Effect of PLGA raw materials on in vitro and in vivo performance of drug-loaded microspheres.

Poly(lactide-co-glycolide) and poly(lactic-co-glycolic acids) (PLGAs) play a critical role in the development of commercial long-acting injectable microsphere formulations. However, very little information is available describing the impact of PLGA manufacturer and monomer distribution along the polymer chain (e.g., glycolic blockiness (Rc) and average lactic block length (LL)) on the degradation and release behavior of PLGA drug carriers in vitro and in vivo. Here, we compared the in vitro and in vivo performance of (a) four leuprolide-loaded microsphere formulations prepared from similar low-molecular-weight acid-capped PLGAs (10-14 kD, i.e., Expansorb® DLG 75-2A, Purasorb® PDLG 7502A, Resomer® RG 752H and Wako® 7515) and (b) two triamcinolone acetonide-loaded (Tr-A) microsphere formulations from similar medium-molecular-weight ester-capped PLGAs (i.e., Expansorb® DLG 75-4E and Resomer® RG 753S). Lupron Depot® and Zilretta® were used as reference commercial products. The six 75/25 PLGAs displayed block lengths that were either above or below values expected from a random copolymer. Drug release and polymer degradation were monitored simultaneously in vitro and in vivo using a cage implant system. The four leuprolide-loaded formulations showed similar release and degradation patterns with some notable differences between each other. Microspheres from the Expansorb® polymer displayed lower LL and higher Rc relative to the other 3 PLGA 75/25 microspheres, and likewise exhibited distinct peptide release and degradation behavior compared to the other 3 formulations. For each formulation, leuprolide release was erosion-controlled up to about 30% release after the initial burst followed by a faster than erosion release phase. In vitro release was similar as that in vivo over the first phase but notably different from the latter release phase, particularly for the most blocky Expansorb® formulation. The Purasorb® and Wako® formulations displayed highly similar performance in release, degradation, and erosion analysis. By contrast, the two ester-capped Expansorb® DLG 75-4E and Resomer® RG 753S used to prepare Tr-A microspheres shared essentially identical LL and higher Rc and behaved similarly although the Expansorb® degraded and released the steroid faster in vivo, suggestive of other factors responsible (e.g., residual monomer). The in vivo release performance for both drugs from the six microsphere formulations was similar to that of the commercial reference products. In summary, this work details information on comparing the similarities and differences in in vitro and in vivo performance of drug-loaded microspheres as a function of manufacturing and microstructural variables of different types of PLGA raw materials utilized and could, therefore, be meaningful in guiding the source control during development and manufacturing of PLGA microsphere-based drug products. Future work will expand the analysis to include a broader range of LL and higher Rc, and add additional important formulation metrics (e.g., thermal analysis, and residual monomer, moisture, and organic solvent levels).

Aqueous remote loading of model cationic peptides in uncapped poly(lactide-co-glycolide) microspheres for long-term controlled release.

Remote loading microencapsulation of peptides into polymer microspheres without organic solvent represents a promising alternative to develop long-acting release depots relative to conventional encapsulation methods. Here, we formulated drug-free microspheres from two kinds of uncapped poly(lactide-co-glycolides) (PLGAs), i.e., ring-opening polymerized Expansorb® DLG 50-2A (50/50, 11.2 kDa) and Expansorb® DLG 75-2A (75/25, 9.0 kDa), and evaluated their potential capacity to remote-load and control the release of two model peptides, leuprolide and octreotide. Degradation and erosion kinetics, release mechanism, and storage stability was also assessed. As control formulations, peptide was loaded in the same PLGA 75/25 polymer by the conventional double emulsion-solvent evaporation method (W/O/W) and remote loaded in polycondensation poly(lactic-co-glycolic acid) 75/25 (Wako 7515, 14.3 kDa). Loading content of 6.7%-8.9% w/w (~ 67%-89% encapsulation efficiency (EE)) was attained for octreotide, and that of 9.5% w/w loading (~ 95% EE) was observed for leuprolide, by the remote loading paradigm. Octreotide and leuprolide were both slowly and continuously released in vitro from the remote-loaded Expansorb® DLG 75-2A MPs for over 56 days, which was highly similar to that observed from traditionally-loaded formulations by W/O/W (8.8% loading, 52.8% EE). The faster release kinetics was observed for the faster degrading PLGA 50/50 remote-loaded Expansorb® DLG 50-2A MPs relative to microspheres from the PLGA 75/25 Expansorb® DLG 75-2A. Despite slight differences in degradation kinetics, the release mechanism of octreotide from the Expansorb® microspheres, whether remote loaded or by W/O/W, was identical as determined by release vs. mass loss curves. Octreotide acylation was also minimal (< ~ 10%) for this polymer. Finally, drug-free Expansorb® DLG 75-2A MPs displayed excellent storage stability over 3 months. Overall, this work offers support for the use of ring-opening Expansorb® PLGA-based microspheres to remote load peptides to create simple and effective long-acting release depots.

