Mechanistic evaluation of the initial burst release of leuprolide from spray-dried PLGA microspheres.

Spray-dried poly(lactic-co-glycolic acid) (PLGA) peptide-loaded microspheres have demonstrated similar long-term in vitro release kinetics compared to those produced by the solvent evaporation method and commercial products. However, the difficult-to-control initial burst release over the first 24 h after administration presents an obstacle to product development and establishing bioequivalence. Currently, detailed information about underlying mechanisms of the initial burst release from microspheres is limited. We investigated the mechanism and extent of initial burst release using 16 previously developed spray-dried microsphere formulations of the hormone drug, leuprolide acetate, with similar composition to the commercial 1-month Lupron Depot® (LD). The burst release kinetics was measured with a previously validated continuous monitoring system as well as traditional sample-and-separate methods. The changes in pore structure and polymer permeability were investigated by SEM imaging and the uptake of a bodipy-dextran probe. In vitro results were compared to pharmacokinetics in rats over the same interval. High-burst, spray-dried microspheres were differentiated in the well-mixed continuous monitoring system but reached an upper limit when measured by the sample-and-separate method. Pore-like occlusions observed by confocal microscopy in some formulations indicated that particle swelling may have contributed to probe diffusion through the polymer phase and showed the extensive internal pore structure of spray-dried particles. Continuous monitoring revealed a rapid primary (1°) phase followed by a constant-rate secondary (2°) release phase, which comprised ∼80% and 20% of the 24-hr release, respectively. The ratio of 1° phase duration (t1°) and the characteristic probe diffusion time (τ) was highly correlated to 1° phase release for spray dried particles. Of the four spray-dried formulations administered in vivo, three spray-dried microspheres with similar polymer density showed nearly ideal linear correlation between in vivo absorption and well-mixed in vitro release kinetics over the first 24 h. By contrast, the more structurally dense LD and a more-dense in-house formulation showed a slight lag phase in vivo relative to in vitro. Furthermore, in vitro dimensionless times (tburst/τ) were highly correlated with pharmacokinetic parameters for spray-dried microspheres but not for LD. While the correlation of increases in effective probe diffusion and 1° phase release strongly suggests diffusion through the polymer matrix as a major release mechanism both in vitro and in vivo, a fixed lower limit for this release fraction implies an alternative release mechanism. Overall, continuous monitoring release and probe diffusion appears to have potential in differentiating between leuprolide formulations and establishing relationships between in vitro release and in vivo absorption during the initial burst period.

Polymer source affects in vitro-in vivo correlation of leuprolide acetate PLGA microspheres.

Poly(lactic-co-glycolic acid) PLGA (release controlling excipient) plays a dominant role on the performance of PLGA based long-acting parenterals. These types of drug products typically exhibit complex multi-phasic in vitro/in vivo release/absorption characteristics. In particular, owing to their large size, charged state, and hydrophilicity, peptide loaded microspheres can exhibit more complex release mechanisms. Accordingly, it is challenging to develop Level A in vitro-in vivo correlations (IVIVCs) for such complex long-acting parenterals. With the objective of gaining a better understanding of how to achieve IVIVCs for peptide loaded PLGA microspheres, formulations with similar as well as different release characteristics were prepared with PLGAs from different sources. Leuprolide acetate was selected as the model drug. Owning to the different physicochemical properties of the PLGAs (such as inherent viscosity, molecular weight and blockiness), the formulations exhibited significant differences in their critical quality attributes (such as particle size, porosity and pore size) and consequently had different in vitro and in vivo performance. Affirmative conventional IVIVCs were developed that were able to predict the in vivo performance using the corresponding in vitro release profiles. In addition, the developed conventional IVIVCs were able to discriminate between formulations with comparable in vitro/in vivo performance and those that had dissimilar in vitro/in vivo performance. The present work provides a comprehensive understanding of the influence of PLGA source variations on IVIVC development and predictability for peptide loaded PLGA microspheres.

