component elements and substantial method steps of administering the formulations disclosed herein. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0071] "Combination" is intended to mean a combination of an active agent with another compound or composition, inert (eg, a detectable agent or label) or active (eg, an adjuvant).

[0072] A "pharmaceutical formulation" is intended to include a combination of an active agent with an inert or active carrier which makes the formulation suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0073] "Pharmaceutically acceptable carrier" refers to any diluent, excipient, or carrier that can be used in the formulations of the present invention. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences," Mack Publishing Company, a standard reference textbook in this field. They are preferably selected according to the intended form of administration, i.e., oral tablets, capsules, elixirs, syrups, etc., consistent with conventional pharmaceutical practice.

[0074] As used herein, the term "sustained release" refers to the ability to release the active ingredient (hCG) over a defined period of time. The term "burst release" refers to a rapid release period of the active ingredient (hCG) into an environment such that continued release over an extended period of time is not sustained.

[0075] As used herein, the terms "subject" or "patient" are used interchangeably to refer to any animal. In some embodiments, the subject may be a mammal; in further embodiments, the subject may be a human, a mouse, or a rat.

[0076] As used herein, "treating" a disease in a subject or "treatment" thereof refers to (1) preventing a symptom or disease from occurring in a subject who is susceptible to the disease or who does not yet exhibit symptoms thereof; (2) inhibiting or halting the development of the disease; or (3) ameliorating or causing regression of the disease or symptoms of the disease. As understood in the art, "treatment" is an approach for obtaining a beneficial or desired result, e.g., a clinical result. For purposes of the current technology, a beneficial or desired result may include, but is not limited to, one or more of the following: alleviation or amelioration of one or more symptoms, whether detectable or undetectable; reduction in the extent of a pathological condition (e.g., a disease); stabilization (i.e., non-worsening) of a pathological condition (e.g., a disease); slowing or delaying the progression of a pathological condition (e.g., a disease); improvement or palliation of a pathological condition (e.g., a disease); and remission (whether partial or complete).

[0077] Unless otherwise specified, all terms used herein (for example, technical and scientific terms) have the same meaning as that commonly understood by those skilled in the art to which the present invention belongs.Terms, for example, those defined in commonly used dictionaries, should be interpreted to have meanings consistent with their meanings in the context of this application and related fields, and it is further understood that unless expressly defined herein, they should not be interpreted in an idealized or overly formal sense.For example, descriptive terms can be used to refer to biological materials (for example, tissues, organoids, samples) that exhibit the characteristics of specific organs, and for example, "liver" can be used to describe liver-derived tissue or liver-like organoids.Unless expressly defined below, such terms should be interpreted according to their general meaning.

[0078] The terminology used in the detailed description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0079] The practice of the present techniques employs conventional techniques, within the skill of one of ordinary skill in the art, unless otherwise indicated.

[0080] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Furthermore, the present disclosure also contemplates that, in some embodiments, any feature or combination of features described herein can be excluded or omitted. For illustration, if the specification describes a composite comprising components A, B, and C, it is expressly intended that any of A, B, or C, or combinations thereof, singly or in any combination, can be omitted or discarded.

[0081] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations that vary (+) or (-) by increments of 1.0 or 0.1, as appropriate, or by variations of + / - 15%, or 10%, or 5%, or 2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about." It is also to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. [Example]

[0082] V. Working Examples The following examples are non-limiting and are illustrative of procedures that may be used in various instances in practicing the present disclosure. Additionally, all references disclosed herein are incorporated by reference in their entirety.

[0083] Example 1 - Comparison of commercially available hCG Ovidrel® ("Serono") obtained from EMD Serono and Dong-A hCG were compared for differences in molecular weight.

[0084] Colloidal Blue Native and SDS page gels were prepared. A gel suitable for silver staining was also prepared but produced unreadable results.

[0085] Under native gel conditions using native samples and running buffer, both Dong-A and Serono prepared samples of API hCG showed the same banding pattern.

[0086] Native PAGE analysis demonstrated that there was no significant difference in molecular weight between hCG from Dong-A and Serono. The molecular weights on native PAGE gels were similar but difficult to read due to the mismatched markers. Under non-native and semi-native conditions (presence of SDS), the molecular weights of hCG from Dong-A and Serono (banding between 44 and 52 kDa) were similar, with a single diffuse band of 50-60 kDa listed (Figure 12).

[0087] Example 2 - Production of PolyActive hCG Microspheres Dong-A hCG was concentrated to a target concentration of 20 mg / ml using PBS buffer + methionine at pH 7.4. A summary of the analysis is provided in Table 1 below.

[0088] [Table 1]

[0089] Protein integrity was maintained during the concentration step, resulting in a protein solution of approximately 20 mg / ml (21.5 mg / ml), which then allows for a protein / polymer ratio of 1.5-2%. Therefore, the final drug product should be in the range of approximately 0.2-0.6 mg hCG in 0.2 cc. Protein integrity was maintained during concentration (Figure 11).

