Efficient Excipients Research Articles

Development of pH‐Responsive Hydrogel Excipient From Karaya Gum for Improved Oral Delivery of Donepezil Hydrochloride in Alzheimer's Disease Treatment

ABSTRACTThe present study focused on fabricating an efficient excipient for donepezil HCl from karaya gum as the base material. The karaya gum was grafted and crosslinked with N‐isopropyl acrylamide and N,N′‐methylene(bis)acrylamide, respectively, using microwave. The successful fabrication of the resulting hydrogel was confirmed and characterized by FTIR, FESEM, and TGA analyses. Remarkably, the synthesized gel exhibited distinct swelling behavior in different pH mediums ranging from 1.2 to 13. As expected, the gel exhibited an impressive 96% release of donepezil, a widely used hydrophilic drug for treating Alzheimer's disease, in a buffer solution of pH 1.2, within a 48‐h timeframe. Kinetic studies further revealed that drug release from the gel aligns with the Higuchi model. Cytotoxicity studies using Drosophila melanogaster as a model system showed the biocompatible nature of the excipient. This characteristic highlights its potential application as a drug carrier for the oral delivery of Donepezil HCl in treating Alzheimer's disease.

Evaluation of different techniques for wet granulation and pelletization processes using milk as innovative pharmaceutical excipient for pediatric use

The present study focused on the use of milk as a novel excipient for the manufacture of pharmaceutical dosage forms specifically designed for the pediatric population. Dairy milks with different fat contents were studied to deliver paracetamol orally. The World Health Organization included milk in the list of GRAS (generally recognized as safe) substances, which together with its taste-masking ability and solubility solving properties, makes it a good candidate as an excipient in formulations containing paracetamol for pediatrics. The influence of the fat content in the milk, the fraction of paracetamol, the type of diluent and drying temperature (considered independent variables) were systematically investigated using a Design of Experiments (DoE) approach for the preparation of granules for oral administration, by wet agglomeration using different processing techniques, enabled the construction of mathematical models reflecting the correlation between the variables. Four different techniques were evaluated: wet granulation by low shear mixer, wet granulation by high shear mixer, wet granulation by fluidized bed, and extrusion and spheronization. The granules and pellets obtained were characterized for size, and size distribution of agglomerates, and complete release of the drug (dependent variables), according to the European Pharmacopoeia. The fraction of fat content in the milk promoted an increase on the dissolution rate of paracetamol. The key finding of the first two process techniques was a migration of paracetamol from powdered agglomerates towards the larger particles, probably due to friction and attrition events, which created a fraction of smaller size granules due to the fragmentation and loss of powder from the larger granules. The study has confirmed the potential of milk to be a novel and efficient excipient that can be used as a liquid binder in various agglomeration techniques to deliver drugs orally.

Alternative Excipients for Protein Stabilization in Protein Therapeutics: Overcoming the Limitations of Polysorbates.

Given their safety and efficiency in protecting protein integrity, polysorbates (PSs) have been the most widely used excipients for the stabilization of protein therapeutics for years. In recent decades, however, there have been numerous reports about visible or sub-visible particles in PS-containing biotherapeutic products, which is a major quality concern for parenteral drugs. Alternative excipients that are safe for parenteral administration, efficient in protecting different protein drugs against various stress conditions, effective in protein stabilization in high-concentrated liquid formulations, stable under the storage conditions for the duration of the product's shelf-life, and compatible with other formulation components and the primary packaging are highly sought after. The aim of this paper is to review potential alternative excipients from different families, including surfactants, carbohydrate- and amino acid-based excipients, synthetic amphiphilic polymers, and ionic liquids that enable protein stabilization. For each category, important characteristics such as the ability to stabilize proteins against thermal and mechanical stresses, current knowledge related to the safety profile for parenteral administration, potential interactions with other formulation components, and primary packaging are debated. Based on the provided information and the detailed discussion thereof, this paper may pave the way for the identification or development of efficient excipients for biotherapeutic protein stabilization.

Cyclodextrin Derivatives as Promising Solubilizers to Enhance the Biological Activity of Rosmarinic Acid.