The impact of leuprolide acetate-loaded calcium phosphate silicate cement to bone regeneration under osteoporotic conditions.

Osteoporosis is detrimental to the health of skeletal structure and significantly increases the risks of bone fracture. Moreover, bone regeneration is adversely impaired by increased osteoclastic activities as a result of osteoporosis. In this study, we developed a novel formulation of injectable bone cement based on calcium phosphate silicate cement (CPSC) and leuprolide acetate (LA). Several combinations of LA-CPSC bone cement were characterized and, it is found that LA could increase the setting time and compressive strength of CPSC in a concentration-dependent manner. Moreover, thein vitroresults revealed that LA-CPSC was biocompatible and able to encourage the osteoblast proliferation via the mTOR signalling pathway. Furthermore, the LA-CPSC was implanted in the osteoporotic rats to evaluate its effectiveness to repair bone fractures under the osteoporotic conditions. The biomarker study and micro-CT analyses indicated that LA-CPSC could effectively reduce the osteoclast activities and promote the bone regeneration. In conclusion, our study demonstrated that LA-CPSC injectable bone cement should be a viable solution to repair bone fractures under the osteoporotic conditions.

A Lipid-Based Depot Formulation with a Novel Non-lamellar Liquid Crystal Forming Lipid.

PURPOSE: Non-lamellar liquid crystal (NLLC)-forming lipids have gained attention as a novel component because of their ability to self-assemble upon contact with body fluids. In this study, a novel NLLC-forming lipid, mono-O-(5, 9, 13-trimethyl-4-tetradecenyl) glycerol ester (C17MGE), and a model drug with a middle molecule weight, leuprolide acetate (LA), were used to confirm the usefulness of C17MGE as an excipient for depot formulations with sustained release properties. METHODS: A self-constructed depot formulation was prepared by mixing C17MGE and different types of phospholipids. The constructed NLLC structure was evaluated using small angle X-ray analysis and cryo-transmission electron microscopy. In vitro release and blood concentration profiles of LA were investigated. RESULTS: The NLLC structure was confirmed by small angle X-ray analysis. LA release was able to be modified by adding different ratios of various phospholipids to C17MGE. Formulations containing 1, 2-dioleoyl-sn-glycero-3-phosphoglycerol sodium salt with a mixing ratio of 12% or 24% (MDOPG12 or MDOPG24, respectively) exhibited sustained release profiles of LA. In addition, the blood concentration of LA was detected over 21 days or more after administration of MDOPG12, and the absolute bioavailability was calculated to be about 100%. CONCLUSIONS: A depot formulation using C17MGE was useful to achieve sustained release of LA.

Initial Leuprolide Acetate Release from Poly(d,l-lactide-co-glycolide) in Situ Forming Implants as Studied by Ultraviolet-Visible Imaging.

The initial drug release from in situ forming implants is affected by factors such as the physicochemical properties of the active pharmaceutical ingredient, the type of the excipients utilized, and the surrounding environment. The feasibility of UV-vis imaging for characterization of the initial behavior of poly(d,l-lactide-co-glycolide) (PLGA)/1-methyl-2-pyrrolidinone (NMP) in situ forming implants was investigated. The in vitro release of leuprolide acetate (LA) and implant formation in real time were monitored using dual-wavelength imaging at 280 and 525 nm, respectively, in matrices based on agarose gel and hyaluronic acid (HA) solution emulating the subcutaneous matrix. Three hours upon injection of the pre-formulation, approximately 15% of the total amount of LA administered was found in the agarose gel, while 5% was released from the implant into the HA solution. Concurrently, more extensive swelling of the implants in the HA solution as compared to implants in the agarose gel was observed. Transport of both LA and the solvent NMP was investigated using UV-vis imaging in a small-scale cell where the geometry of the formulation was controlled, showing a linear correlation between drug release and solvent escape. Light microscopy showed that the microstructures of the resulting implants in agarose gel and HA solution were different, which may be attributed to the different solvent exchange rates. UV imaging was also used to examine the interaction of LA with the release medium by characterizing the diffusion of LA in agarose gel, HA solution, and phosphate buffered saline. The reduced LA diffusivity in HA solution as compared to agarose gel and the LA distribution coefficient in the agarose gel-HA system indicated the presence of interactions between LA and HA. Our findings show that the external environment affects the solvent exchange kinetics for in situ forming implants in vitro, resulting in different types of initial release behavior. UV-vis imaging in combination with biorelevant matrices may offer an interesting approach in the development of in situ forming implant delivery systems.