[0090] Concentrated hCG was introduced into the primary emulsion containing the polymer-containing organic solution (PolyActive™ polymer (1 g / 9 g dichloromethane)) using a positive displacement pipette, and then homogenized at 19,000 rpm at room temperature for 30 seconds. This primary emulsion was then mixed with PBS PVA 7% for 5 minutes, then PBS + methionine was added for 5 hours, and then washed 5 times. The same process was repeated using placebo (PBS + methionine)-loaded microspheres.

[0091] Microspheres were produced according to this method for a variety of polymers, examples of which are listed below: PolyAvctive™ PEG1500 series: PEG / PBT weight ratio = 70 / 30, 75 / 25, 80 / 20, 90 / 10 PolyActive™ PEG1000 series: PEG / PBT weight ratio = 70 / 30, 67 / 33 PolyActive™ PEG2000 series: PEG / PBT weight ratio = 60 / 40, 75 / 25, 80 / 20

[0092] The resulting particles were sized using a Mastersizer as described in Table 2.

[0093] [Table 2]

[0094] Microscopic observations confirmed the results obtained using the Mastersizer. No significant morphology differences were observed, except for the microsphere formulation with a weight ratio of 1500 / 90 / 10, which gave large, ovoid microsphere shapes. Based on size distribution, the formulations in Table 3, generally in the size range of about 50 to 100 μm without any additional processing, were selected for further analysis.

[0095] [Table 3]

[0096] Example 3 - Analysis of the release profile of PolyActive hCG microspheres Microspheres were prepared according to the method of Example 2. A series of formulations listed in Table 4 were tested via an in vitro release assay.

[0097] [Table 4]

[0098] Samples and controls were incubated with 0.05% NaN3 and 0.05% Tween 20 in PBS at pH 7.4 at 37°C. Preliminary cumulative and normalized release profiles conducted for up to 14 days were determined (Figures 2-3). Formulations 695-01-0008, 695-01-0010, and 695-01-0011 all demonstrated low to no burst release. hCG remained substantially pure for the control formulation (Figure 13) and decreased by only about 5% over 14 days, as shown by Table 5.

[0099] [Table 5]

[0100] Formulation 695-01-0011 ("Formulation 11") demonstrated a release profile suitable for sustained-release hCG administration. Theoretical release profiles were determined (Figures 5-9) for varying hCG doses of Formulation 11 based on its measured in vitro release profile (Figure 4), assuming the following: bioavailability = 0.5; volume of distribution = 5.5 L; absorption half-life = 8 hours; terminal elimination half-life = 24 hours; maximum microsphere density of 15% w / v (150 mg / ml) for injectability using a 23-25G needle; 2% drug loading; 1 cc injection delivers 3 mg of hCG; and single injection: 3 mg of hCG in 1 ml formulated as "Formulation 11" PolyActive microspheres. Table 6 shows how the half-life of hCG and dosing interval result in the minimum sustained hCG level in the blood, in ng / ml, for a product with 3 mg of hCG in Formulation 11.

[0101] [Table 6]

[0102] C min and C max The ratio between the two is also large. 1 / 2 = 24 and 36 hours. Plasma levels are proportional to the administered dose and elimination half-life. For example, formulations with lower release rates, even up to 4 or 6 weeks for biweekly dosing, have lower C max / C min This may provide a more efficient use of ratios and doses, reducing the need for higher loading % hCG.

[0103] Therefore, additional rounds of in vitro release were investigated by varying the parameters of Functional Formulation 11. The proposed variations are listed in Table 7. Further investigations included blending PolyActive™ PEG 1500 75 / 25 and PolyActive™ PEG 1500 70 / 30 in weight ratios of 1:4, 1:1, 1.5:1, or 4:1 to mimic PolyActive™ PEG 1500 71 / 29, 72.5 / 27.5, 73 / 27, and 74 / 26, respectively.

[0104] [Table 7]

[0105] Second and third rounds of in vitro release assays were performed on additional samples listed in Table 8. A number of formulations—695-01-0021 (at least 4 to 6 weeks of release), 695-01-0025, 695-01-0031, 695-01-0032, 695-01-0033 (potentially optimal), and 695-01-0034—exhibited suitable hCG release profiles with sustained release durations ranging from 1 week to over 50 days (FIGS. 10, 14-16). Further analysis of these formulations is provided in Table 8.

[0106] [Table 8]

[0107] Example 4 - Production of hCG sustained-release microspheres composed of SynBiosys [PCL-co-PEG-co-PCL]-b-[PLLA] multiblock copolymer This example describes the preparation and characterization of hCG sustained-release microspheres prepared from SynBiosys [poly(ε-caprolactone)-co-polyethylene glycol-co-poly(ε-caprolactone)]-b-[poly(L-lactide)] (PCL-co-PEG-co-PCL]-b-[PLLA]) multiblock copolymers.

[0108] hCG (Dong-A Pharmaceutical Co., Ltd.; Korea) was concentrated to a target concentration of 30 mg / ml using PBS buffer + methionine at pH 7.4 and an Amicon Ultra-15 vial with a polyethersulfone membrane with a 10 kDa cutoff size. The hCG concentration of the concentrated solution was 30.7 mg / ml hCG as determined by RP-UPLC. Visual examination of the concentrated protein solution showed the absence of insoluble particles. The integrity of the hCG was maintained during the concentration step. RP-UPLC analysis (Figure 17, UPLC chromatogram) confirmed the absence of aggregates, and there was no sign of degradation.