Mechanistic analysis of the photolytic decomposition of solid-state S-nitroso-N-acetylpenicillamine.

S-Nitroso-N-acetylpenicillamine (SNAP) is among the most common nitric oxide (NO)-donor molecules and its solid-state photolytic decomposition has potential for inhaled nitric oxide (iNO) therapy. The photochemical NO release kinetics and mechanism were investigated by exposing solid-state SNAP to a narrow-band LED as a function of nominal wavelength and intensity of incident light. The photolytic efficiency, decomposition products, and the photolytic pathways of the SNAP were examined. The maximum light penetration depth through the solid layer of SNAP was determined by an optical microscope and found to be within 100-200 µm, depending on the wavelength of light. The photolysis of solid-state SNAP to generate NO along with the stable thiyl (RS·) radical was confirmed using Electron Spin Resonance (ESR) spectroscopy. The fate of the RS· radical in the solid phase was studied both in the presence and absence of O2 using NMR, IR, ESR, and UPLC-MS. The changes in the morphology of SNAP due to its photolysis were examined using PXRD and SEM. The stable thiyl radical formed from the photolysis of solid SNAP was found to be reactive with another adjacent thiyl radical to form a disulfide (RSSR) or with oxygen to form various sulfonyl and sulfonyl peroxyl radicals {RS(O)xO·, x = 0 to 7}. However, the thiyl radical did not recombine with NO to reform the SNAP. From the PXRD data, it was found that the SNAP loses its crystallinity by generating the NO after photolysis. The initial release of NO during photolysis was increased with increased intensity of light, whereas the maximum light penetration depth was unaffected by light intensity. The knowledge gained about the photochemical reactions of SNAP may provide important insight in designing portable photoinduced NO-releasing devices for iNO therapy.

Aqueous remote loading of setmelanotide in poly(lactic-co-glycolic acid) microspheres for long-term obesity treatment.

Setmelanotide (Imcivree™) was developed as a daily injectable therapeutic peptide for the treatment of rare forms of syndromic obesity, such as POMC deficiency and leptin receptor deficiency. The important option of poly(lactic-co-glycolic acid) (PLGA) controlled release microspheres has become more attractive for this class of drugs upon the discovery that net positively charged peptides can be remote-loaded rapidly from aqueous peptide solution into blank microspheres at high loading and encapsulation efficiency. Here we sought to remote-load setmelanotide in PLGA microspheres and examine its potential for long-term controlled release and body weight control. The influence of PLGA microsphere porosity was investigated with respect to morphology, drug loading, and in vitro release profiles. Increased density of the microspheres inhibited the progress of encapsulation of the dicationic peptide. A diet-induced obese murine model was then used to determine the pharmacokinetic profile and to evaluate long-term efficacy of an optimal formulation. Remote loaded PLGA formulations encapsulated setmelanotide as high as ∼63% (∼6.3% w/w loading) and exhibited slow and continuous peptide release over ∼6 weeks in vitro largely independent of microsphere porosity. The obtained in vivo release pattern from deconvolution of the pharmacokinetics after subcutaneous microsphere injection was consistent with the in vitro release profile but with a lower initial burst release and overall slightly faster release rate. After a single injection of remote-loaded setmelanotide, continuous long-term inhibition of food intake and body weight control was observed over 17 and 30 days, respectively. The improvement in body weight control over drug-free microsphere vehicle-treated control groups matched the observed PK profile. This study provides the first report of long-acting release formulation for 1-month controlled release of setmelanotide and body weight control in a diet induced obese murine model, and supports the further development of long-acting treatment options for obese patients.

In vitro performance of composition-equivalent PLGA microspheres encapsulating exenatide acetate by solvent evaporation.