Physical-Chemical Characterization of Octreotide Encapsulated in Commercial Glucose-Star PLGA Microspheres.

Sandostatin LAR (SLAR) is an injectable long-acting release (LAR) microsphere formulation for octreotide based on a biodegradeable glucose star copolymer of d,l-lactic and glycolic acids (PLGA-glu), which is primarily used for the treatment of patients with acromegaly. There currently is no generic SLAR approved in the United States despite expiration of patent coverage. To understand better this important formulation, SLAR was assessed for its composition and physical-chemical properties. Octreotide release kinetics was monitored under physiological conditions over 56 days together with several bioerosion parameters [mass loss, water uptake, pH of release media, polymer molecular weight (Mw), and confocal microscopy after BODIPY uptake]. A significant increase in the amount of released peptide occurred after day 14. After 1 day of incubation in PBST, octreotide was not extractable completely from SLAR during 2 h of the extraction process, but complete extraction was accomplished after 24 h, which suggested that strong and noncovalent PLGA-octreotide interactions occurred beginning in the initial release phase. Leuprolide is considered as a cationic peptide competitor for octreotide-PLGA interactions and its presence in the release medium resulted in more continuous octreotide release from SLAR, which was linearly correlated with the mass loss from the polymer (i.e., an indication of erosion-controlled release). These data strongly suggest that octreotide forms a salt with acid end groups of linear PLGA chains that are either present as impurities in, and/or produced by the degradation of, the PLGA-Glu. This salt is expected to catalyze octreotide acylation and extend peptide release beyond that driven by erosion control. The characterization studies of physicochemical properties of SLAR described here could be useful for the development and regulatory evaluation of generic octreotide microspheres as well as new polymer formulations, in which the polymer strongly interacts with encapsulated peptides.

Hydrogen-Bonded Films for Zero-Order Release of Leuprolide.

Leuprolide has been widely used in androgen deprivation therapy for the treatment of advanced prostate cancer, but its use is still limited due to its short half-life. Herein, hydrogen-bonded layer-by-layer films are fabricated from PEGylated leuprolide (PEG-LEU) and tannic acid (TA). Because of its dynamic nature, the film disintegrates gradually in water and releases PEG-LEU and TA. The in vitro release profile indicated perfect zero-order kinetics, which is explained by the unique release mechanism. When implanted subcutaneously in male rats, the films maintain a constant serum drug level. For a 60-bilayer film, the serum drug level is maintained constant for ≈24 days. No initial burst release is observed, suggesting that the in vivo release also follows zero-order kinetics. Initially, an increase in the level of serum testosterone is induced by the released drug, followed by testosterone suppression to a constant level below the castrate level, which could be maintained as long as a constant serum drug level is maintained. Since the new drug carriers avoid an initial burst release of the drug and maintain a constant serum drug level and hence a constant serum testosterone level below the castrate level, these carriers are highly promising for androgen deprivation therapy.

Solid lipid nanocarriers diffuse effectively through mucus and enter intestinal cells - but where is my peptide?