[0109] Approximately 0.35 g of concentrated hCG solution was added to a solution of 0.50 g of polymer in 2.92 g of dichloromethane (DCM), and then homogenized at 22,000 rpm for 40 seconds to obtain a water-in-oil (W / O) primary emulsion. This primary emulsion was then emulsified with a 4.0% aqueous PVA solution containing 5.0 w / v% NaCl using a continuous flow reactor, thereby forming a water-in-oil-in-water (W / O / W) double emulsion. The W / O / W emulsion was stirred at room temperature for 3 hours to allow extraction and evaporation of dichloromethane. After solvent evaporation was completed, the hCG microspheres were collected by filtration and freeze-dried to obtain dry hCG microspheres.

[0110] Using the above procedure, microspheres were prepared from blends of SynBiosys 50[PCL-co-PEG1000-co-PCL]2000-b-[PLLA]4000 and SynBiosys 30[PCL-co-PEG3000-co-PCL]4000-b-[PLLA]4000 at different formulations and process parameter settings (polymer blend ratio, polymer concentration, CP:DP ratio).

[0111] SynBiosys 50[PCL-co-PEG1000-co-PCL]2000-b-[PLLA]4000 (also abbreviated as 50CP10C20-LL40) is a multiblock copolymer consisting of a hydrophilic [PCL-co-PEG1000-co-PCL] prepolymer segment (A) with a molecular weight of 2000 g / mol (containing 50 mol% polyethylene glycol with a molecular weight of 1000 g / mol) and a semicrystalline poly(L-lactide) prepolymer segment (B) with a molecular weight of 4000 g / mol that has been chain-extended with 1,4-butane diisocyanate in a 50 / 50 wt% block ratio. SynBiosys 30[PCL-co-PEG3000-co-PCL]4000-b-[PLLA]4000 (also abbreviated as 30CP30C40-LL40) is a multiblock copolymer composed of a hydrophilic [PCL-co-PEG3000-co-PCL] prepolymer segment (A) with a molecular weight of 4000 g / mol (containing 75 mol% polyethylene glycol with a molecular weight of 3000 g / mol) and a semicrystalline poly(L-lactide) prepolymer segment (B) with a molecular weight of 4000 g / mol that has been chain-extended with 1,4-butanediisocyanate in a 30 / 70 wt% block ratio.

[0112] The resulting hCG microspheres were characterized for their particle size distribution using a Coulter Counter Multisizer III. The mean particle size varied from 21 to 48 μm (Table 9).

[0113] [Table 9]

[0114] Microscopic examination of the hCG microspheres, assessed by scanning electron microscopy using a JEOL JCM-5000 Neoscope, confirmed the results using the Coulter Counter. All hCG microspheres had similar morphological characteristics, i.e., spherically shaped microparticles with smooth surfaces (Figure 18).

[0115] The hCG content and encapsulation efficiency (EE) of all microspheres were determined by hydrolysis of the polymer in 0.1 M NaOH, mixing the hydrolysate with 100 mM phosphate buffer pH 7.4, and subsequent analysis of the hCG concentration by RP-UPLC. The hCG content of the hCG microsphere batches varied from 1.3% to 1.8%, representing encapsulation efficiencies of 75% to 95% (Table 9).

[0116] In vitro release kinetics were determined by incubating hCG microspheres in PBS buffer, pH 7.4, containing 0.01% Tween-20 and 0.01% sodium azide, at 37°C, routinely sampling, and analyzing hCG concentrations by RP-UPLC. Cumulative release profiles were determined for at least 4 weeks (Figure 19). All formulations demonstrated low to no burst release followed by a sustained release of hCG. Some formulations exhibited a lag time followed by an irregular release pattern (JA16044 and JA16045). JA16043 demonstrated the most promising release kinetics, as it had no burst, a linear release over 1 week, and a recovery rate of >75%. JA16-043 was further analyzed for the integrity of released hCG. The concentration of intact hCG was determined by RP-UPLC. As shown in Figure 20, hCG was released almost completely in its intact form from the hCG microspheres.

[0117] Example 5 - Synthesis of SynBiosys20[PCL-PEG3000-PCL]-b-[PDO] multiblock copolymer The [poly(ε-caprolactone)-co-polyethylene glycol-co-poly(ε-caprolactone)]-b-[poly(L-lactide)] multiblock copolymer used in Example 4 is known to degrade relatively slowly. Multiblock copolymers composed of poly(p-dioxanone)-based crystalline blocks are known to degrade more quickly, which could be beneficial in preventing polymer carrier accumulation during repeated subcutaneous administration.

[0118] This example describes the synthesis and characterization of a [poly(ε-caprolactone)-co-polyethylene glycol-co-poly(ε-caprolactone)]-b-[poly(p-dioxanone)] multiblock copolymer based on PEG3000 and a block ratio of 20 / 80 wt %.