The once-weekly Bydureon® (Bdn) PLGA microsphere formulation encapsulating the GLP-1 receptor agonist, exenatide acetate, is an important complex injectable product prepared by coacervation for the treatment of type 2 diabetic patients. Encapsulation by coacervation is useful to minimize an undesirable initial burst of exenatide, but it suffers from manufacturing difficulties such as process scale-up and batch-to-batch variations. Herein we prepared exenatide acetate-PLGA formulations of similar compositions using the desirable alternative double emulsion-solvent evaporation technique. After screening several process variables, we varied the PLGA concentration, the hardening temperature, and the collected particle size range, and determined the resulting drug and sucrose loading, initial burst release, in vitro retention kinetics, and peptide degradation profiles using Bdn as a positive control. All formulations exhibited a triphasic release profile with a burst, lag, and rapid release phase, although the burst release was greatly decreased to <5% for some. Marked differences were observed in the peptide degradation profiles, particularly the oxidized and acylated fractions, when the polymer concentration was varied. For one optimal formulation, the release and peptide degradation profiles were similar to Bdn microspheres, albeit with an induction time shift of one week, likely due to the slightly higher Mw of PLGA in Bdn. These results highlight the effects of key manufacturing variables on drug release and stability in composition-equivalent microspheres encapsulating exenatide acetate and indicate the potential of manufacturing the microsphere component of Bdn by solvent evaporation.

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.

Gelatin methacryloyl hydrogel as an injectable scaffold with multi-therapeutic effects to promote antimicrobial disinfection and angiogenesis for regenerative endodontics.

Regenerative endodontics represents a paradigm shift in dental pulp therapy for necrotic young permanent teeth. However, there are still challenges associated with attaining maximum root canal disinfection while supporting angiogenesis and preserving resident stem cells viability and differentiation capacity. Here, we developed a hydrogel system by incorporating antibiotic-eluting fiber-based microparticles in gelatin methacryloyl (GelMA) hydrogel to gather antimicrobial and angiogenic properties while prompting minimum cell toxicity. Minocycline (MINO) or clindamycin (CLIN) was introduced into a polymer solution and electrospun into fibers, which were further cryomilled to attain MINO- or CLIN-eluting fibrous microparticles. To obtain hydrogels with multi-therapeutic effects, MINO- or CLIN-eluting microparticles were suspended in GelMA at distinct concentrations. The engineered hydrogels demonstrated antibiotic-dependent swelling and degradability while inhibiting bacterial growth with minimum toxicity in dental-derived stem cells. Notably, compared to MINO, CLIN hydrogels enhanced the formation of capillary-like networks of endothelial cells in vitro and the presence of widespread vascularization with functioning blood vessels in vivo. Our data shed new light onto the clinical potential of antibiotic-eluting gelatin methacryloyl hydrogel as an injectable scaffold with multi-therapeutic effects to promote antimicrobial disinfection and angiogenesis for regenerative endodontics.

Influence of encapsulation variables on formation of leuprolide-loaded PLGA microspheres.

Emulsion-based solvent evaporation microencapsulation methods for producing PLGA microspheres are complex often leading to empirical optimization. This study aimed to develop a more detailed understanding of the effects of process variables on the complex emulsification processes during encapsulation of leuprolide in PLGA microspheres using a high-shear rotor-stator mixer. Following extensive analysis of previously developed formulation conditions that yield microspheres of equivalent composition to the commercial 1-month Lupron Depot, multiple variables during the formation of primary and secondary emulsion were investigated with the aid of dimensional analysis, including: rotor speed (ω) and time (t), dispersed phase fraction (Φ) and continuous phase viscosity (µc). The dimensionless Sauter mean diameter (d3,2) of primary emulsion was observed to be proportional to the product of several key dimensionless groups (Φ1,We,Re,ω1t1) raised to the appropriate power indices. A new dimensionless group (Θ ) (surface energy/energy input) was used to rationalize insertion of a proportionate time dependence in the scaling of the d3,2. The dimensionless d3,2 of secondary emulsion was found proportional to the product of three dimensionless groups ( [Formula: see text] ) raised to the appropriate power indices. The increased viscosity of the primary emulsion, decreased secondary water phase volume and reduced second homogenization time each elevated encapsulation efficiency of peptide by reducing drug leakage to the outer water phase. These results could be useful for dimensional analysis and improving manufacturing of PLGA microspheres by the solvent evaporation method.

