Biopharmaceutical Characterization Research Articles

Physicochemical and biopharmaceutical characterization of amorphous solid dispersion of nobiletin, a citrus polymethoxylated flavone, with improved hepatoprotective effects

The present study aimed to develop an amorphous solid dispersion (SD) of nobiletin (NOB), a citrus polymethoxylated flavone, with the aim of improving its biopharmaceutical and hepatoprotective properties. SD formulation of NOB (NOB/SD) was prepared by wet-milling and subsequent freeze drying, and its stability and dissolution properties were characterized. The hepatoprotective effects and pharmacokinetic behavior of orally dosed NOB/SD were evaluated in rats. During the storage of NOB/SD for 4weeks under accelerated conditions, there were no significant transitions in the appearance, particle size, and amorphousity of wet-milled NOB. In comparison with crystalline NOB, the NOB/SD exhibited significant improvement in the dissolution with a 10-fold higher dissolution rate. In a rat model of acute liver injury, repeated treatment with NOB/SD (2mg NOB/kg) every 4h led to marked attenuation of hepatic damage as evidenced by decreased ALT and AST, surrogate biomarkers for hepatic injury; however, crystalline NOB was found to be less effective. After oral administration of NOB/SD (2mg NOB/kg) in rats, compared with crystalline NOB, improved pharmacokinetic behavior was observed with increases of bioavailability and hepatic delivery by ca. 7- and 6-fold, respectively, possibly leading to better hepatoprotection. Given the improved physicochemical and biopharmaceutical properties, the SD formulation strategy might be efficacious for enhancing the therapeutic potential of NOB.

PH-sensitive microparticles for oral drug delivery based on alginate/oligochitosan/Eudragit® L100-55 “sandwich” polyelectrolyte complex

The primary objective of this study was to investigate the influence of the oligochitosan–Eudragit® L100-55 polyelectrolyte complex (OCH–EL PEC) on the pH-sensitivity of Eudragit® L100-55-treated alginate–oligochitosan microparticles. In order to achieve this, three types of naproxen-loaded microparticles were prepared under mild and environmentally friendly conditions using a custom made device with coaxial air flow: Ca-alginate (Ca-ALG), alginate–oligochitosan (ALG–OCH) and alginate–oligochitosan–Eudragit® L100-55 (ALG–OCH–EL) microparticles. After drying, the microparticles were subjected to microscopic analysis, and physicochemical and biopharmaceutical characterization. The non-covalent interaction between OCH and EL and the formation of OCH–EL PEC during the preparation procedure of the particles were verified by thermal and FT-IR analysis. The obtained particles exhibited acceptable sphericity and surface roughness due to the presence of the drug crystals (Ca-ALG particles) and OCH–EL PEC (ALG–OCH–EL particles). It was found that reinforcement of the ALG–OCH particles with OCH–EL PEC had no significant effect on the relatively high encapsulation efficiencies (>74.4%). The results of drug release studies confirmed the ability of ALG–OCH PEC to sustain drug release at pH 6.8 and 7.4. However, this PEC showed enhanced sensitivity to an acidic environment and to simulated intestinal fluid (pH 6.8) after prior exposure to an acidic medium. Additional treatment of ALG–OCH particles with EL and formation of “sandwich” ALG–OCH–EL PEC was essential not only to improve stability and decrease drug release in acidic medium, but also to achieve sustained release after the pH of dissolution medium was raised to 6.8. The obtained results suggested that ALG–OCH–EL microparticles have promising potential as pH-sensitive multiparticulate drug carriers for oral delivery of NSAIDs.