Peptides are therapeutic molecules with high potential to treat a wide variety of diseases. They are large hydrophilic compounds for which absorption is limited by the intestinal epithelial border covered by mucus. This study aimed to evaluate the potential of Hydrophobic Ion Pairing combined with Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers (NLC) to improve peptide transport across the intestinal border using Caco-2 cell monolayers (enterocyte-like model) and Caco-2/HT29-MTX co-cultured monolayers (mucin-secreting model). A Hydrophobic Ion Pair (HIP) was formed between Leuprolide (LEU), a model peptide, and sodium docusate. The marked increase in peptide lipophilicity enabled high encapsulation efficiencies in both NLC (84%) and SLN (85%). After co-incubation with the nanoparticles, confocal microscopy images of the cell monolayers demonstrated particles internalization and ability to cross mucus. Flow cytometry measurements confirmed that 82% of incubated SLN and 99% of NLC were internalized by Caco-2 cells. However, LEU transport across cell monolayers was not improved by the nanocarriers. Indeed, combination of particles platelet-shape and HIP low stability in the transport medium led to LEU burst release in this environment. Improvement of peptide lipidization should maintain encapsulation and enable benefit from nanocarriers enhanced intestinal transport.

Accelerated Forced Degradation of Therapeutic Peptides in Levitated Microdroplets.

PURPOSE: Forced degradation is critical to probe the stabilities and chemical reactivities of therapeutic peptides. Typically performed in bulk followed by LC-UV or LC-MS analysis, this traditional workflow consists of a reaction/analysis sequence and usually requires half a day to several days to form and measure the desired amounts of degradants. A faster method is needed to study peptide degradation in a shorter time in order to speed up the drug development process. METHODS: In the new rapid method developed in this study, peptide degradation occurs in levitated aqueous microdroplets using the Leidenfrost effect. RESULTS: This two-minute reaction/analysis workflow allows major degradation pathways of Buserelin, Octreotide, Desmopressin and Leuprorelin to be studied. The reactions include deamidation, disulfide bond cleavage, ether cleavage, peptide bond hydrolysis, and oxidation. CONCLUSIONS: The accelerated forced degradation method requires a minimal amount of therapeutic peptide per stress condition, and the appropriate extent of degradation can be readily generated in seconds by adjusting the droplet levitation time. Levitated microdroplets should be applicable in pharmaceutical development to rapidly determine the intrinsic stability of therapeutic peptides and to aid formulation development by screening the effects of excipients on the stability of the peptides. Graphical abstract.

Effect of Manufacturing Variables and Raw Materials on the Composition-Equivalent PLGA Microspheres for 1-Month Controlled Release of Leuprolide.

The 1-month Lupron Depot (LD) is a 75/25 acid-capped poly(lactic-co-glycolic acid) (PLGA) microsphere product encapsulating water-soluble leuprolide acetate with no generic products available in the U.S. Composition-equivalent PLGA microsphere formulations to the LD as a function of raw material and manufacturing variables were developed by using the solvent evaporation encapsulation method. The following variables were adjusted: polymer supplier/polymerization type, gelatin supplier/bloom number, polymer concentration, first homogenization speed and time, volume of primary water phase, second homogenization time, volume of secondary water phase, and stirring rate. The loading and encapsulation efficiency (EE) of leuprolide and gelatin were determined to identify a large number of composition-equivalent formulations within a ±10% specification of the LD. Key physical-chemical properties of the formulations (e.g., morphology, particle size distribution, glass transition temperature (Tg), residual moisture and solvent, and porosity) were characterized to determine the effect of manufacturing variables on the product attributes. The EE of gelatin across all formulations prepared (101 ± 1%) was observed to be much higher than the EE of leuprolide (57 ± 1%). Judicious adjustment of polymer concentration, second homogenization time, and volume of second water phase was key to achieving high EE of leuprolide, although EE higher than 70% was not easily achievable owing to the difficulty of emulsifying highly viscous primary emulsion into homogeneous small droplets that could prevent peptide loss during the second homogenization under the conditions and equipment used. The in vitro release kinetics of the formulations was highly similar to the LD in a zero-order manner after ∼20% initial burst release, indicating a critical role of the composition on peptide release in this formulation. The characterization of composition-equivalent formulations described here could be useful for further development of generic leuprolide PLGA microspheres and for guiding decisions on the influence of process variables on product physicochemical attributes and release performance.

