madman
Super Moderator
Long-acting microspheres of Human Chorionic Gonadotropin hormone: In-vitro and in-vivo evaluation (2021)
Manoj A. Pawar, Lalitkumar K. Vora, Prasad Kompella, Venkata Kishan Pokuri, Pradeep R. Vavia
ABSTRACT
Human Chorionic Gonadotropin (hCG) hormone is used to cause ovulation, treat infertility in women, and increase sperm count in men. Conventional hCG solution formulations require multiple administration of hCG per week and cause patient noncompliance. The long-acting PLGA depot microspheres (MS) approach with hCG can improve patient compliance, increase the efficacy of hCG with a lower total dose and improve quality of life. Therefore, hCG was encapsulated by a modified double emulsion solvent evaporation technique within PLGA MS by high-speed homogenizer and industrially scalable in-line homogenizer, respectively. MS was characterized for particle size, encapsulation efficiency (EE), surface morphology, and in-vitro release. The spherical, dense, nonporous microspheres were obtained with a size of 58.88 ± 0.18 µm. Microspheres showed high EE (77.4% ± 5.9%) with low initial burst release (12.82% ± 2.07%). Circular Dichroism and SDS-PAGE analysis indicated good stability and structural integrity of hCG in the microspheres. Its bioactivity was proven further by a bioassay study in immature Wistar rats. Pharmacokinetic analysis showed that the hCG PLGA MS maintained serum hCG concentration up to 13 days compared to multiple injections of a marketed conventional parenteral injectable formulation of hCG. Thus, it can be ascertained that the hCG PLGA MS may have great potential for clinical use in long-term therapy.
1. Introduction
Human Chorionic Gonadotropin (hCG) is a glycoprotein hormone mainly produced by the placental syncytiotrophoblast cells (Cha et al., 2015; de Medeiros and Norman, 2009). hCG is a heterodimer comprised of the α- and specific β- subunits having a molecular weight of 37,000 Da with 237 amino acids. In the entire structure of hCG, 70% of the structure comprises the protein chain and the remaining 30% carbohydrate units (de Medeiros and Norman, 2009; Keay et al., 2004). Generally, hCG is used to induce ovulation and luteal phase support in in-vitro fertilization (IVF) treatment (5000–10,000 IU as a single dose). In men, hCG is used to induce spermatogenesis and pre-pubertal cryptorchism (250–1500 IU, 2 times a week for 6 weeks) (Cha et al., 2015; Chan et al., 2003; Mannaerts et al., 1998). The hCG conventional solution formulations have been given via subcutaneous (SC) and intramuscular (IM) route; however, the SC route of administration permits self-administration to the patients (Moraloǧlu Üçkardes¸ler et al., 2009). Also, hCG has been used for the prevention of recurrent miscarriage (RM). RM is the loss of three or more consecutive pregnancies, and worldwide 15% to 20% of pregnancies have been ended with miscarriage, and 1 to 3% affected by RM (Morley et al., 2013). hCG plays a vital role in implanting an embryo by secreting the progesterone from the corpus luteum (Carp, 2010; Morley et al., 2013).