Improvement of Intestinal Absorption of Forsythoside A and Chlorogenic Acid by Different Carboxymethyl Chitosan and Chito-oligosaccharide, Application to Flos Lonicerae - Fructus Forsythiae Herb Couple Preparations

The current study aims to investigate the effect of chitosan derivatives on the intestinal absorption and bioavailabilities of forsythoside A (FTA) and Chlorogenic acid (CHA), the major active components in Flos Lonicerae - Fructus Forsythiae herb couple. Biopharmaceutics and pharmacokinetics properties of the two compounds have been characterized in vitro, in situ as well as in rats. Based on the identified biopharmaceutics characteristics of the two compounds, the effect of chitosan derivatives as an absorption enhancer on the intestinal absorption and pharmacokinetics of FTA and CHA in pure compound form as well as extract form were investigated in vitro, in situ and in vivo. Both FTA and CHA demonstrated very limited intestinal permeabilities, leading to oral bioavailabilities being only 0.50% and 0.13% in rats, respectively. Results from both in vitro, in situ as well as in vivo studies consistently indicated that Chito-oligosaccharide (COS) at dosage of 25 mg/kg could enhance intestinal permeabilities significantly as well as the in vivo bioavailabilities of both FTA and CHA than CMCs in Flos Lonicerae - Fructus Forsythiae herb couple preparations, and was safe for gastrointestine from morphological observation. Besides, treatment with Flos Lonicerae - Fructus Forsythiae herb couple preparations with COS at the dosage of 25 mg/kg prevented MDCK damage after influenza virus propagation, which was significantly better than control. The current findings not only identified the usefulness of COS for the improved delivery of Flos Lonicerae - Fructus Forsythiae preparations but also demonstrated the importance of biopharmaceutical characterization in the dosage form development of traditional Chinese medicine.

C&EN Webinars - Orthogonal Techniques for Advanced Characterization of Biopharmaceuticals

C&EN Webinars - Orthogonal Techniques for Advanced Characterization of Biopharmaceuticals

Synthesis, biopharmaceutical characterization, and antimicrobial study of novel azo dyes of 7-hydroxy-4-methylcoumarin

Herein, we report the synthesis and biopharmaceutical characterization of coumarin based azo compounds. The novel coumarin based azo compounds were obtained by the coupling of bis-coumarin (2Z,2′Z)-2,2′-[ethane-1,2-diylbis(azan-1-yl-1-ylidene)]bis(4-methylchroman-7-ol) with diazotized aromatic amines. The compounds were fully characterized using spectroscopic analytical method and tested for their antibacterial and antifungal activity. A correlation of structure and activities relationship of these compounds with respect to molecular modeling, Lipinski rule of five, drug-likeness, toxicity profiles, and other physico-chemical properties of drugs are described and verified experimentally.

CE‐MS for the analysis of intact proteins 2010–2012

Since its introduction in 1987, CE-MS has become an increasingly important technique for the analysis of biomolecules. Since our previous update on CE-MS methods within the field of intact protein analysis (Electrophoresis 2011, 32, 66-82), a variety of interesting methodological improvements and applications have been reported in literature. Therefore, this article presents an overview of the development and application of CE-MS for intact protein analysis as published between June 2010 and June 2012. The article is divided in sections that treat CE coupled to MS through ESI, MALDI, and ICP ionization, respectively. In the section about CE-ESI-MS, technological developments with respect to CE-MS interfacing, prevention of protein adsorption, and chip-based CE-MS are treated in more detail. Novel interfacing strategies and the development of improved capillary coating strategies appeared to be the major developments. Furthermore, in all sections, the applicability of CE-MS for intact protein analysis is demonstrated by representative examples, including important developments in the fields of biopharmaceutical characterization and the analysis of proteins in biological samples. Finally, some general conclusions and future perspectives are given.

A preclinical pharmacokinetic modeling approach to the biopharmaceutical characterization of immediate and microsphere-based sustained release pulmonary formulations of rifampicin