[0119] Molecular weight (M) of approximately 4000 g / mol n Poly(ε-caprolactone)-co-PEG3000-co-poly(ε-caprolactone) prepolymer (abbreviated as PCL-PEG3000-PCL) with a molecular weight (M) of approximately 2500 g / mol was prepared by ring-opening polymerization of ε-caprolactone using polyethylene glycol (PEG3000) with a molecular weight of 3000 g / mol as an initiator and stannous octoate as a catalyst. n Poly(p-dioxanone) prepolymer (abbreviated as PDO) with α-dimethylformamide (α-dimethylformamide) was synthesized by ring-opening polymerization of p-dioxanone using 1,4-butanediol as initiator and stannous octoate as catalyst. The molecular weight of the prepolymer was 1 The product was analyzed by H-NMR.

[0120] [PCL-PEG3000-PCL]-b-[PDO] multiblock copolymers with a block ratio of 20 / 80 wt%, abbreviated as 20[PCL-PEG3000-PCL]-b-[PDO], were prepared by chain extension of PCL-PEG3000-PCL prepolymer with PDO prepolymer in p-dioxane using 1,4-butanediisocyanate as a chain extender, followed by freeze-drying or precipitation to remove p-dioxane.

[0121] The polymer 1 The polymers were analyzed for polymer composition by H-NMR, intrinsic viscosity (Ubbelohde, chloroform), residual p-dioxane content (gas chromatography), and thermal characteristics by modulated differential scanning calorimetry. Table 10 lists the characteristics of various [poly(ε-caprolactone)-co-PEG-co-poly(ε-caprolactone)]-b-[poly(p-dioxanone)] multiblock copolymers.

[0122] [Table 10]

[0123] Example 6 - Production of hCG sustained-release microspheres composed of SynBiosys20[PCL-PEG3000-PCL]-b-[PDO] multiblock copolymer hCG (Dong-A Pharmaceutical Co., Ltd., Korea) was concentrated to 30 mg / ml as described in Example 4. 1.5 g of 20[PCL-PEG3000-PCL]-b-[PDO] multiblock copolymer (RCP-1557) was dissolved in dichloromethane to a concentration of 15 wt%. 0.73 g of concentrated hCG solution was added to the polymer solution and homogenized at 22,000 rpm for 40 seconds to obtain a water-in-oil (W / O) primary emulsion. The primary emulsion was then emulsified with a 4.0% aqueous PVA solution containing 5.0 w / v% NaCl via membrane emulsification using a membrane with 20 μm pores, thereby forming a water-in-oil-in-water (W / O / W) double emulsion. The W / O / W emulsion was stirred at room temperature for 3 hours to allow for extraction and evaporation of dichloromethane. After solvent evaporation was complete, the hCG microspheres were collected by filtration and freeze-dried to obtain dry hCG microspheres.

[0124] hCG microparticles characterized using the method described in Example 4 were spherical with a smooth surface morphology (Figure 21), had a mean particle size of 38 μm, and a narrow particle size distribution (CV = 14-18%). The hCG content varied from 1.33 to 1.62 wt%, representing encapsulation efficiencies of 67 to 86% (Table 10). All microparticles released rhCG gradually and largely intact over an 11-week period without any significant burst release (Figure 22).

[0125] [Table 11]

[0126] Batches JA16101 and JA16102 were combined into one batch for further testing of in vivo pharmacokinetics / pharmacodynamics.

[0127] Example 7 - Pilot Pharmacokinetic / Pharmacodynamic Study of hCG Sustained-Release Microspheres in Young Adult Cynomolgus Monkeys An in vivo pharmacokinetic / pharmacodynamic study using the hCG sustained-release microspheres recovered from JA16101 and JA16102 (Example 5) was conducted in five healthy young adult male cynomolgus monkeys.

[0128] For the duration of the study, all monkeys were treated with a GnRH antagonist (Cetrorelix 250 μg) every three days to suppress pituitary function and endogenous testosterone production. All monkeys were pretreated with Cetrorelix (days -5 and -2) and evaluated for both hCG and testosterone levels. Two monkeys received daily subcutaneous injections of 3 μg of hCG for the duration of the study (control group): one monkey received Ovidrel and the other received Dong-A hCG. Three monkeys received a single dose of hCG sustained-release microspheres (200 μg, 600 μg, and 1200 μg r-hCG) subcutaneously (hCG-MSP group).

[0129] For both groups, blood samples were obtained frequently over the first 24 hours and at regular intervals thereafter. The resulting serum was evaluated for hCG by ELISA (LLOQ = 0.5 ng / ml) and for testosterone levels by LC / MS / MS (LLOQ = 0.25 ng / ml) until hCG and testosterone levels fell to low levels.

[0130] Monkeys in the control group had an initial rise in serum hCG levels, which was accompanied by a corresponding rise in serum testosterone levels (Figure 23). Serum levels reached a steady state by day 3. However, both serum hCG and serum testosterone levels later eventually declined to near zero despite continued daily injections of hCG.

[0131] A binding inhibition assay was used to confirm the presence of anti-drug antibodies (ADA) to hCG. Spiking recovery assays on samples collected on day 55 from control monkeys confirmed that the decline in serum hCG and serum testosterone levels accompanied the ADA response to hCG. Spiking of both 5 and 55 ng / nL reference standards to treated monkey serum resulted in complete inhibition of hCG recovery, as opposed to over 80% recovery when untreated or pre-treated monkey serum was spiked using an ELISA assay.