In vitro degradation and erosion behavior of commercial PLGAs used for controlled drug delivery.

Copolymers of lactic (or lactide) and glycolic (or glycolide) acids (PLGAs) are among the most commonly used materials in biomedical applications, such as parenteral controlled drug delivery, due to their biocompatibility, predictable degradation rate, and ease of processing. Besides manufacturing variables of drug delivery vehicles, changes in PLGA raw material properties can affect product behavior. Accordingly, an in-depth understanding of polymer-related "critical quality attributes" can improve selection and predictability of PLGA performance. Here, we selected 19 different PLGAs from five manufacturers to form drug-free films, submillimeter implants, and microspheres and evaluated differences in their water uptake, degradation, and erosion during in vitro incubation as a function of L/G ratio, polymerization method, molecular weight, end-capping, and geometry. Uncapped PLGA 50/50 films from different manufacturers with similar molecular weights and higher glycolic unit blockiness and/or block length values showed faster initial degradation rates. Geometrically, larger implants of 75/25, uncapped PLGA showed higher water uptake and faster degradation rates in the first week compared to microspheres of the same polymers, likely due to enhanced effects of acid-catalyzed degradation from PLGA acidic byproducts unable to escape as efficiently from larger geometries. Manufacturer differences such as increased residual monomer appeared to increase water uptake and degradation in uncapped 50/50 PLGA films and poly(lactide) implants. This dataset of different polymer manufacturers could be useful in selecting desired PLGAs for controlled release applications or comparing differences in behavior during product development, and these techniques to further compare differences in less reported properties such as sequence distribution may be useful for future analyses of PLGA performance in drug delivery.

Fenretinide combines perturbation of signaling kinases, cell-extracellular matrix interactions and matrix metalloproteinase activation to inhibit invasion in oral squamous cell carcinoma cells.

Basement membrane invasion defines malignant transformation of surface premalignancy. Treatment of oral squamous cell carcinoma (OSCC) cells with the synthetic vitamin A derivative, fenretinide (4HPR), induces numerous cancer-preventive effects including suppression of basement membrane invasion, elimination of anchorage-independent growth, disruption of actin cytoskeletal components and inhibition of the invasion-enabling focal adhesive kinase. The purpose of this study was to elucidate 4HPR's effects on additional invasion-relevant mechanisms including matrix metalloproteinase (MMP) activation and function, cell-extracellular matrix (ECM) attachments and interaction with a kinase that is essential for the epithelial-myoepithelial transformation i.e. c-Jun NH2-terminal kinase (JNK). Our data revealed that 4HPR binds with high affinity to the ATP-binding site of all three JNK isoforms with concurrent suppression of kinase function. Additional studies showed 4HPR treatment inhibited both OSCC cell-ECM adhesion and MMP activation and function. JNK downregulation and induced expression studies confirmed that the JNK3 isoform conveyed that largest impact on OSCC migration and invasion. Biodegradable polymeric implants formulated to preserve 4HPR's function and bioavailability were employed to assess 4HPR's chemopreventive impact on an OSCC tumor induction model. These studies revealed 4HPR local delivery significantly inhibited OSCC tumor size, mitotic indices and expression of the endothelial marker, erythroblast transformation-specific-related gene with concurrent increases in tumor apoptosis (cleaved caspase-3). Collectively, these data show that 4HPR suppresses invasion at multiple sites including 'outside-in' signaling, cell-ECM interactions and suppression of MMPs. These functions are also essential for physiologic function. Regulation is therefore essential and reinforces the pharmacologic advantage of local delivery chemopreventive formulations. .

Mapping in vivo microclimate pH distribution in exenatide-encapsulated PLGA microspheres.