Rosmarinic acid (RA) is a natural antioxidant with neuroprotective properties; however, its preventive and therapeutic use is limited due to its slight solubility and poor permeability. This study aimed to improve RA physicochemical properties by systems formation with cyclodextrins (CDs): hydroxypropyl-α-CD (HP-α-CD), HP-β-CD, and HP-γ-CD, which were prepared by the solvent evaporation (s.e.) method. The interactions between components were determined by X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC) and Fourier Transform infrared spectroscopy (FTIR). The sites of interaction between RA and CDs were suggested as a result of in silico studies focused on assessing the interaction between molecules. The impact of amorphous systems formation on water solubility, dissolution rate, gastrointestinal (GIT) permeability, and biological activity was studied. RA solubility was increased from 5.869 mg/mL to 113.027 mg/mL, 179.840 mg/mL, and 194.354 mg/mL by systems formation with HP-α-CD, HP-β-CD, and HP-γ-CD, respectively. During apparent solubility studies, the systems provided an acceleration of RA dissolution. Poor RA GIT permeability at pH 4.5 and 5.8, determined by parallel artificial membrane permeability assay (PAMPA system), was increased; RA–HP-γ-CD s.e. indicated the greatest improvement (at pH 4.5 from Papp 6.901 × 10−7 cm/s to 1.085 × 10−6 cm/s and at pH 5.8 from 5.019 × 10−7 cm/s to 9.680 × 10−7 cm/s). Antioxidant activity, which was determined by DPPH, ABTS, CUPRAC, and FRAP methods, was ameliorated by systems; the greatest results were obtained for RA–HP-γ-CD s.e. The inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) was increased from 36.876% for AChE and 13.68% for BChE to a maximum inhibition of the enzyme (plateau), and enabled reaching IC50 values for both enzymes by all systems. CDs are efficient excipients for improving RA physicochemical and biological properties. HP-γ-CD was the greatest one with potential for future food or dietary supplement applications.

A compatibility study between rice starch and some non-steroidal anti-inflammatories

The objective of this work was to mix three milled drugs – Ibuprofen, Naproxen, and Ketoprofen – with rice starch in different proportions and then evaluate its use as an excipient. Differential scanning calorimetry and X-ray powder diffraction verified that the starch was amorphized after the milling process. The samples exhibited similar thermal decomposition profiles, as observed by thermogravimetry. In addition, mid-infrared spectroscopy elucidated the milling process did not alter the functional groups of the starch or the drugs, even in the presence of ethanol. The morphology of the starch granules, as observed by the scanning electron microscope, confirmed decreased particles size and only a physical mixture for the systems. The presence of ethanol in the milling process did not influence the properties of starch or the active ingredients. Therefore, rice starch can be considered as an efficient excipient for these drugs.

Stability of Proteins in Carbohydrates and Other Additives during Freezing: The Human Growth Hormone as a Case Study.

Molecular dynamics is here used to elucidate the mechanism of protein stabilization by carbohydrates and other additives during freezing. More specifically, we used molecular dynamics simulations to obtain a quantitative estimation of the capability of various cryoprotectants to preserve a model protein, the human growth hormone, against freezing stresses. Three mechanisms were investigated, preferential exclusion, water replacement, and vitrification. Model simulations were finally validated upon experimental data in terms of the ability of excipients to prevent protein aggregation. Overall, we found that the preferential exclusion and vitrification mechanisms are important during the whole freezing process, while water replacement becomes dominant only toward the end of the cryoconcentration phase. The disaccharides were found to be the most efficient excipients, in regard to both preferential exclusion and water replacement. Moreover, sugars were in general more efficient than other excipients, such as glycine or sorbitol.

Application of cyclodextrins in antibody microparticles: potentials for antibody protection in spray drying

Objectives: Dry powder formulations are extensively used to improve the stability of antibodies. Spray drying is one of important methods for protein drying. This study investigated the effects of trehalose, hydroxypropyl beta cyclodextrin (HPBCD) and beta cyclodextrin (BCD) on the stability and particle properties of spray-dried IgG.Methods: D-optimal design was employed for both experimental design and analysis and optimization of the variables. The size and aerodynamic behavior of particles were determined using laser light scattering and glass twin impinger, respectively. In addition, stability, ratio of beta sheets and morphology of antibody were analyzed using size exclusion chromatography, IR spectroscopy and electron microscopy, respectively.Results: Particle properties and antibody stability were significantly improved in the presence of HPBCD. In addition, particle aerodynamic behavior, in terms of fine-particle fraction (FPF), enhanced up to 52.23%. Furthermore, antibody was better preserved not only during spray drying, but also during long-term storage. In contrast, application of BCD resulted in the formation of larger particles. Although trehalose caused inappropriate aerodynamic property, it efficiently decreased antibody aggregation.Conclusion: HPBCD is an efficient excipient for the development of inhalable protein formulations. In this regard, optimal particle property and antibody stability was obtained with proper combination of cyclodextrins and simple sugars, such as trehalose.

Acyclic Cucurbit[n]uril-Type Molecular Container Enables Systemic Delivery of Effective Doses of Albendazole for Treatment of SK-OV-3 Xenograft Tumors.