Hydrophobic H-bond pairing: A novel approach to improve membrane permeability.

The aim of the present study was to develop hydrophobic H-bond pairs (HHPs) of leuprolide (LEU) with non-ionic surfactants to improve its membrane permeability. LEU was lipidized via hydrophobic H-bond pairing (HHP) with the sucrose esters (SEs) sucrose laurate HLB 15 (SLA-15), sucrose palmitate HLB 16 (SPA-16), sucrose stearate HLB 11 (SST-11) and sucrose stearate HLB 15 (SST-15). HHPs were evaluated regarding precipitation efficiency in water, zeta potential, log Pn-octanol/water and dissociation behavior at various pH over time. Cytotoxic potential of HHPs of LEU with SST-11 was investigated on Caco-2 cells. Subsequently, ex vivo permeation studies were carried out across freshly excised Sprague-Dawley rat intestinal mucosa. At a molar ratio of LEU to SEs of 1:≥1 a precipitation efficiency of above 50% was achieved. Zeta potential of complexes was neither influenced by the type nor the amount of added surfactants. Log Pn-octanol/water of LEU was up to 250-fold increased due to HHP utilizing SST-11. Dissociation studies showed that HHPs of LEU with SST-11 dissociate up to 20% in gastrointestinal (GI) pH conditions within 4 h. Moreover, HHPs of LEU with SST-11 exhibited no cytotoxicity. Ex vivo permeation studies revealed 2-fold improved membrane permeation of HHPs of LEU with SST-11 compared to free LEU. Findings of this study show that HHP can be considered as a promising strategy to improve membrane permeation.

In vitro-in vivo correlation of parenteral PLGA microspheres: Effect of variable burst release.

Development of IVIVCs is a very complicated process, especially for complex drug products such as parenteral PLGA microspheres with multiphasic drug release characteristics. Specifically, microspheres that exhibit an initial burst release phase are even more challenging since the in vitro and in vivo burst release phases may not be comparable if drug absorption is rate-limiting at this stage. Therefore, the objectives of the present work were: 1) to investigate the predictability of developed IVIVCs for the in vivo burst release phase based on the in vitro burst release phase of the formulations; and 2) to evaluate the impact of variable burst release on the predictability of the developed IVIVCs for two different types of microsphere-based drug products. Accordingly, Risperdal Consta® (Risperidone) and Lupron Depot® (Leuprolide acetate, LA) were selected as model products. Compositionally equivalent risperidone and LA formulations with variable burst release phases were prepared with manufacturing process changes (such as solvent systems and mixing methods). The prepared microspheres exhibited differences in critical physicochemical properties (such as particle size, porosity, average pore diameter, and drug distribution) and hence differences in their in vitro release characteristics (such as variable burst release and release rate). The in vitro and in vivo (rabbit model (intramuscular injection) burst release were similar for the risperidone microspheres but were significantly different for the LA microspheres. This had an impact on the complexity of the developed IVIVC models. Level A IVIVCs with the ability to predict various types of burst release were developed using time scaling and shifting factors. Moreover, it was observed that IVIVCs developed using formulations with less variation in burst release had better predictability and vice-versa. Thus, the present research has provided a comprehensive understanding of the impact of the burst release phase on the development, complexity, and predictability of IVIVCs for complex parenteral microspheres containing a variety of therapeutic molecules.

Development of Level A in vitro-in vivo correlations for peptide loaded PLGA microspheres.