A rifampicin-hydroxypropyl-beta-cyclodextrin (RIF-HPCD) complex solution and two RIF-loaded PLGA microspheres with slow or fast release rates were nebulized into the rat lungs for a comparative biopharmaceutical evaluation. A pharmacokinetic model was applied to model systemic RIF concentrations and to predict the RIF concentrations in the lung epithelial lining fluid (ELF). With intravenous RIF and nebulized RIF-HPCD, plasma profiles and predicted RIF ELF profiles were superimposed indicating that RIF diffused almost instantaneously through the broncho-alveolar barrier. 5h post administration RIF ELF predicted concentrations were in agreement with experimental concentrations determined using the broncho-alveolar lavage (BAL) sampling method. Microsphere formulations resulted in different plasma concentration profiles, demonstrating RIF sustained release. The PK model predicted the ELF concentrations to be much higher with microspheres than with nebulized and IV RIF, over a prolonged time period, which was confirmed by BAL sampling. In conclusion this work demonstrated the benefit of using sustained-release microspheres administered as aerosols to maintain, over a prolonged time period, high levels of pulmonary concentrations of drugs characterized by a rapid absorption through the broncho-alveolar barrier. Moreover, PK modeling was a useful tool to build concentration-versus-time profiles in non-readily accessible ELF compartment and to assess the biopharmaceutical properties of aerosol formulations for lung delivery.

Biopharmaceutical characterization of decursin and their derivatives for drug discovery

Angelica gigas Nakai and its components are known to have neuroprotective, antiplatelet, and anticancer activities. The present study evaluated the in vitro and in vivo biopharmaceutical characterization of Angelica gigas component substances, including decursin (the main substance), decursinol angelate (decursin isomer), JH714 (ether form of decursin) and epoxide decursin (epoxide form of decursin). Decursin, decursinol angelate and JH714 exhibited acceptable metabolic stability (>50%) in liver microsomes from human and higher bound fraction (>90%) in human plasma operating ultrafiltration. Decursin and decursinol angelate in CYP1A2 and CYP2C19 indicated less than 50% CYP activity, suggesting inhibition of the CYP isoforms using Vivid® CYP screening kit. JH714 only showed an apparent permeability coefficient of <10 × 10−6 cm/s in MDCK cells, suggesting that it is poorly absorbed. Blood brain barrier permeability was examined after oral administration to male Sprague–Dawley (SD) rats, and pharmacokinetic studies were performed after oral and intravenous administration of 10 mg/kg compounds. Decursin, decursinol angelate and JH714 showed ratios of compound concentration in brain with respect to plasma (Cbrain/Cplasma) of >1.5, suggesting good brain/plasma ratio at 0.5, 1, 3, and 5 h. In contrast, Cbrain/Cplasma was <0.5 for epoxide decursin. For all test compounds, >1.5% of the dose remained in GI tract after 8 h, and the excretion rate in urine was <0.5% which suggests that gastro intestinal tract may be major site of disposition following oral administration. Finally, these results may be useful for the design of dosage regimens of decursin and its derivatives.

Ultraviolet Resonance Raman spectroscopy used to study formulations of salmon calcitonin, a starch–peptide conjugate and TGF-β3

Ultraviolet Resonance Raman (UVRR) spectroscopy with excitation at 244nm was investigated here as a possible useful tool for fast characterization of biopharmaceuticals. Studies were performed on three protein drugs: salmon calcitonin (sCT), starch–peptide conjugate, and transforming growth factor-β3 (TGF-β3) adsorbed onto solid granules of tricalcium phosphate (TCP). Secondary structure of sCT was investigated for solutions of 0.5mg/mL up to 200mg/mL, regardless of the turbidity or aggregation states. An increase in β-sheet content was detected when sCT solutions aggregated. UVRR spectroscopy also detected a small amount of residual organic solvent in a starch–peptide conjugate solution containing only 40μg/mL of peptide. UVRR spectroscopy was then used to characterize a protein, TGF-β3, adsorbed onto solid granules of TCP at 50 and 250μg/cm3. This study shows that UVRR is suitable to characterize the protein formulations in a broad range of concentrations, in liquid, aggregated, and solid states.