Approximately, 40-70% of active pharmaceutical ingredients (API) are severely limited by their extremely poor aqueous solubility, and consequently, there is a high demand for excipients that can be used to formulate clinically relevant doses of these drug candidates. Here, proof-of-concept studies demonstrate the potential of our recently discovered acyclic cucurbit[n]uril-type molecular container Motor1 (M1) as a solubilizing agent for insoluble drugs. M1 did not induce significant rates of mutations in various Salmonella typhimurium test strains during the Ames test, suggesting low genotoxicity. M1 also has low risk of causing cardiac toxicity in humans since it did not inhibit the human Ether-à-go-go-Related Gene channel as tested on transfected CHO cell lines via patch clamp analysis. Albendazole (ABZ) is a widely used antihelminthic agent but that has also shown promising efficacy against cancerous cells in vitro. However, due to its low aqueous solubility (2.7 μM) and poor pharmacokinetics, ABZ is clinically limited as an anticancer agent. Here we investigated the potential of M1 as a solubilizing excipient for ABZ formulation. A pharmacokinetic study indicated that ABZ escapes the peritoneal cavity resulting in 78% absolute bioavailability, while its active intermediate metabolite, albendazole sulfoxide, achieved 43% absolute bioavailability. The daily dosing of 681 mg/kg M1 complexed with 3.2 mg/kg of ABZ for 14 days did not result in significant weight loss or pathology in Swiss Webster mice. In vivo efficacy studies using this M1·ABZ inclusion complex showed significant decreases in tumor growth rates and increases in survival of mice bearing SK-OV-3 xenograft tumors. In conclusion, we provide substantial new evidence demonstrating that M1 is a safe and efficient excipient that enables in vivo parenteral delivery of poorly water-soluble APIs.

Rational use of antioxidants in solid oral pharmaceutical preparations

Antioxidants are currently used as efficient excipients that delay or inhibit the oxidation process of molecules. Excipients are often associated with adverse reactions. Stability studies can guide the search for solutions that minimize or delay the processes of degradation. The ability to predict oxidation reactions in different drugs is important. Methods: This study was conducted to assess the rational use of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), sodium metabisulfite (SMB), propyl gallate (PG) and cysteine (CYS) in tablet formulations of simvastatin and ketoconazole. These antioxidants were evaluated according to stability parameters and the relationship between efficiency of the antioxidant and chemical structure of the drugs. Results were compared with DPPH tests and computational simulations. BHT was most efficient regarding simvastatin stability, and the most effective BHT concentrations for maintaining stability were 0.5 and 0.1%. In relation to ketoconazole, SMB was most efficient for maintaining content and dissolution profile. The evaluation by DPPH showed that the largest percentage of absorbance reduction was observed for PG, while SMB proved most efficient and had lower consumption of DPPH. The same pattern was observed, albeit with lower efficiency, for the other lipophilic antioxidants such as BHT and BHA. The results of the molecular modeling study demonstrated that electronic properties obtained were correlated with antioxidant activity in solution, being useful for the rational development of liquid pharmaceutical formulations but not for solid oral formulations. This study demonstrated the importance of considering stability parameters and molecular modeling to elucidate the chemical phenomena involved in antioxidant activity, being useful for the rational use of antioxidants in the development of pharmaceutical formulations.

Structure‐guided Encapsulation of Bovine Serum Albumin in Poly(DL‐lactic‐co‐glycolic) Acid

Protein stability is a limiting factor when encapsulating proteins in polymeric matrices for sustained release. We present an approach to stabilize protein structure upon encapsulation in hydrophobic, synthetic polymers. Bovine serum albumin (BSA) was encapsulated in the biocompatible polymer poly(DL-lactic-co-glycolic) acid (PLGA) using a non-aqueous approach. Fourier-transform infrared (FTIR) spectroscopy was used as a non-invasive structural probe to guide the optimization of the encapsulation process. The secondary structure of BSA was determined under relevant conditions from the amide I region after resolution enhancement by Gaussian curve-fitting. The α-helix content was used to describe the structural integrity of BSA. BSA was co-lyophilized with twelve different polyol lyoprotectants to identify efficient excipients for structural preservation upon freeze-drying. Sorbitol, glucose, trehalose and lactitol largely prevented lyophilization-induced structural alterations in BSA. However, only formulations with trehalose allowed the formation of a homogeneous fine suspension in the organic solvent methylene chloride, and were therefore used in the encapsulation procedure. This fine suspension is essential in drug-delivery devices to avoid burst release. Exposure of the BSA-trehalose co-lyophilizate to conditions relevant to the encapsulation process did not result in any further structural alterations. The encapsulation process was guided using FTIR spectroscopy as a non-invasive probe and, by selecting the appropriate conditions, we were able to preserve protein secondary structure in all the steps before and during the encapsulation in PLGA.