After SC and IM administration of hCG conventional formulation, it has an average 32–33 h elimination half-life irrespective of the treatment regimen (Mannaerts et al., 1998). For the long-term treatment in the RM, repeated doses of hCG solutions are required to maintain the effective concentration. Frequent administration of the conventional solution injectable formulation of hCG by SC or IM route is very inconvenient for patients. Polymeric-based long-acting drug delivery systems are widely used to encapsulate small molecules, proteins, and peptides (Yu et al., 2018; Zhou et al., 2020). These injectable polymeric long-acting delivery systems provide the controlled release of drugs and therapeutic proteins for a long time (weeks or months) after a single dose of injection. It improves patient compliance (Keles et al., 2015; Kim and Pack, 2006; L.K. Vora et al., 2017). Most of the long-acting depot formulations are made up of the biodegradable and biocompatible poly (lactic-co-glycolic acid) polymers (PLGA), which are approved by the Food and Drug Administration (FDA) (Hirenkumar and Steven, 2011; Li et al., 2018a; Park et al., 2019). The PLGA polymers are available in different grades based on the monomer composition (lactic acid and glycolic acid) and molecular weights, which are responsible for tailoring the drug release from the delivery system (Kohno et al., 2020; Wang et al., 2019). PLGA polymer hydrolyses into lactic acid and glycolic acid, which are biocompatible in nature and rapidly cleared from the body (Keles et al., 2015; Li et al., 2018a; Park et al., 2019). The first PLGA based long-acting depot formulation, Lupron depot was approved by USFDA in 1989, and after that, only 19 formulations are available in the market (Okada, 1997; Park et al., 2019). Most of the PLGA based depot formulations are available in the form of microparticles, implants, and in-situ gel forming implants. Microparticles are advantageous over others due to the ease of administration (Fredenberg et al., 2011; Guo et al., 2015; Park et al., 2019). Currently available marketed depot formulations, in which only one protein-containing PLGA microparticles formulation Nutropin® Depot was in clinical use, but this formulation has not been available in the market since 2004. The main reason for the withdrawal of Nutropin® Depot is a manufacturing difficulty. The maintenance of the integrity of protein structure, high initial burst release from the microparticles, and scale-up of the microparticles are the major challenges for manufacturing of long-acting PLGA depot formulation of proteins (Kang et al., 2014; Kim and Pack, 2006; Park et al., 2019; Xiao et al., 2020). Previously, hCG-loaded PLGA microspheres have been developed as a single unit antifertility vaccine by double emulsion technique by a conventional method using a mechanical stirrer. Zhu et al studied the effect of some preparative parameters such as stirring rate of mechanical stirrer, the composition of inner aqueous phase and oil phase and performed the in-vitro release study and in-vivo immunization in rats (Zhu et al., 2001). However, more detailed studies will be needed to understand the manufacturing difficulties of hCG loaded PLGA microspheres preparation concerning scalability, along with the stability of the protein during the process and in-vivo pharmacokinetic behavior of the microspheres. In-line homogenizer approaches were used for the scale-up production of the drug-loaded PLGA microparticles to avoid the lack of batch-to-batch reproducibility of conventional PLGA microparticles generation technique. Recently, one research paper has been published in a similar area of continuous in-line homogenization process for scale-up production, where naltrexone-loaded PLGA microparticles were prepared, wherein Sharifi et al., mainly focused on the encapsulation of small molecules by emulsification-extraction technique (Sharifi et al., 2020).
The main objective of the current work was to develop the hCGloaded PLGA microspheres (hCG PLGA MS) system by modified double emulsion technique using high-speed homogenizer and industrially scalable in-line homogenizer, respectively. The developed hCG PLGA microspheres were investigated for the in-vitro characterization concerning particle size, encapsulation efficiency, surface morphology by scanning electron microscopy, and in-vitro release. The integrity, degradation, and bioactive nature of hCG in the PLGA microspheres were confirmed by Circular Dichroism (CD), SDS-PAGE and bioassay study in immature Wistar rats, respectively. A pharmacokinetic study of hCG PLGA microspheres was performed in Wistar male rats to understand the controlled release behavior of the developed system.
2.13. Pharmacokinetic study
The pharmacokinetic study of hCG PLGA microspheres was investigated in male Wistar rats. For this study, male Wistar rats were used instead of immature rats considering the safety of animals and the sensitivity of the analytical technique. The animal ethics committee of the Institute of Chemical Technology, Mumbai, approved the study protocol (Protocol no. ICT/IAEC/2017/P43). Twelve male Wistar rats were divided into four groups (n = 3/group). The animals were acclimatized for 7 days before the initiation of the study. The food and water were provided and, the 12 h light and 12 h dark cycles were maintained throughout the study. Before dosing, animals fasted for 12 h, and the blood was collected at a 0-time point from all the groups of animals for the blank reading. Group-I animals received normal saline solution as a control, Group-II animals received 5000 IU hCG PLGA microspheres formulation (B-5), Group-III animals received 5000 IU hCG PLGA microspheres formulation (B-7), and Group-IV animals received 5000 IU hCG marketed injection formulation, subcutaneously in the neck using a regular disposable syringe with 24 G hypodermic needle. The animals were anesthetized and, the blood samples were removed (500 µL) from the retro-orbital plexus region with heparinized glass capillary at 0.5, 1, 2, 3, 5, 7, 10, 13, and 15 days. The blood samples were centrifuged at 5000 rpm for 10 min and, the serum from blood samples was isolated. The concentration of hCG in the serum samples was measured by the ELISA method using hCG ELISA kits (Calbiotech Inc. USA). The pharmacokinetic data were analyzed by non-compartmental analysis (extravascular drug administration) using the PKSolver add-in program in Microsoft Excel (Microsoft Corporation, Redmond, WA, USA).