The pH inside the aqueous pores of poly(lactic-co-glycolic acid) (PLGA) microspheres, often termed microclimate pH (µpH), has been widely evaluated in vitro and shown to commonly be deleterious to pH-labile encapsulated drug molecules. However, whether the in vitro µpH is representative of the actual in vivo values has long been remained a largely unresolved issue. Herein we quantitatively mapped, for the first time, the in vivo µpH distribution kinetics inside degrading PLGA microspheres by combining two previously validated techniques, a cage implant system and confocal laser scanning microscopy. PLGA (50/50, Mw = 24-38 kDa, acid-end capped and ester-capped) microsphere formulations with and without encapsulating exenatide, a pH-labile peptide that is known to be unstable when pH > 4.5, were administered to rats subcutaneously via cage implants for up to 6 weeks. The results were compared with two different in vitro conditions. Strikingly, the in vivo µpH developed similarly to the low microsphere concentration in vitro condition with 1-µm nylon bags but very different from conventional high microsphere concentration sample-and-separate conditions. Improved maintenance of stable external pH in the release media for the former condition may have been one important factor. Stability of exenatide remaining inside microspheres was evaluated by mass spectrometry and found that it was steadily degraded primarily via pH-dependent acylation with a trend that slightly paralleled the changes in µpH. This methodology may be useful to elucidate pH-triggered instability of PLGA encapsulated drugs in vivo and for improving in vivo-predictive in vitro conditions for assessing general PLGA microsphere performance.

Efficient aqueous remote loading of peptides in poly(lactic-co-glycolic acid).

Poly(lactic-co-glycolic acid) (PLGA) long-acting release depots are effective for extending the duration of action of peptide drugs. We describe efficient organic-solvent-free remote encapsulation based on the capacity of common uncapped PLGA to bind and absorb into the polymer phase net positively charged peptides from aqueous solution after short exposure at modest temperature. Leuprolide encapsulated by this approach in low-molecular-weight PLGA 75/25 microspheres slowly and continuously released peptide for over 56 days in vitro and suppressed testosterone production in rats in an equivalent manner as the 1-month Lupron Depot®. The technique is generalizable to encapsulate a number of net cationic peptides of various size, including octreotide, with competitive loading and encapsulation efficiencies to traditional methods. In certain cases, in vitro and in vivo performance of remote-loaded PLGA microspheres exceeded that relative to marketed products. Remote absorption encapsulation further removes the need for a critical organic solvent removal step after encapsulation, allowing for simple and cost-effective sterilization of the drug-free microspheres before encapsulation of the peptide.

Optimizing zinc-HisTag coordination remote loading of proteins in PLGA microspheres.

Metal-HisTag coordination remote loading (MHCRL) of proteins in PLGA microspheres was previously developed to provide a useful tool for discovery and preclinical development of controlled release protein formulations. Here we describe optimization of MHCRL, including (1) reducing thermal stress, (2) decreasing the complexity and duration of the procedure, (3) increasing loading capacity, (4) increasing the penetration depth of protein, and (5) improving the release profile. Directly encapsulating ZnCO3as a Zn2+source for HisTag coordination, rather than remotely loading Zn2+, increased the Zn content ∼6-fold. Microspheres with directly encapsulated ZnCO3more deeply encapsulated green fluorescent protein and more efficiently encapsulated human serum albumin at protein loading solutions concentrations ≥100 µg/mL than remotely loaded Zn2+microspheres. Tributyl acetylcitrate plasticized microspheres in terms of decreasingTg, but led to a decrease in protein encapsulation efficiency. As such, the plasticizer was not deemed useful. The loading/healing cycles were reduced in time and temperature from 48 h/42 h at 43 °C to 2 h/6h at 37 °C while maintaining strong encapsulation efficiency, resulting in significantly improved protein stability. Immunoreactive protein was slowly released for months following a modest burst release. The improved microspheres and shorter, low-temperature encapsulation could be a valuable asset to drug discovery scientists interested in controlled release of delicate and/or costly biologic candidates.

Effect of Surface Interactions on Microsphere Loading in Dissolving Microneedle Patches.

Microneedle (MN) patches enable simple self-administration of drugs via the skin. In this study, we sought to deliver drug-loaded microspheres (MSs) using MN patches and found that the poly(lactic-co-glycolic acid) (PLGA) MSs failed to localize in the MN tips during fabrication, thereby decreasing their delivered dose and delivery efficiency into skin. We determined that surface interactions between the hydrophobic MSs and the poly(dimethylsiloxane) (PDMS) mold caused MSs to adhere to the mold surface during casting in aqueous formulations, with hydrophobic interactions largely responsible for adhesion. Further studies with polystyrene MSs that similarly carry a negative charge like the PLGA MSs demonstrated both repulsive electrostatic interactions as well as adhesive hydrophobic interactions. Reducing hydrophobic interactions by addition of a surfactant or modifying mold surface properties increased MS loading into MN tips and delivery into porcine skin ex vivo by 3-fold. We conclude that surface interactions affect the loading of hydrophobic MSs into MN patches during aqueous fabrication procedures and that their modulation with the surfactant can increase loading and delivery efficiency.