Peptide loaded PLGA microsphere products are more complex in terms of manufacturing, drug release characteristics as well as release mechanism compared to small molecule loaded PLGA microsphere products. This is due to the complex structure of peptides, their hydrophilicity, charged state, large size and potential for instability. Moreover, therapeutic peptides are highly potent and therefore, any unintended change in the microsphere release profile may lead to undesirable side effects and toxicity. Accordingly, the objectives of the present work were: 1) to evaluate the impact of minor manufacturing changes on the quality and performance of peptide microspheres; and 2) to investigate the feasibility of developing Level A in vitro-in vivo correlations (IVIVCs) for peptide microspheres. Compositionally equivalent leuprolide acetate (LA) microspheres prepared with minor manufacturing changes (solvent system/homogenization speed) showed significant differences in their physicochemical properties (such as pore size, total porosity, particle size and surface distribution of peptide on the prepared microspheres). This, in turn, resulted in significant alteration in the release characteristics. Peptide-polymer interaction, in vitro degradation and microsphere morphology studies were conducted to facilitate understanding of the differences in the drug release characteristics. A rabbit model was used to determine the pharmacokinetic profiles of all the prepared formulations. The obtained in vivo release profiles showed the same rank order as the in vitro release profiles but with low burst release and overall faster in vivo release rates. The low in vivo burst release is considered to be due to the masking effect of the absorption phase from the intramuscular site, and this complicated the development of an IVIVC. Despite these challenges, an affirmative Level A IVIVC over the entire release profile was successfully developed in a rabbit model for peptide microspheres for the first time. The developed IVIVC was also predictive of the RLD product, Lupron Depot®. This work highlights the feasibility of developing IVIVCs for complex parenteral drug products such as peptide microspheres. In conclusion, these results indicate the sensitivity of peptide release, and hence, the safety and efficacy of highly potent peptide microspheres, to minor manufacturing changes. Accordingly, development of IVIVCs for such complex drug products is highly desirable.

In vivo evaluation of solid lipid microparticles and hybrid polymer-lipid microparticles for sustained delivery of leuprolide.

This study aims to investigate the potential of solid lipid microparticles (MP) and hybrid polymer-lipid MPs for sustained delivery of a peptide drug, leuprolide. A peptide-phospholipid complex was prepared to increase the compatibility of the peptide with triglyceride (TG) and poly (lactide-co-glycolide) (PLGA). Peptide loaded solid lipid MPs, PLGA MPs, and hybrid MPs were prepared using a spray drying method and characterized in terms of particle size, morphology and encapsulation efficiency. The pharmacokinetics and pharmacodynamics of leuprolide after subcutaneous injection of spray-dried MPs were evaluated in rats. Spray-dried MPs were spherical ranging in size from 4 µm to 10 µm, which are suitable for injection. After subcutaneous administration of reconstituted MPs, leuprolide could be detected in plasma samples of rats for one to two months, depending on the formulation and dose. Sustained release of leuprolide from PLGA MPs and glyceryl tristearate (TG18) MPs was observed over one month, with a chemical castration effect of 25 and 30 days, respectively. The bioavailability of leuprolide from PLGA-TG18 hybrid MPs was approximately four times higher than that from TG18 MP and PLGA MP alone using the same dose of leuprolide (6 mg/kg). Chemical castration in rats was observed over 30 and 60 days after injection of the PLGA-TG18 hybrid MP with a dose of 3 mg/kg and 6 mg/kg leuprolide, respectively. Additionally, a much lower Cmax was observed for the hybrid MP group. In conclusion, spray-dried PLGA-triglyceride hybrid MPs can be used as better carriers than other MPs for subcutaneous delivery of peptide drugs due to the synergetic effect of lipids and PLGA for sustained drug release from the spray-dried MP.

In-vitro evaluation of solid lipid nanoparticles: Ability to encapsulate, release and ensure effective protection of peptides in the gastrointestinal tract.