3.12. Pharmacokinetic study
The mean serum level of hCG from the conventional marketed injection formulation and hCG PLGA microspheres after subcutaneous administration in Wistar rats is depicted in Fig. 7. The serum concentration of hCG from the hCG PLGA microspheres of B-5 (0.0161 ± 0.006 IU/mL) and B-7 batch (0.0137 ± 0.003 IU/mL) maintained the level up to 13th day compared to the conventional marketed injection formulation of hCG (7th day 0.026 ± 0.002 IU/mL). This result showed that the microspheres had sustained release potential. The rapid absorption of conventional marketed injection formulation showed the higher Cmax 27.71 IU/mL ± 0.58 IU/mL with a half-life (t1/2) of 0.65 ± 0.02 days and the mean residence time (MRT) of 1.039 ± 0.004 days. However, hCG PLGA microspheres showed the decreased Cmax (B-5: 6.11 ± 0.94 IU/ mL, and B-7: 4.89 IU/mL ± 0.37 IU/mL) with increased t1/2 (B-5: 0.86 ± 0.06 days, and B-7:0.97 ± 0.14 days) and MRT (B-5: 3.81 ± 0.16 days, and B-7: 4.04 ± 0.16 days), indicated the controlled release of hCG from the microspheres. The MRT of hCG PLGA microspheres was increased by 3.66 (B-5) and 3.88 (B-7) times compared to the conventional marketed injection formulation of hCG. The hCG PLGA microsphere formulation was also well-tolerated in animals without any noticeable side effects. The hCG level dropping after 13 days, means faster release than in-vitro release profile in PBS pH 7.4, mainly because the in vivo degradation of PLGA was quicker than that in vitro owing to the local enzymatic environment and the foreign body response (Chu et al., 2006). The AUC0-t of hCG PLGA microspheres (B-5: 18.41 ± 1.82 IU/mL*d, and B-7:16.33 ± 2.18 IU/mL*d) were lower than the AUC0-t of conventional injection formulation of hCG (27.58 ± 1.60 IU/mL*d). This might be observed due to the immediate absorption of hCG from the conventional injection solution formulation than the hCG PLGA microspheres. Despite higher AUC0-t in the conventional injection formulation, the in-vivo serum concentration was observed till 7 days; however, hCG PLGA microspheres maintained the in-vivo serum concentration up to 13 days. The lower AUC0-t in the hCG PLGA microspheres might be due to the presence of residual hCG, which leads to its incomplete absorption. Alternatively, due to the slow release of the hCG from the microspheres, after a certain time, the concentration was not detectable because of method LOD. In general, pharmacokinetics results indicate that it is possible to develop long-acting PLGA microsphere formulations for hCG.
4. Conclusion
The hCG PLGA MS was developed to deliver a therapeutic concentration of the hCG after the parenteral administration. The process of manufacturing microspheres was studied using a high-speed homogenizer and industrially scalable in-line homogenizer. In the in-line homogenizer, the flow of primary emulsion and the stabilizer solution were optimized by using a peristaltic pump, and the process was automated to prevent manual errors. The effect of process and formulation parameters were optimized concerning particle size, encapsulation efficiency, in-vitro release. The SDS-PAGE, circular dichroism, and bioassay study confirmed the maintenance of structural integrity and bioactive nature of hCG in the microspheres system. The sustained release potential of hCG PLGA microspheres was confirmed by pharmacokinetic studies in rats. The developed hCG PLGA microspheres can improve patient compliance by reducing the dosing frequency and serve as a promising approach in the treatment of infertility.