Metal-HisTag coordination for remote loading of very small quantities of biomacromolecules into PLGA microspheres.

Challenges to discovery and preclinical development of long-acting release systems for protein therapeutics include protein instability, use of organic solvents during encapsulation, specialized equipment and personnel, and high costs of proteins. We sought to overcome these issues by combining remote-loading self-healing encapsulation with binding HisTag protein to transition metal ions. Porous, drug-free self-healing microspheres of copolymers of lactic and glycolic acids with high molecular weight dextran sulfate and immobilized divalent transition metal (M2+) ions were placed in the presence of proteins with or without HisTags to bind the protein in the pores of the polymer before healing the surface pores with modest temperature. Using human serum albumin, insulin-like growth factor 1, and granulocyte-macrophage colony-stimulating factor (GM-CSF), encapsulated efficiencies of immunoreactive protein relative to nonencapsulation protein solutions increased from ~41%, ~23%, and ~9%, respectively, without Zn2+ and HisTags to ~100%, ~83%, and ~75% with Zn2+ and HisTags. These three proteins were continuously released in immunoreactive form over seven to ten weeks to 73%-100% complete release, and GM-CSF showed bioactivity >95% relative to immunoreactive protein throughout the release interval. Increased encapsulation efficiencies were also found with other divalent transition metals ions (Co2+, Cu2+, Ni2+, and Zn2+), but not with Ca2+. Ethylenediaminetetraacetic acid was found to interfere with this process, reverting encapsulation efficiency back to Zn2+-free levels. These results indicate that M2+-immobilized self-healing microspheres can be prepared for simple and efficient encapsulation by simple mixing in aqueous solutions. These formulations provide slow and continuous release of immunoreactive proteins of diverse types by using a amount of protein (e.g., <10 µg), which may be highly useful in the discovery and early preclinical development phase of new protein active pharmaceutical ingredients, allowing for improved translation to further development of potent proteins for local delivery.

Core-shell microneedle patch for six-month controlled-release contraceptive delivery.

There is a tremendous need for simple-to-administer, long-acting contraception, which can increase access to improved family planning. Microneedle (MN) patches enable simple self-administration and have previously been formulated for 1-2 months' controlled release of contraceptive hormone using monolithic polymer/drug MN designs having first-order release kinetics. To achieve zero-order release, we developed a novel core-shell MN patch where the shell acts as a rate-controlling membrane to delay release of a contraceptive hormone, levonorgestrel (LNG), for 6 months. In this approach, LNG was encapsulated in a poly(lactide-co-glycolide) (PLGA) core surrounded by a poly(l-lactide) (PLLA) shell and a poly(D,L-lactide) (PLA) cap that were fabricated by sequential casting into a MN mold. Upon application to skin, the core-shell MNs utilized an effervescent interface to separate from the patch backing within 1 min. The core-shell design limited the initial 24 h burst release of LNG to 5.8 ± 0.5% and achieved roughly zero-order LNG release for 6.2 ± 0.1 months in vitro. A monolithic MN patch formulated with the same LNG and PLGA core, but without the rate-controlling PLLA shell and PLA cap had a larger LNG burst release of 22.6 ± 2.0% and achieved LNG release for just 2.1 ± 0.2 months. This study provides the first core-shell MN patch for controlled months-long drug release and supports the development of long-acting contraception using a simple-to-administer, twice-per-year MN patch.

Minimizing the initial burst of octreotide acetate from glucose star PLGA microspheres prepared by the solvent evaporation method.