[0132] For all three monkeys treated with hCG sustained-release microspheres, hCG levels increased in a near-linear and dose-dependent manner. Serum hCG levels over 48 hours confirmed the presence of a near-zero burst, even at the highest dose (Figure 24, Panel A). Serum hCG and serum testosterone levels over the duration of the study are graphed in Figure 24, Panels B and C). Figure 25 shows serum hCG and serum testosterone on the same graph for each individual monkey receiving hCG sustained-release microspheres representing 200 μg (Panel A), 600 μg (Panel B), and 1200 μg hCG (Panel C). These graphs demonstrate clear similarities in serum hCG and serum testosterone levels throughout the study. Furthermore, they demonstrate that serum hCG is released steadily until a significant decline occurs at approximately 14 days. Spike recovery analysis of the serum of monkeys treated with hCG sustained-release microspheres collected on day 33 confirmed that the decline in serum hCG levels was caused by an inactivated ADA response, as in the control group.

[0133] [Table 12]

[0134] In summary, this pilot study demonstrates that a single subcutaneous injection of hCG sustained-release microspheres provides a dose-dependent sustained release of hCG with minimal burst in cynomolgus monkeys, and that the hCG released from the microspheres retains its bioactivity and induces a testosterone response.

[0135] However, due to limitations of the primate model for formulation of anti-drug antibodies (ADA) against recombinant human proteins, it was not possible to assess the pharmacokinetics over the full duration of release.

[0136] Because anti-drug antibodies are known to form against hCG in animals after repeated exposure, the formation of ADAs against hCG in this study was not surprising. Collectively, these observations indicate that the drop in hCG levels does not reflect a problem with the formulation, but is simply a limitation of the animal model for evaluating sustained release of a human protein. The occurrence of inactive ADAs is not expected in humans and is unlikely to reduce therapeutic efficacy. Human males have very low, but detectable, naturally occurring levels of hCG in adulthood and are not expected to mount an immune response to administration of hCG. In summary, a single subcutaneous injection of hCG sustained-release microspheres provides a dose-dependent sustained release of hCG with minimal burst in cynomolgus monkeys.

[0137] Example 8 - Ovidrel-based hCG microspheres composed of SynBiosys multiblock copolymer 20 [PCL-PEG3000-PCL]-b-[PDO] This example describes the preparation and characterization of hCG sustained-release microspheres using Ovidrel as an alternative hCG source. Various batches (RCP-1801, RCP-1803, RC1811, and RCP-1814) of 20[PCL-PEG3000-PCL]-b-[PDO] synthesized as described in Example 5 were used.

[0138] hCG solution (Ovidrel, Serono) was concentrated to 30 mg / ml as described in Example 4. The integrity of hCG was maintained during the concentration step. SEC-UPLC analysis confirmed the absence of aggregates and hCG degradation. hCG sustained-release microspheres were manufactured at a 1.5 g scale following the general procedure described in Example 5, while varying key formulation and process parameters (Table 13).

[0139] All hCG microspheres were characterized for particle size distribution by laser diffraction. The particles had a narrow particle size distribution, with a mean particle size of 38 to 49 μm (Table 14). Microscopic examination by SEM showed that all microparticles had a smooth surface morphology.

[0140] The hCG UPLC method was optimized for maximum resolution between intact hCG and its degradation products, primarily consisting of its subunits. The method was performed on a Waters Acquity H-Class UPLC system equipped with a photodiode array (PDA) detector and a fluorescence detector. hCG integrity was determined by comparing the concentration of intact protein to the total concentration of all hCG-related compounds. An example of a typical chromatogram containing intact hCG, α and β subunits, soluble aggregates, and protein fragments is shown in Figure 26.

[0141] [Table 13]

[0142] [Table 14]

[0143] The hCG content, determined by extraction of hCG from the microparticles and analysis of the hCG concentration by the optimized SEC-UPLC method, varied from 0.88% (EE 44%) to 1.93% (EE 97%) (Table 14).

[0144] In vitro release kinetics was analyzed using an optimized method with a higher buffer capacity, which allows for more accurate identification and quantification of hCG and hCG integrity. hCG microspheres were incubated at 37°C in vials containing 1.0 ml of 100 mM phosphate buffer, pH 7.4, containing 0.025% Tween-20 and 0.02% sodium azide, and placed in a shaking thermostatic water bath. Samples were collected twice weekly, with a 2-day sampling interval of no more than one week. At each sampling time point, the samples were centrifuged, and 0.85 ml of the supernatant was removed for analysis. The samples were washed twice with fresh IVR medium, and the removed volume was replaced with fresh PBS buffer. Total and intact hCG content in the in vitro release samples was determined by SEC-UPLC. The integrity of the released rhCG was established only for samples collected after the 2-day sampling interval to ensure minimal degradation of the released rhCG (in solution).