Manoj A. Pawar, Lalitkumar K. Vora, Prasad Kompella, Venkata Kishan Pokuri, Pradeep R. Vavia
ABSTRACT
Human Chorionic Gonadotropin (hCG) hormone is used to cause ovulation, treat infertility in women, and increase sperm count in men. Conventional hCG solution formulations require multiple administration of hCG per week and cause patient noncompliance. The long-acting PLGA depot microspheres (MS) approach with hCG can improve patient compliance, increase the efficacy of hCG with a lower total dose and improve quality of life. Therefore, hCG was encapsulated by a modified double emulsion solvent evaporation technique within PLGA MS by high-speed homogenizer and industrially scalable in-line homogenizer, respectively. MS was characterized for particle size, encapsulation efficiency (EE), surface morphology, and in-vitro release. The spherical, dense, nonporous microspheres were obtained with a size of 58.88 ± 0.18 µm. Microspheres showed high EE (77.4% ± 5.9%) with low initial burst release (12.82% ± 2.07%). Circular Dichroism and SDS-PAGE analysis indicated good stability and structural integrity of hCG in the microspheres. Its bioactivity was proven further by a bioassay study in immature Wistar rats. Pharmacokinetic analysis showed that the hCG PLGA MS maintained serum hCG concentration up to 13 days compared to multiple injections of a marketed conventional parenteral injectable formulation of hCG. Thus, it can be ascertained that the hCG PLGA MS may have great potential for clinical use in long-term therapy.
1. Introduction
Human Chorionic Gonadotropin (hCG) is a glycoprotein hormone mainly produced by the placental syncytiotrophoblast cells (Cha et al., 2015; de Medeiros and Norman, 2009). hCG is a heterodimer comprised of the α- and specific β- subunits having a molecular weight of 37,000 Da with 237 amino acids. In the entire structure of hCG, 70% of the structure comprises the protein chain and the remaining 30% carbohydrate units (de Medeiros and Norman, 2009; Keay et al., 2004). Generally, hCG is used to induce ovulation and luteal phase support in in-vitro fertilization (IVF) treatment (5000–10,000 IU as a single dose). In men, hCG is used to induce spermatogenesis and pre-pubertal cryptorchism (250–1500 IU, 2 times a week for 6 weeks) (Cha et al., 2015; Chan et al., 2003; Mannaerts et al., 1998). The hCG conventional solution formulations have been given via subcutaneous (SC) and intramuscular (IM) route; however, the SC route of administration permits self-administration to the patients (Moraloǧlu Üçkardes¸ler et al., 2009). Also, hCG has been used for the prevention of recurrent miscarriage (RM). RM is the loss of three or more consecutive pregnancies, and worldwide 15% to 20% of pregnancies have been ended with miscarriage, and 1 to 3% affected by RM (Morley et al., 2013). hCG plays a vital role in implanting an embryo by secreting the progesterone from the corpus luteum (Carp, 2010; Morley et al., 2013).