Sandostatin long-acting release (SLAR) depot for 1-month controlled release of octreotide is a somatostatin analogue product that has been used extensively in the pharmacological treatment of acromegaly. The complexities in the SLAR coacervation manufacturing processes and the use of a unique glucose-starpoly(lactic-co-glycolic acid) (PLGA-glu) may have contributed to the lack of US FDA-approved generic products referencing SLAR in the USA. To address this challenge, we encapsulated octreotide acetate by the commonly used solvent evaporation method in microspheres of a similar composition to SLAR, including the use of a comparable PLGA-glu. Based on our previous study that identified key formulation variables to prepare octreotide acetate/PLGA-glu microspheres, including lowering initial peptide pH and introducing an annealing step post loading, here we added NaCl to the external water phase to further improve the formulation. The resulting microspheres exhibited highly similar release and stability performance in vitro to SLAR, including an exceptionally low initial burst. The very low initial burst was also confirmed by pharmacokinetics in rats. Full erosion behavior analysis (polymer MW, water uptake and mass loss) revealed a slightly faster degradation of SLAR than the solvent evaporation formulations. Analysis of kinetics of dry Tg of the formulations reflected (a) the elevated residual solvent in SLAR and was not duplicated in the solvent evaporation formulations, and (b) the slightly higher Tg of peptide loaded formulations relative to than blank microspheres, consistent with the interaction of the acetate salt of octreotide with linear PLGA chains in the PLGA-glu. These data indicate that it is possible to prepare peptide loaded microspheres by the solvent evaporation method with extraordinarily similar performance to microspheres, such as those in SLAR, that are prepared by the low-burst release coacervation method.

Characterization of commercial PLGAs by NMR spectroscopy.

Poly(lactic-co-glycolic acid) (PLGA) is among the most common of biodegradable polymers studied in various biomedical applications such as drug delivery and tissue engineering. To facilitate the understanding of the often overlooked impact of PLGA microstructure on important factors affecting PLGA performance, we measured four key parameters of 17 commonly used commercial PLGA polymers (Expansorb®, Resomer®, Purasorb®, Lactel®, and Wako®) by NMR spectroscopy. 1HNMR and 13CNMR spectra were used to determine lactic to glycolic ratio (L/G ratio), polymer end-capping, glycolic blockiness (Rc), and average glycolic and lactic block lengths (LG and LL). In PLGAs with a labeled L/G ratio of 50/50 and acid end-capping, the actual lactic content slightly decreased as molecular weight increased in both Expansorb® and Resomer®. Whether or not acid- or ester-, termination of these PLGAs was confirmed to be consistent with their brand labels. Moreover, in the ester end-capped 75/25 L/G ratio group, the blockiness value (Rc) of Resomer® RG 756S (Rc: 1.7) was highest in its group; whereas for the 50/50 acid end-capped group, Expansorb® DLG 50-2A (Rc: 1.9) displayed notably higher values than their counterparts. Expansorb® 50-2E (LL: 2.5, LG: 2.6) and Resomer® RG 502 (LL: 2.6, LG: 2.5) showed the lowest block lengths, suggesting they may undergo a steadier hydrolytic process compared to random, heterogeneously distributed PLGA.

Development and characterization of composition-equivalent formulations to the Sandostatin LAR® by the solvent evaporation method.

Sandostatin long-acting release® (SLAR) is a long-acting injectable somatostatin analogue formulation composed of octreotide encapsulated in glucose-initiated poly(lactic-co-glycolic acid) (PLGA) microspheres. Despite the end of patent protection, SLAR remains resistant to generic competition likely due to complexity of production process, the uniqueness of the glucose star polymer, and the instability of octreotide in the formulation. Here, we describe development of glucose-PLGA-based composition-equivalent to SLAR formulations prepared by double emulsion-solvent evaporation method and the effect of variations in encapsulation variables on release kinetics and other formulation characteristics. The following encapsulation variables were adjusted at constant theoretical loading of 7.0% peptide: PLGA concentration, pH of inner water phase, and stirring rate. After final drying, the microspheres were examined with and without annealing at 50 °C under vacuum for 3 days. The loading and encapsulation efficiency (EE) of octreotide acetate, manufacturing yield, and in vitro drug release kinetics in PBStc (10 mM phosphate-buffered saline (PBS) with 1% triethyl citrate and 0.02% sodium azide at pH 7.4) were determined by UPLC. The in vitro release and acylation kinetics of octreotide for the solvent evaporation formulations prepared were similar to SLAR although the initial burst was slightly higher. Key formulation steps identified to maximize microsphere yield and minimize residual solvent and initial burst release included (a) addition of acetic acid to the peptide before preparation and (b) annealing the microspheres under vacuum after drying. Controlled release octreotide formulations prepared and investigated in this study could provide a better understanding of the effect of production variables on release performance and supply information useful for making progress in manufacturing of SLAR generic equivalents.