[0145] Using the optimized in vitro release assay, hCG was released from the hCG sustained-release microparticles significantly faster than with the older method. Figure 27 shows the cumulative release kinetics of 20[PCL-PEG3000-PCL]-b-[PDO]-based hCG sustained-release microspheres prepared according to Table 13. Except for MS18-035, which was prepared using a low polymer concentration of 12.5%, all formulations showed similar in vitro hCG release kinetics, characterized by a low burst release of rhCG followed by a nearly linear release, with a total duration of release ranging from 1 to 4 weeks and approximately 5 weeks. The integrity of the released rhCG, as measured by SEC-UPLC, varied from 85 to 99% (Table 15).

[0146] [Table 15]

[0147] In summary, it can be concluded that the microencapsulation process is robust and reproducible, resulting in hCG sustained-release microspheres with a narrow particle size distribution, an average hCG content of approximately 1.62% by weight, an acceptable EE of >80%, good integrity of the encapsulated hCG (>86%), and a sigmoidal in vitro release profile with a duration of approximately 5 weeks.

[0148] Example 9 - Bioactivity of encapsulated hCG and hCG released from hCG-MSP The bioactivity of encapsulated and released hCG was measured in a mouse MA-10 Leydig cell bioassay. Test samples were generated from three representative lots (MS18-031, MS18-037, and MS18-038) of 20[PCL-PEG3000-PCL]-b-[PDO]-based hCG sustained-release microspheres prepared as described in Example 8. The sustained-release microspheres were subjected to an in vitro release assay to release hCG and were collected after 2 hours, 23 days, and 37 days. Furthermore, to evaluate the stability of encapsulated rhCG, hCG was extracted from a sustained-release microsphere lot that had been frozen and stored for 5 months and tested in a Leydig cell bioassay.

[0149] The bioactivity of released rhCG from each hCG-MSP lot was measured using an hCG bioassay, which measures hCG-induced production of progesterone in the murine Leydig cell tumor line MA-10 via the ENZO Progesterone Enzyme-Linked Immunosorbent Assay (ELISA) kit, and compared to the bioactivity of Ovidrel (recombinant chorionic gonadotropin).

[0150] The total hCG concentration and percent intact hCG of each test sample measured by high performance liquid chromatography (SEC-UPLC) method are listed in Table 16.

[0151] [Table 16]

[0152] The measured levels of progesterone induced by the various hCG samples are summarized in Table 17.

[0153] [Table 17]

[0154] The percent difference in the ability of the hCG test samples to induce progesterone production in MA-10 cells was calculated relative to the reference standard, see Table 18 and Figure 28 (Panels A and B).

[0155] [Table 18]

[0156] For IVR samples, the average of the three lots at each sampling time was calculated (Table 19, plotted in Figure 28 (Panel C)).

[0157] [Table 19]

[0158] The data demonstrate that hCG extracted or released from microspheres can induce progesterone production by murine MA-10 Leydig cells. For extracted hCG, biological activity was similar to that of the reference standard (>90%), suggesting that Ovidrel in hCG-MSP maintained its potency when stored frozen for 5.5 months. Furthermore, the extraction solvent did not attenuate hCG integrity, nor did it interfere with the hCG bioassay. For hCG released from hCG-MSP, progesterone responses were maintained across the early, mid, and late release time points. Progesterone responses were greater than 75% in eight of nine release samples and greater than 84% in six of nine samples. While there was a decrease in progesterone response over time, this did not occur for all hCG microsphere batches; for example, batch MS18-037 demonstrated no change in activity across the three time points.

[0159] In summary, this study demonstrates that hCG can be encapsulated in microspheres and released over time from hCG sustained-release microspheres while maintaining a pharmacologically important portion of its biological activity.

[0160] Example 10 - 2-AB glycan mapping of microencapsulated hCG hCG is a highly glycosylated and sialylated molecule. hCG sialylation is a CQA that affects receptor interaction, signal transduction, pharmacokinetics, and in vivo exposure. The linker of the sugar moiety to the hCG molecule, particularly the terminal sialic acid, is potentially unstable. To investigate whether encapsulation of hCG into microspheres via a water-in-oil-in-water process affected the sialylation level or general glycosylation, and whether this subsequently affected the pharmacokinetics of the protein without necessarily altering its in vitro bioactivity, the sialylation and general glycosylation levels of microencapsulated hCG were analyzed by 2-AB glycan mapping characterization. 20[PCL-PEG3000-PCL]-b-[PDO]-based hCG-MSP (MS18-037) was prepared as described in Example 8. hCG was extracted according to the procedure described in Example 7, and the concentration and integrity of the extracted hCG were determined by SEC-UPLC (Table 20). Several controls were included to account for possible matrix interference with the extraction buffer (i.e., PBS:MeOH (67:33) with 0.2% SDS) or the potential effects of vacuum concentration required for sample preparation.

[0161] [Table 20]

[0162] Sixty-nine different glycan species were monitored for r-hCG. The abundance of each species was assessed with respect to the total area and expressed as relative abundance. The major species are summarized in Figure 29, Panel A. All N-glycan species were further detailed by grouping them according to their structural characteristics. The branching, galactosylation, fucosylation, and sialylation distributions were obtained and are presented in Figure 29, Panel B.