Evaluation of nanoparticle tracking analysis (NTA) in the characterization of therapeutic antibodies and seasonal influenza vaccines: pros and cons

Nanoparticle tracking analysis (NTA) is a relatively new technique for the size characterization of particles between 40 and 1000 nm. Recent studies have shown an increasing interest in NTA as a complementary technique in the field of pharmaceutical sciences. In this review, the applications of NTA for the characterization of biopharmaceuticals are discussed, and procedures are proposed for the determination of size distributions. The size distribution of monoclonal antibody (mAb) aggregates in samples produced from different clones in the presence of cell culture medium is presented as an example in addition to that of seasonal influenza vaccines. This review also discusses techniques such as fluorescence microscopy with Nile Red staining, micro-flow imaging, 90° light scattering, and flow field-flow fractionation (FFF) and concludes that NTA is a suitable technique for a wide range of applications in the field of biopharmaceuticals.

In vivo Biopharmaceutical Characterisation of a New Formulation Containing the Antiepileptic Drug Lamotrigine in Comparison to Plain and Dispersible/Chewable Lamotrigine Tablets

Two studies in healthy male volunteers were carried out to evaluate the pharmacokinetic profile of a new divisible formulation of lamotrigine (CAS 84057-84-1, Plexxo, Lamotrigin Desitin) in plasma in comparison to plain or dispersible lamotrigine tablets. The plasma pharmacokinetics of lamotrigine were analysed after administration of single doses of 100 mg lamotrigine given as one tablet of the new formulation and either the plain or the dispersible reference formulation in two separate studies. In each study the data of 24 subjects were analysed according to the study protocol. Venous blood samples were taken at appropriate intervals up to 120 h after dosing. Concentrations of lamotrigine were determined in plasma by a validated HPLC method using UV detection. In both studies, mean plasma concentration-time profiles of the new lamotrigine formulation and both reference formulations ran nearly in parallel. The pharmacokinetic mean data calculated from different subject groups of the two studies were very similar. The mean ratios of the main pharmacokinetic parameters and the corresponding 90% confidence intervals of AUC(0-t), AUC(0-inf) and C(max) were 103% (99.7-105.7), 103% (99.6-107.3) and 101% (95.2-106.6) for the comparison with the plain lamotrigine tablet and 100% (98.0-102.8), 100% (96.5-102.8) and 102% (99.1-105.3) for the comparison with the dispersible/chewable tablet, respectively. The most frequently reported adverse events possibly related to the administration of lamotrigine were headache and dizziness in both studies. It is concluded that the new divisible lamotrigine formulation is bioequivalent with regard to rate and extent of absorption to both the plain reference lamotrigine product and to the dispersible/chewable reference product.

Biopharmaceutical Characterization of Carbamazepine Immediate Release Tablets. In vitro-in vivo comparison

A growing concern for the biopharmaceutical characterization of pharmaceutical products increased the interest in the evaluation and identification of physicochemical properties of drugs and dosage forms that govern its biological performance. In vitro and in vivo characteristics of two carbamazepine (CAS 298-46-4) immediate release tablets were investigated and compared in order to establish level A in vitro-in vivo correlation. An in vivo study was conducted as a controlled, two-way, complete cross-over, single dose, pharmacokinetic trial in 18 subjects. The in vitro study was performed using various dissolution media in order to evaluate their potential influence on drug release and distinguish the set of experimental conditions relevant to the in vivo behavior of the investigated drug products. Beside significant differences among in vitro release profiles, the in vivo data indicated bioequivalence of the two formulations. Although a high level of correlation between in vivo and in vitro data was observed in some media, there was no single in vitro-in vivo correlation model applicable to both investigated products. The obtained results add to the existing debate on the rationale for the use of surfactants in drug release media and their in vivo relevance, emphasizing the importance of in vitro dissolution testing in addition to in vivo bioequivalence testing.