After SC and IM administration of hCG conventional formulation, it has an average 32–33 h elimination half-life irrespective of the treatment regimen (Mannaerts et al., 1998). For the long-term treatment in the RM, repeated doses of hCG solutions are required to maintain the effective concentration. Frequent administration of the conventional solution injectable formulation of hCG by SC or IM route is very inconvenient for patients. Polymeric-based long-acting drug delivery systems are widely used to encapsulate small molecules, proteins, and peptides (Yu et al., 2018; Zhou et al., 2020). These injectable polymeric long-acting delivery systems provide the controlled release of drugs and therapeutic proteins for a long time (weeks or months) after a single dose of injection. It improves patient compliance (Keles et al., 2015; Kim and Pack, 2006; L.K. Vora et al., 2017). Most of the long-acting depot formulations are made up of the biodegradable and biocompatible poly (lactic-co-glycolic acid) polymers (PLGA), which are approved by the Food and Drug Administration (FDA) (Hirenkumar and Steven, 2011; Li et al., 2018a; Park et al., 2019). The PLGA polymers are available in different grades based on the monomer composition (lactic acid and glycolic acid) and molecular weights, which are responsible for tailoring the drug release from the delivery system (Kohno et al., 2020; Wang et al., 2019). PLGA polymer hydrolyses into lactic acid and glycolic acid, which are biocompatible in nature and rapidly cleared from the body (Keles et al., 2015; Li et al., 2018a; Park et al., 2019). The first PLGA based long-acting depot formulation, Lupron depot was approved by USFDA in 1989, and after that, only 19 formulations are available in the market (Okada, 1997; Park et al., 2019). Most of the PLGA based depot formulations are available in the form of microparticles, implants, and in-situ gel forming implants. Microparticles are advantageous over others due to the ease of administration (Fredenberg et al., 2011; Guo et al., 2015; Park et al., 2019). Currently available marketed depot formulations, in which only one protein-containing PLGA microparticles formulation Nutropin® Depot was in clinical use, but this formulation has not been available in the market since 2004. The main reason for the withdrawal of Nutropin® Depot is a manufacturing difficulty. The maintenance of the integrity of protein structure, high initial burst release from the microparticles, and scale-up of the microparticles are the major challenges for manufacturing of long-acting PLGA depot formulation of proteins (Kang et al., 2014; Kim and Pack, 2006; Park et al., 2019; Xiao et al., 2020). Previously, hCG-loaded PLGA microspheres have been developed as a single unit antifertility vaccine by double emulsion technique by a conventional method using a mechanical stirrer. Zhu et al studied the effect of some preparative parameters such as stirring rate of mechanical stirrer, the composition of inner aqueous phase and oil phase and performed the in-vitro release study and in-vivo immunization in rats (Zhu et al., 2001). However, more detailed studies will be needed to understand the manufacturing difficulties of hCG loaded PLGA microspheres preparation concerning scalability, along with the stability of the protein during the process and in-vivo pharmacokinetic behavior of the microspheres. In-line homogenizer approaches were used for the scale-up production of the drug-loaded PLGA microparticles to avoid the lack of batch-to-batch reproducibility of conventional PLGA microparticles generation technique. Recently, one research paper has been published in a similar area of continuous in-line homogenization process for scale-up production, where naltrexone-loaded PLGA microparticles were prepared, wherein Sharifi et al., mainly focused on the encapsulation of small molecules by emulsification-extraction technique (Sharifi et al., 2020).
The main objective of the current work was to develop the hCGloaded PLGA microspheres (hCG PLGA MS) system by modified double emulsion technique using high-speed homogenizer and industrially scalable in-line homogenizer, respectively. The developed hCG PLGA microspheres were investigated for the in-vitro characterization concerning particle size, encapsulation efficiency, surface morphology by scanning electron microscopy, and in-vitro release. The integrity, degradation, and bioactive nature of hCG in the PLGA microspheres were confirmed by Circular Dichroism (CD), SDS-PAGE and bioassay study in immature Wistar rats, respectively. A pharmacokinetic study of hCG PLGA microspheres was performed in Wistar male rats to understand the controlled release behavior of the developed system.
2.13. Pharmacokinetic study
The pharmacokinetic study of hCG PLGA microspheres was investigated in male Wistar rats. For this study, male Wistar rats were used instead of immature rats considering the safety of animals and the sensitivity of the analytical technique. The animal ethics committee of the Institute of Chemical Technology, Mumbai, approved the study protocol (Protocol no. ICT/IAEC/2017/P43). Twelve male Wistar rats were divided into four groups (n = 3/group). The animals were acclimatized for 7 days before the initiation of the study. The food and water were provided and, the 12 h light and 12 h dark cycles were maintained throughout the study. Before dosing, animals fasted for 12 h, and the blood was collected at a 0-time point from all the groups of animals for the blank reading. Group-I animals received normal saline solution as a control, Group-II animals received 5000 IU hCG PLGA microspheres formulation (B-5), Group-III animals received 5000 IU hCG PLGA microspheres formulation (B-7), and Group-IV animals received 5000 IU hCG marketed injection formulation, subcutaneously in the neck using a regular disposable syringe with 24 G hypodermic needle. The animals were anesthetized and, the blood samples were removed (500 µL) from the retro-orbital plexus region with heparinized glass capillary at 0.5, 1, 2, 3, 5, 7, 10, 13, and 15 days. The blood samples were centrifuged at 5000 rpm for 10 min and, the serum from blood samples was isolated. The concentration of hCG in the serum samples was measured by the ELISA method using hCG ELISA kits (Calbiotech Inc. USA). The pharmacokinetic data were analyzed by non-compartmental analysis (extravascular drug administration) using the PKSolver add-in program in Microsoft Excel (Microsoft Corporation, Redmond, WA, USA).