Peptides are rarely orally administrated due to rapid degradation in the gastrointestinal tract and low absorption at the epithelial border. The objective of this study was to encapsulate a model water-soluble peptide in biodegradable and biocompatible solid lipid-based nanoparticles, i.e. Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers (NLC) in order to protect it from metabolic degradation. Leuprolide (LEU) and a LEU-docusate Hydrophobic Ion Pair (HIP) were encapsulated in SLN and NLC by High Pressure Homogenization. The particles were characterized regarding their Encapsulation Efficiency (EE), size, morphology, peptide release in FaSSIF-V2, and protective effect towards proteases. Nanoparticles of 120 nm with platelet structures were obtained. Formation of HIP led to a significant increase in LEU EE. Particle size was moderately affected by the presence of simulated fluids. Nonetheless, an important burst release was observed upon dispersion in FaSSIF-V2. NLC were able to improve LEU-HIP resistance to enzymatic degradation induced by trypsin but presented no advantages in presence of α-chymotrypsin. SLN provided no protection regarding both proteases. Despite an increased amount of encapsulated peptide in solid lipid-based nanoparticles following HIP formation, the important specific surface area linked to their platelet structures resulted in an important peptide release upon dispersion in FaSSIF-V2 and limited protection towards enzymatic degradation.

Self-emulsifying drug delivery systems: Impact of stability of hydrophobic ion pairs on drug release.

The aim of this study was to evaluate the impact of stability of hydrophobic ion pairs (HIPs) in gastrointestinal (GI) fluids on their release from self-emulsifying drug delivery systems (SEDDS). HIPs of leuprolide (LEU), insulin (INS) and bovine serum albumin (BSA) were formed using various mono- and di-carboxylate surfactants i.e. sodium deoxycholate (SDC), sodium dodecanoate (SDD), sodium stearoyl glutamate (SSG) and pamoic acid di-sodium salt (PAM). HIPs were evaluated regarding precipitation efficiency, log Pn-butanol/water and dissociation behavior at various pH and ionic strength. Solubility studies of these HIPs were accomplished to identify suitable solvents for the formulation of SEDDS. Subsequently, HIPs were incorporated into SEDDS followed by characterization regarding zeta potential, stability and log DSEDDS/release medium. Independent from the type of (poly)peptides, PAM showed most efficient HIP properties among tested surfactants. The highest encapsulation efficiency with PAM was achieved at molar ratios of 1:1 for LEU, 1:3 for INS and 1:50 for BSA and log Pn-butanol/water of HIPs were increased at least 2.5 units. Dissociation studies showed that LEU-PAM, INS-PAM, BSA-PAM complexes were dissociated within 6 h up to 25%, 60% and 85% in GI fluids, respectively. These HIPs were successfully incorporated into SEDDS exhibiting negative zeta potential and high stability for 4 h. Log DSEDDS/release medium of LEU-PAM, INS-PAM, BSA-PAM complexes were 2.4 ±â€¯0.7, 2.1 ±â€¯0.62 and 1.6 ±â€¯0.45, respectively. Findings of this study showed that stability of HIPs has great impact on log DSEDDS/release medium and consequently on their release from SEDDS.

Separation and identification of acylated leuprorelin inside PLGA microspheres.

Studies have shown that the N-terminus and lysine side residue of peptides are prone to acylation in poly(d,l-lactide-co-glycolide) (PLGA) microspheres. Peptides such as leuprorelin lack a free N-terminus or lysine and only contain serine, arginine, and tyrosine as nucleophilic residues. The purpose of this study was to detect potential acylation impurities and determine their corresponding acylation sites in commercial leuprorelin-loaded PLGA microspheres. Commercial samples from three vendors were selected as targets for our study. The high-performance liquid chromatography (HPLC) conditions of the European Pharmacopoeia were used to separate and collect impurities. HPLC-tandem mass spectrometry (HPLC-MS/MS) was applied to confirm both the structure and acylation sites of the impurities. Our study demonstrated that impurities originating from both degradation of microspheres and synthesis of leuprorelin were well separated and identified using these HPLC conditions. HPLC-MS/MS analysis of acylated leuprorelin showed that diglycoyl, lactoyl-glycoyl, dilactoyl, and monolactoyl groups were conjugated to serine in leuprorelin-loaded PLGA microspheres. This is the first report showing serine to be the acylation site in peptide-loaded PLGA microspheres. Separation and identification of acylated leuprorelin derivatives will assist in minimising acylation and guiding the development of quality control for commercial leuprorelin-loaded PLGA microspheres.