Identification of penetration enhancers for testosterone transdermal delivery from spray formulations

The goal of this study was to identify a suitable penetration enhancer-containing formulation for the transdermal delivery of testosterone from a spray. The first step involved in vitro measurement of drug flux from a 1:1 ethanol/water saturated solution across hairless rat skin, which had been pre-treated with a series of penetration enhancers. Isopropyl myristate (IPM) was found to be the most efficient excipient, increasing testosterone transport by more than a factor of 5. The enhancing ability of IPM was also apparent when the drug was formulated in 3:1 ethanol/propylene glycol, a more compatible vehicle for use in a spray. IPM was then incorporated into this formulation directly (as opposed to being used to pre-treat the skin) over a range of concentrations from 10–25% v/v, and testosterone transport was evaluated when delivered from either a solution, or from a mechanical spray, or from an aerosol (which also contained 50% v/v propellant). At the highest level of enhancer, the flux was improved 2.5-fold from both the spray and the aerosol, relative to a control. However, these formulations were far from optimally conceived, in that the amount of drug which eventually contacted the skin represented only ∼10% of the pulverized quantity from the spray, and ~40% of that from the aerosol. Repeated application, especially from the aerosol, improved matters somewhat, but further work is clearly required before the concept can be developed for practical application.

Effect of excipients on the stability and transport of recombinant human epidermal growth factor (rhEGF) across Caco-2 cell monolayers.

The effect of sixteen excipients on the transport of recombinant human epidermal growth factor (rhEGF) across Caco-2 cell monolayers was examined at 37 degrees C. The apparent apical to basolateral (A-B) permeability (Papp) of 30 microM rhEGF was 8.15 x 10(-7) cm/sec, indicative of a poor level of absorption in the GI tract. The Papp was 1.7- and 6.3-fold greater than the Papp in the basolateral to apical (B-A) direction and the A-B permeability of mannitol, respectively, and decreased dramatically to a negligible level at 4 degrees C, consistent with a receptor mediated transcytosis of rhEGF. The stability of rhEGF was very poor, undergoing more than 85% degradation in 2 h in the transport medium at 37 degrees C. A significant increase in the Papp could be achieved by the addition of certain excipients, as exemplified by 23, 21, 20 and 16-fold increases, in the presence of sodium taurochenodeoxycholate (NaTCDC), sodium taurodeoxycholate (NaTDC), sodium glycodeoxycholate (NaGDC) and sodium laurylsulfate (SLS) (all at a concentration of 1% w/v), respectively. A significant increase in stability could also be achieved by the addition of some of the excipients, as represented by 1% SLS, which nearly completely stabilized the rhEGF. Unfortunately, however, an increase in the Papp of rhEGF could not be achieved without a simultaneous and extensive decrease in the integrity of the cell membranes. Thus, more efficient excipients, that specifically enhance the permeation of rhEGF and do not alter the membrane integrity, should be pursued in order to safely enhance the permeation of rhEGF.

Stabilization of α-chymotrypsin at the CH 2Cl 2/water interface and upon water-in-oil-in-water encapsulation in PLGA microspheres

Protein inactivation and aggregation are serious drawbacks in the encapsulation of proteins in bioerodible polymers by water-in-oil-in-water (w/o/w) encapsulation. The model protein α-chymotrypsin was employed to investigate whether its stabilization towards the major stress factors in the w/o/w encapsulation procedure would allow for the encapsulation and release of non-aggregated and active protein. Due to the formation of amorphous aggregates α-chymotrypsin is an excellent sensor to probe unfolding events. Furthermore, its enzymatic activity is highly sensitive towards the presence of organic solvents. α-Chymotrypsin in aqueous solution showed substantial aggregation and activity loss when it was homogenized with CH 2Cl 2 due to adsorption to the interface. Its w/o/w encapsulation in poly(lactic-co-glycolic)acid (PLGA) microspheres caused formation of 35% non-covalent aggregates and reduced the specific activity by 14%. Screening for efficient excipients revealed that co-dissolving the protein with maltose and polyethylene glycol (PEG, M w 5000) in the first aqueous phase reduced interface-induced protein aggregation and inactivation. Employing these excipients during encapsulation led to a reduction in α-chymotrypsin inactivation (10%) and aggregation (12%). Optimizing the effect of PEG by also dissolving the excipient in the organic phase prior to encapsulation further decreased the amount of non-covalent aggregates to 7% and loss in activity to 5%. The data obtained demonstrate that the w/o emulsification step is the main stress-factor in the w/o/w encapsulation procedure but subsequent encapsulation steps also cause some protein aggregation.