3.12. Pharmacokinetic study
The mean serum level of hCG from the conventional marketed injection formulation and hCG PLGA microspheres after subcutaneous administration in Wistar rats is depicted in Fig. 7. The serum concentration of hCG from the hCG PLGA microspheres of B-5 (0.0161 ± 0.006 IU/mL) and B-7 batch (0.0137 ± 0.003 IU/mL) maintained the level up to 13th day compared to the conventional marketed injection formulation of hCG (7th day 0.026 ± 0.002 IU/mL). This result showed that the microspheres had sustained release potential. The rapid absorption of conventional marketed injection formulation showed the higher Cmax 27.71 IU/mL ± 0.58 IU/mL with a half-life (t1/2) of 0.65 ± 0.02 days and the mean residence time (MRT) of 1.039 ± 0.004 days. However, hCG PLGA microspheres showed the decreased Cmax (B-5: 6.11 ± 0.94 IU/ mL, and B-7: 4.89 IU/mL ± 0.37 IU/mL) with increased t1/2 (B-5: 0.86 ± 0.06 days, and B-7:0.97 ± 0.14 days) and MRT (B-5: 3.81 ± 0.16 days, and B-7: 4.04 ± 0.16 days), indicated the controlled release of hCG from the microspheres. The MRT of hCG PLGA microspheres was increased by 3.66 (B-5) and 3.88 (B-7) times compared to the conventional marketed injection formulation of hCG. The hCG PLGA microsphere formulation was also well-tolerated in animals without any noticeable side effects. The hCG level dropping after 13 days, means faster release than in-vitro release profile in PBS pH 7.4, mainly because the in vivo degradation of PLGA was quicker than that in vitro owing to the local enzymatic environment and the foreign body response (Chu et al., 2006). The AUC0-t of hCG PLGA microspheres (B-5: 18.41 ± 1.82 IU/mL*d, and B-7:16.33 ± 2.18 IU/mL*d) were lower than the AUC0-t of conventional injection formulation of hCG (27.58 ± 1.60 IU/mL*d). This might be observed due to the immediate absorption of hCG from the conventional injection solution formulation than the hCG PLGA microspheres. Despite higher AUC0-t in the conventional injection formulation, the in-vivo serum concentration was observed till 7 days; however, hCG PLGA microspheres maintained the in-vivo serum concentration up to 13 days. The lower AUC0-t in the hCG PLGA microspheres might be due to the presence of residual hCG, which leads to its incomplete absorption. Alternatively, due to the slow release of the hCG from the microspheres, after a certain time, the concentration was not detectable because of method LOD. In general, pharmacokinetics results indicate that it is possible to develop long-acting PLGA microsphere formulations for hCG.
4. Conclusion
The hCG PLGA MS was developed to deliver a therapeutic concentration of the hCG after the parenteral administration. The process of manufacturing microspheres was studied using a high-speed homogenizer and industrially scalable in-line homogenizer. In the in-line homogenizer, the flow of primary emulsion and the stabilizer solution were optimized by using a peristaltic pump, and the process was automated to prevent manual errors. The effect of process and formulation parameters were optimized concerning particle size, encapsulation efficiency, in-vitro release. The SDS-PAGE, circular dichroism, and bioassay study confirmed the maintenance of structural integrity and bioactive nature of hCG in the microspheres system. The sustained release potential of hCG PLGA microspheres was confirmed by pharmacokinetic studies in rats. The developed hCG PLGA microspheres can improve patient compliance by reducing the dosing frequency and serve as a promising approach in the treatment of infertility.