[0163] In practicality experiments using RHS, matrix effects were observed primarily on fucosylated species, but also partially on sialylated species, considering the application and interpretation of 2-AB glycan mapping from extracted material (as shown by the values ​​for RHS relative to RHS in extraction buffer in Figure 29, panel B). Regarding sialylation (known as a critical quality attribute for hCG activity and potency), the levels observed in the "sample" of hCG extracted from MSP were comparable to the "related control in extraction buffer," indicating no effect of encapsulation on the sialylation level relative to the respective control. The differences compared to Ovidrel ("reference" as a baseline without added extraction buffer) perfectly aligned with the matrix effects observed in practicality experiments, indicating no additional interference. In summary, encapsulation / extraction did not alter the sialylation profile relative to the related control. Regarding fucosylation, some differences were clearly related to matrix effects. Considering the standardization of the extraction procedure when orthogonally checked, a certain level of variation was observed between the two samples extracted from MSP. All other glycan species were perfectly aligned.

[0164] The methods and compositions defined herein are representative of preferred embodiments, are illustrative, and do not limit the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon review of this specification and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention described as illustrative herein can preferably be practiced in the absence of any element or elements, or limitation or limitations, not specifically disclosed herein as essential. Thus, for example, in each instance herein, in an embodiment or example of the invention, any of the terms "comprising," "consisting essentially of," and "consisting of" can be replaced with either of the other two terms herein. Also, the terms "comprising," "including," "containing," etc., should be read broadly and without limitation. The methods and processes described as illustrative herein can preferably be practiced in different orders of steps, and are not necessarily limited to the order of steps set forth in the specification or claims. Also, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Under no circumstances may the patent be construed as limited to any specific examples or embodiments or methods specifically disclosed herein.

[0165] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings that fall within the generic disclosure also form part of the invention. This includes the generic description of the invention with a condition or negative limitation that excludes any subject matter from the genus, regardless of whether the excluded material is specifically recited herein.

[0166] Other embodiments are within the scope of the following claims. Furthermore, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

1. 1. A sustained-release human chorionic gonadotropin (hCG) pharmaceutical composition comprising: (a) hCG; and (b) multi-block copolymer microspheres; the multi-block copolymer comprises polyethylene glycol (PEG), the hCG is present in the microspheres, and the microspheres provide a sustained release of the hCG, wherein less than 1 / 7 of the hCG in the microspheres is released in the first 24 hours after administration; The pharmaceutical composition, wherein the multi-block copolymer is a [poly(ε-caprolactone)-co-polyethylene glycol-co-poly(ε-caprolactone)]-b-[poly(L-lactide)] multi-block copolymer.

2. 10. The pharmaceutical composition of claim 1, wherein the microspheres release from about less than 3% to about 40% of the hCG, based on the total weight of the hCG present in the microspheres, within about 24 hours.

3. The multi-block copolymer comprises a multi-block copolymer comprising at least one hydrolyzable prepolymer (A) segment and at least one hydrolyzable prepolymer (B) segment, and the multi-block copolymer has a T of 37° C. or less under physiological conditions. ｇ and T of 110 to 250°C ｍ wherein the segments are linked by a multifunctional chain extender, the segments are randomly distributed on the polymer chain, and the prepolymer (A) segments comprise polyethylene glycol.

4. 4. The pharmaceutical composition of claim 3, wherein the content of prepolymer (A) in the multi-block copolymer is from about 10% to about 90%, from about 30% to about 75%, or from about 50% to about 70% based on the total weight of the multi-block copolymer.

5. The prepolymer (A) segment has an M of about 500 g / mol or more, about 700 g / mol or more, about 1000 g / mol or more, about 2000 g / mol or more, about 3000 g / mol or more, or about 4000 g / mol or more. ｎ 5. The pharmaceutical composition according to claim 3 or 4, comprising:

6. 6. The pharmaceutical composition of claim 3, wherein the prepolymer (B) segment comprises poly(L-lactide) having an Mn of about 1000 g / mol or more, about 2000 g / mol or more, about 3000 g / mol or more, or about 4000 g / mol or more.

7. 7. The pharmaceutical composition according to claim 3, wherein the content of the prepolymer (B) in the multi-block copolymer is from about 10% to about 90%, from about 25% to about 70%, or from about 30% to about 50%, based on the total weight of the multi-block copolymer.

8. The pharmaceutical composition according to any one of claims 3 to 7, wherein the polyfunctional chain extender is a difunctional aliphatic chain extender, a diisocyanate, or 1,4-butane diisocyanate.

9. 9. The pharmaceutical composition of any one of claims 3 to 8, wherein the polyethylene glycol has a Mn of about 150 to about 5000 g / mol, about 200 g / mol to about 1500 g / mol, about 600 to about 1000 g / mol, about 400 to about 3000 g / mol, about 600 to about 1500 g / mol, about 600 to about 5000 g / mol, or about 1000 to about 3000 g / mol.

10. 10. The pharmaceutical composition of claim 3, wherein the multi-block copolymer has a swelling ratio under physiological conditions of about 1 to about 4, about 1 to about 2, or about 1 to about 1.

5.