Biopharmaceutical Characterization of Ciprofloxacin HCl–Ferrous Sulfate Interaction

The ciprofloxacin-iron interaction, resulting in a lower bioavailability, is well documented in vivo; however, a mechanistic explanation supported by experimental data of this interaction is missing. In the present study, ciprofloxacin hydrochloride (HCl) and ferrous sulfate interaction was simulated in vitro by performing solubility and dissolution studies in the reactive media containing ferrous sulfate. Characterization of the precipitate formed indicated its probable chemical structure as Fe(SO(4) (2-) )(2) (Cl(-) )(2) (ciprofloxacin)(2) × (H(2) O)(n) , where n is up to 12 molecules of water. The solubility of this complex in water was estimated to be approximately 2 mg/mL, being about 20-fold lower than the solubility of ciprofloxacin HCl. The solubility of the complex was used as input parameter for an in silico modeling by GastroPlus™ and the resulting predicted plasma time curves were in good agreement with the in vivo data. These results strongly indicate that ciprofloxacin-iron interaction in vivo is caused by the formation of a low soluble complex. This interaction was also simulated by in vitro dissolution, in which a mini scale apparatus provided more biorelevant results than the standard dissolution apparatus, probably because the drug concentrations in the mini apparatus were higher and, thus, closer to the conditions encountered in vivo.

Petra, Osiris and Molinspiration (POM) together as a successful support in drug design: antibacterial activity and biopharmaceutical characterization of some azo Schiff bases

Herein, we report the design and calculated molecular properties of ten existing azo Schiff bases 5a–h and 7a, b. On the basis of a hypothetical antibacterial pharmacophore, the structures are designed to interact with Gram-positive and Gram-negative bacteria. The in vitro biological evaluation of these compounds allowed us to point out new potential non-nucleoside hits, with MIC values in the range of 2–8 μg/ml active against Staphylococcus aureus and Bacillus subtilis. In contrast to Gram positive strains, no activity is noted in favor of Gram negative strains (Klebsiella pneumonia, Pseudomonas aeruginosa and Escherichia coli). The antibacterial activity of these azo Schiff bases is discussed on the basis of theoretical calculations by using Petra, Osiris and Molinspiration (POM) model. Petra, Osiris and Molinspiration (POM) together as a successful support in drug design: antibacterial activity and biopharmaceutical characterization of some azo schiff bases.

The Utility of Hydrogen/Deuterium Exchange Mass Spectrometry in Biopharmaceutical Comparability Studies

The function, efficacy, and safety of protein biopharmaceuticals are tied to their three-dimensional structure. The analysis and verification of this higher-order structure are critical in demonstrating manufacturing consistency and in establishing the absence of structural changes in response to changes in production. It is, therefore, essential to have reliable, high-resolution and high sensitivity biophysical tools capable of interrogating protein structure and conformation. Here, we demonstrate the use of hydrogen/deuterium exchange mass spectrometry (H/DX-MS) in biopharmaceutical comparability studies. H/DX-MS measurements can be conducted with good precision, consume only picomoles of protein, interrogate nearly the entire molecule with peptide level resolution, and can be completed in a few days. Structural comparability or lack of comparability was monitored for different preparations of interferon-β-1a. We present specific graphical formats for the display of H/DX-MS data that aid in rapidly making both the qualitative (visual) and quantitative assessment of comparability. H/DX-MS is capable of making significant contributions in biopharmaceutical characterization by providing more informative and confidant comparability assessments of protein higher-order structures than are currently available within the biopharmaceutical industry.

Synthesis, biopharmaceutical characterization, antimicrobial and antioxidant activities of 1-(4′- O-β- d-glucopyranosyloxy-2′-hydroxyphenyl)-3-aryl-propane-1,3-diones

This research communication is toward the investigation of the antibacterial, antifungal and antioxidant activities of the synthesized compounds 1-(4′- O-β- d-glucopyranosyloxy-2′-hydroxyphenyl)-3-aryl-propane-1,3-diones ( 5a) –( 5h). These compounds have been obtained by the interaction of α-acetobromoglucose with 1-(2′,4′-dihydroxyphenyl)-3-aryl-propane-1,3-diones ( 3a) –( 3h) under anhydrous condition and at lower temperature. The structures of these newly synthesized O-β- d-glucopyranosides were established on the basis of chemical, elemental, and spectral analyses. Further, the compounds ( 5b), ( 5c), ( 5d) and ( 5g) showed potent antibacterial and antifungal activity. A good correlation was obtained between the theoretical predictions of bioavailability using Lipinski’s rule-of-five and experimental verification.