Peptide release from SEDDS containing hydrophobic ion pair therapeutic peptides measured by Taylor dispersion analysis.

Therapeutic peptides are facing an increasing interest as drugs for the treatment of many diseases. The challenge in the administration of such drugs, due to inherent properties of these peptides, is to make them bioavailable. Self-emulsifying drug delivery systems (SEDDS) are considered a suitable and promising strategy to deliver the peptides and increase their bioavailability. However, to enter into the SEDDS nanodroplets, the peptides must be made hydrophobic by complexation with surfactants (formation of hydrophobic ion pair, HIP). The aim of this work is to assess the possibility to quantify the amount of released peptides and of the remaining docusate/peptide HIP in the nanodroplets by Taylor Dispersion Analysis (TDA) on two therapeutic peptides (leuprorelin and desmopressin). It also clearly demonstrates that the logP value of the peptide has a strong influence on the extent of HIP inside of the SEDDS nanodroplets. For instance leuprorelin-docusate complex (logP = 3) was 100% inside of the nanodroplets at low ionic strength, while for desmopressin-docusate complex (logP = 0.5) only 30% were able to enter the nanodroplets. It was also shown that an increase in the ionic strength of the release media allowed to increase the amount of released peptide up to 80% for leuprorelin and 100% for desmopressin, at physiological ionic strength. TDA experiments allowed to determine the partitioning coefficient, logD value, of the peptide between the SEDDS and continuous aqueous phases. In conclusion, this work demonstrates that TDA is a rapid, straightforward and useful technique for developing SEDDS formulations.

Reverse Engineering the 1-Month Lupron Depot®.

The 1-month Lupron Depot® (LD) encapsulating water-soluble leuprolide in poly(lactic-co-glycolic acid) (PLGA) microspheres is a benchmark product upon which modern long-acting release products are often compared. Despite expiration of patent coverage, no generic product for the LD has been approved in the USA, likely due to the complexity of components and manufacturing processes involved in the product. Here, we describe the reverse engineering of the LD composition and important product attributes. Specific attributes analyzed for microspheres were as follows: leuprolide content by three methods; gelatin content, type, and molecular weight distribution; PLGA content, lactic acid/glycolic acid ratio, and molecular weight distribution; mannitol content; in vitro drug release; residual solvent and moisture content; particle size distribution and morphology; and glass transition temperature. For the diluent, composition, viscosity, and specific gravity were analyzed. Analyzed contents of the formulation and the determined PLGA characteristics matched well with the official numbers stated in the package insert and those found in literature, respectively. The gelatin was identified as type B consistent with ~ 300 bloom. The 11-µm volume-median microspheres in the LD slowly released the drug in vitro in a zero-order manner after ~ 23% initial burst release. Very low content of residual moisture (< 0.5%) and methylene chloride (< 1 ppm) in the product indicates in-water drying is capable of removing solvents to extremely low levels during manufacturing. The rigorous approach of reverse engineering described here may be useful for development of generic leuprolide-PLGA microspheres as well as other new and generic PLGA microsphere formulations.

A catalytic one-step synthesis of peptide thioacids: the synthesis of leuprorelin via iterative peptide-fragment coupling reactions.

A catalytic one-step synthesis of peptide thioacids was developed. The oxygen-sulfur atom exchange reaction converted the carboxy group at the C-terminus of the peptides into a thiocarboxy group with suppressed epimerization. This method was successfully applied to the synthesis of the peptide drug leuprorelin via an iterative fragment-coupling protocol.