Preservation of lysozyme structure and function upon encapsulation and release from poly(lactic- co-glycolic) acid microspheres prepared by the water-in-oil-in-water method

When proteins are encapsulated in bioerodible polymers by water-in-oil-in-water (w/o/w) encapsulation techniques, inactivation and aggregation are serious drawbacks hampering their sustained delivery. Hen egg-white lysozyme was employed to investigate whether stabilizing it towards the major stress factors in the w/o/w encapsulation procedure would allow for the encapsulation and release of structurally unperturbed, non-aggregated, and active protein. When it was encapsulated in poly(lactic- co-glycolic) acid (PLGA) microspheres without stabilizing additives, lysozyme showed substantial loss in activity and aggregation. It has been shown that by co-dissolving various sugars and polyhydric alcohols with lysozyme in the first aqueous buffer, interface-induced lysozyme aggregation and inactivation can be minimized in the first emulsification step [J. Pharm. Pharmacol. 53 (2001) 1217]. Herein, it was found that those excipients, which were efficient in preventing interface-induced structural perturbations, were also efficient in minimizing lyophilization-induced structural perturbations (e.g. lactulose). The efficient excipients identified also reduced structural perturbations upon lysozyme encapsulation in PLGA microspheres and this led to reduced lysozyme inactivation and aggregation. However, the data obtained also show that later steps in the encapsulation procedure are detrimental to lysozyme activity. Lysozyme inactivation was completely prevented only by employing the efficient excipients in the second aqueous phase also. In summary, protein aggregation and inactivation were minimized by rationally selecting excipients efficient in stabilizing lysozyme against the major stress factors of w/o/w encapsulation.

Investigation of protein/carbohydrate interactions in the dried state. 2. Diffuse reflectance FTIR studies

Upon freeze-drying in the absence of lyoprotectants, Fourier transform infrared (FTIR) spectroscopy has detected changes in the secondary structures of proteins. Such FTIR studies have been typically conducted using protein/KBr pellets, where additional protein degradation could potentially occur due to pressure effects and partial dissolution of the chaotropic KBr. Diffuse reflectance FTIR spectroscopy, in which no sample preparation is necessary, was evaluated as an alternative spectroscopic method to examine protein structure upon freeze-drying. The therapeutic proteins recombinant human deoxyribonuclease I (rh-DNase) and recombinant human insulin like growth factor I (rh-IGF-I) were freeze-dried with mannitol, sucrose, trehalose, and two molecular weight dextrans (69 and 503 kDa) separately, at concentrations ranging from 0 to 100% (w/w). Upon freeze-drying, rh-DNase and rh-IGF-I underwent significant changes in their secondary structure. For both proteins, the presence of intermolecular β-sheets due to aggregation was detected and the α-helix content decreased significantly. The addition of carbohydrates to the formulations inhibited the protein secondary structure rearrangement in a concentration-dependent manner. Sucrose and trehalose appeared to be the most efficient excipients in preventing secondary structure changes. The conformational changes observed for both proteins appeared to be reversible upon rehydration.

Osmotic properties of sulfobutylether and hydroxypropyl cyclodextrins.

The purpose of this study was to determine the osmolality of sulfobutylether (SBE) and hydroxypropyl (HP) derivatives of cyclodextrins (CDs) via vapor pressure osmometry (VPO) and freezing point depression (FPD). (SBE) and HP-CDs are efficient excipients capable of solubilizing and stabilizing poorly water-soluble drugs in parenteral formulations. (SBE)-CDs have also been used as solubility enhancers and osmotic agents for the sustained release of poorly water-soluble drugs from osmotic pump tablets. The knowledge of the CD's osmolality in solution or inside such tablets would allow one to further characterize the release mechanisms. Experiments were conducted at 37 degrees C with eight types of HP and (SBE)-CDs. The aqueous solutions ranged from 0.005-0.350 mol(-1). Methods were developed to allow the measurement of high osmolalities using a vapor pressure osmometer or a differential scanning calorimeter. The osmolality calculations from the VPO and FPD measurements correlated well. The osmolality of (SBE)-CDs was significantly higher than the osmolality of HP-CDs and increased with the total degree of substitution (TDS). All CDs showed deviations from ideality at high concentrations. Empirical correlations of osmolality with concentration and TDS allowed the prediction of osmolality over a wide concentration range. This study also gave some useful insights into the behavior of CD derivatives in solution.