Surface analysis for compositional, chemical and structural imaging in pharmaceutics with mass spectrometry: A ToF-SIMS perspective

We review the application of time-of-flight secondary-ion mass spectrometry (ToF-SIMS) for the surface chemical identification and distribution analysis (mapping) of pharmaceutically relevant materials. Specifically we explore the characterization of both solid state pharmaceuticals and bio-pharmaceuticals by ToF-SIMS; highlighting specific case studies concerning the distribution and stability of pharmaceutical actives within solid matrices, the face-specific properties of pharmaceutical crystals and elucidation of the structure/conformation of adsorbed proteins. Finally, potential future applications of ToF-SIMS in pharmaceutics are detailed.

A review on worldwide essential software resources in pharmacy

Softwares are the collection of computer programs and related data that provide the instructions telling a computer what to do and thereby help to process the data as wanted. These programs are designed to address general and special purpose applications. Softwares used in pharmaceutical sciences cover wide subject areas such as pharmacology, pharmaceutical chemistry, pharmaceutics, pharmacognosy, pharmaceutical biotechnology, etc. Softwares used in pharmacology are mainly related to minimize the efforts needed in determining pharmacokinetic principles of particular drug in individuals, pathways of drug, and consequently, its adverse reactions. Applications of softwares in pharmaceutical chemistry are to elucidate various physiological properties of drugs and to predict activity values for new compounds within certain limits. They may be enormous assistance to those trying to generate the large databases from massive efforts in drug research. However, softwares used in pharmaceutics help for predicting the dissolution rate, biopharmaceutical characterization, accurate and precise stability profile, etc. of formulated dosage form. Softwares used in pharmacognosy give information on herb activity, interactions, mechanisms of action and supporting data underlying the use of herb for health. Wide applications of software in pharmaceutical biotechnology help to increase the predictability of results, identify genes, elucidate protein structure, identify genome responsible for expression of particular characteristics, etc. Hence, in the present article, we have enlisted the subject wise different software names, websites and their features used in the milieu of pharmacy.

Review Soybeans as bioreactors for biopharmaceuticals and industrial proteins

Plants present various advantages for the production of biomolecules, including low risk of contamination with prions, viruses and other pathogens, scalability, low production costs, and available agronomical systems. Plants are also versatile vehicles for the production of recombinant molecules because they allow protein expression in various organs, such as tubers and seeds, which naturally accumulate large amounts of protein. Among crop plants, soybean is an excellent protein producer. Soybean plants are also a good source of abundant and cheap biomass and can be cultivated under controlled greenhouse conditions. Under containment, the plant cycle can be manipulated and the final seed yield can be maximized for large-scale protein production within a small and controlled area. Exploitation of specific regulatory sequences capable of directing and accumulating recombinant proteins in protein storage vacuoles in soybean seeds, associated with recently developed biological research tools and purification systems, has great potential to accelerate preliminary characterization of plant-derived biopharmaceuticals and industrial macromolecules. This is an important step in the development of genetically engineered products that are inexpensive and safe for medicinal, food and other uses.

Capillary electrophoresis–mass spectrometry for the analysis of intact proteins 2007–2010

CE coupled to MS has proven to be a powerful analytical tool for the characterization of intact proteins, as it combines the high separation efficiency of CE with the selectivity of MS. This review provides an overview of the development and application of CE-MS methods within the field of intact protein analysis as published between January 2007 and June 2010. Ongoing technological developments with respect to CE-MS interfacing, capillary coatings for CE-MS, coupling of CIEF with MS and chip-based CE-MS are treated. Furthermore, CE-MS of intact proteins involving ESI, MALDI and ICP ionization is outlined and overviews of the use of the various CE-MS methods are provided by tables. Representative examples illustrate the applicability of CE-MS for the characterization of proteins, including glycoproteins, biopharmaceuticals, protein-ligand complexes, biomarkers and dietary proteins. It is concluded that CE-MS is a valuable technique with high potential for intact protein analysis, providing useful information on protein identity and purity, including modifications and degradation products.