11. A sustained-release human chorionic gonadotropin (hCG) pharmaceutical composition, comprising: (a) hCG; and (b) multi-block copolymer microspheres; the multi-block copolymer comprises polyethylene glycol (PEG), the hCG is present in the microspheres, and the microspheres provide a sustained release of the hCG, wherein less than 1 / 7 of the hCG in the microspheres is released in the first 24 hours after administration; The pharmaceutical composition, wherein the multi-block copolymer is a [poly(ε-caprolactone)-co-polyethylene glycol-co-poly(ε-caprolactone)]-b-[poly(p-dioxanone)] multi-block copolymer.

12. the multi-block copolymer comprises at least one amorphous hydrolyzable prepolymer (A) segment and at least one semi-crystalline hydrolyzable prepolymer (B) segment; said multiblock copolymer under physiological conditions has a T of 37° C. or less ｇ and T of 50 to 110°C ｍ having - the segments are linked by a polyfunctional chain extender; - the segments are randomly distributed on the polymer chain; the prepolymer (B) segment comprises an X-Y-X triblock copolymer; wherein Y is a polymerization initiator and X is a poly(p-dioxanone) segment having a block length represented by 7 or more p-dioxanone monomer units; and 12. The pharmaceutical composition of claim 11, wherein at least a portion of the prepolymer (A) segments are derived from polyethylene glycol.

13. 13. The pharmaceutical composition of claim 12, wherein X is a poly(p-dioxanone) segment having a block length represented by about 7 to about 35, about 8 to about 30, about 9 to about 25, about 10 to about 20, or about 12 to about 15 p-dioxanone monomer units.

14. 14. The pharmaceutical composition of claim 12 or 13, wherein about 30% or more, about 40 to about 95%, about 50 to about 90%, or about 60 to about 85%, based on the total weight of the prepolymer (A), is derived from polyethylene glycol.

15. The pharmaceutical composition according to claim 13 or 14, wherein about 70% or more, about 80% or more, or about 90% or more, based on the total weight of the prepolymer (B) segments, is poly(p-dioxanone).

16. The prepolymer (B) segment has a number average molecular weight M of about 1300 to about 7200 g / mol, about 1300 to about 5000 g / mol, about 1500 to about 4500 g / mol, about 2000 to about 4000 g / mol, or about 2200 to about 3000 g / mol. ｎ The pharmaceutical composition according to any one of claims 12 to 15, comprising:

17. 17. The pharmaceutical composition of any one of claims 12 to 16, wherein the prepolymer (B) segment has a weight average molecular weight Mw of about 1800 to about 10,080 g / mol, about 1800 to about 7,000 g / mol, about 2,100 to about 6,300 g / mol, about 2,600 to about 5,600 g / mol, or about 3,000 to about 4,200 g / mol.

18. The prepolymer (B) has a T of less than about 0° C., less than about −20° C., or less than about −40° C. ｇ The pharmaceutical composition according to any one of claims 12 to 17, comprising:

19. The prepolymer (B) may be cured at a T in the range of about 60 to about 100°C, or in the range of about 75 to about 95°C. ｍ The pharmaceutical composition according to any one of claims 12 to 18, comprising:

20. The polyethylene glycol (PEG) has an M of about 150 to about 5000 g / mol. ｎ The pharmaceutical composition according to any one of claims 12 to 19, comprising:

21. The pharmaceutical composition according to any one of claims 12 to 20, wherein the chain extender is a difunctional aliphatic chain extender, a diisocyanate, or 1,4-butane diisocyanate.

22. The multi-block copolymer is １ R ２ ｎ R ３ ) ｑ ] ｒ [(R ４ ｐ R ５ R ６ ｐ )] ｓ is represented by During the ceremony, R １ , and R ３ are respectively 【Chemistry 1】 and R ２ teeth, 【Chemistry 2】 and R ４ and R ６ are respectively 【Transformation 3】 and n is the repetition R ２ the number of moieties is 20 to 115, 35 to 100, or 45 to 85; p is a repetition R ４ and R ６ the number of moieties is 7 or more, 7 to 35, 10 to 20, or 10 to 14; q is (R １ R ２ ｎ R ３ ) the number average molecular weight of the block is 1000 to 7000 g / mol, 3000 to 5000 g / mol, or 3800 to 4200 g / mol; r / s is the ratio of prepolymer (A) segments to prepolymer (B) segments, and is 0.10 to 1.0, 0.15 to 0.50, or 0.20 to 0.30; The pharmaceutical composition according to any one of claims 12 to 21.

23. 23. The pharmaceutical composition according to claim 22, wherein n is 63 to 73, p is 10 to 14, q is 3800 to 4200, and r / s is 0.15 to 0.

35.

24. 24. The pharmaceutical composition of any one of claims 1 to 23, wherein the microspheres have a release profile that allows for sustained release for about 14 to about 50 days.

25. The pharmaceutical composition according to any one of claims 1 to 24, further comprising at least one pharmaceutically acceptable excipient.

26. The pharmaceutical composition according to claims 1 to 25, wherein the pharmaceutical composition is for intradermal, intramuscular, transdermal, or subcutaneous administration.

27. 26. The pharmaceutical composition of any one of claims 1 to 25, wherein the pharmaceutical composition is used in the treatment of an indication selected from the group consisting of hypogonadism, cryptorchidism, luteal phase maintenance, contraception, pituitary disorders, breast cancer, and weight loss.

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