Evaluation of Pharmacokinetic and Pharmacodynamic (PK/PD) of Novel Fluorenylmethoxycarbonyl- Phenylalanine Antimicrobial Agent.

OBJECTIVE: To assess the pharmacokinetic profile, in-vivo toxicity, and efficacy of 9-Fluorenylmethoxycarbonyl-L-phenylalanine (Fmoc-F) as a potential antibacterial agent, with a focus on its suitability for clinical translation. METHODS: An RP-HPLC-based bio-analytical method was developed and qualified to quantify Fmoc-F levels in mouse plasma for pharmacokinetic analysis. Oral bioavailability was determined, and in-vivo toxicity was evaluated following intra-peritoneal administration. Efficacy was assessed by measuring the reduction in Staphylococcus aureus burden and survival rates in BALB/c mice. RESULTS: The RP-HPLC method is highly sensitive, detecting as low as 0.8 µg mL-1 (~ 2 µM) of Fmoc-F in blood plasma. This study revealed that Fmoc-F has an oral bioavailability of 65 ± 18% and suitable pharmacokinetic profile. Further, we showed that intra-peritoneal administration of Fmoc-F is well tolerated by BALB/c mice and Fmoc-F treatment (100 mg/kg, i.p.) significantly reduces Staphylococcus aureus burden from visceral organs in BALB/c mice but falls short in enhancing survival rates at higher bacterial loads. CONCLUSIONS: The study provides crucial insights into the pharmacokinetic and pharmacodynamic properties of Fmoc-F. The compound displayed favourable oral bioavailability and in-vivo tolerance. Its significant reduction of bacterial burden underscores its potential as a treatment for systemic infections. However, limited effectiveness for severe infections, short half-life, and inflammatory response at higher doses need to be addressed for its clinical application.

Effect of capsaicin on breast cancer resistance protein (BCRP/Abcg2) and pharmacokinetics of probe substrates in rats.

Breast cancer resistance protein (BCRP/Abcg2 in human, Bcrp/Abcg2 in rat), a member of the ATP-binding cassette (ABC) transporter family, acts as an efflux pump for xenobiotics, with ability to transport various drugs out of cells. Capsaicin may have the potential to modulate the function of Bcrp transport. This study was to evaluate the effects of capsaicin on the pharmacokinetics of sulfasalazine, a Bcrp substrate, in rats and investigate the mechanism of this food-drug interaction.The rats were pre-treated with 5% carboxymethylcellulose sodium (vehicle), capsaicin (3, 8, 25 mg/kg) and cyclosporine A (10 mg/kg) by gastric gavage for 7 days. On day 7, blood, liver and intestine samples were collected after sulfasalazine administered. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to study the effects of capsaicin on the pharmacokinetics of sulfasalazine in rats. RT-PCR and western blotting were used to study the mechanism in biomolecules in rats, respectively.Compared with vehicle group, AUC0-∞ of sulfasalazine in rats were increased by 1.5-folds, 1.6-folds and 1.7-folds in 3, 8 and 25 mg/kg/d capsaicin pre-treated groups. At the same time, the CL/F in rats were decreased by 33%, 38% and 42% in the three groups. In addition, we found Bcrp mRNA levels and protein expressions in rat livers and intestines were decreased in 3, 8 and 25 mg/kg/d capsaicin-treated groups.Our study demonstrated that long-term ingestion of capsaicin significantly enhanced the AUC of sulfasalazine involved down-regulate Bcrp gene and protein expression in rat liver and intestine.

Identification and Characterization of Rotigotine Degradation Products by HPLC Coupled DAD and CAD Detectors and HRMS Through Q-Orbitrap and Electrospray Ionization.

Rotigotine (RTG) is a dopamine agonist used in the treatment of Parkinson's disease. As it is susceptible to oxidation, stability studies must be carefully designed for the identification and characterization of all possible degradation products. Here, RTG degradation was evaluated according to the International Conference on Harmonization guidelines under various stress conditions, including acidic and basic hydrolysis, oxidative, metallic, photolytic, and thermal conditions. Additionally, more severe stress conditions were applied to induce RTG degradation. Significant degradation was only observed under oxidative and photolytic conditions. The samples were analyzed by high performance liquid chromatography coupled to photodiode array detectors, charged aerosol, and high-resolution mass spectrometry. Chromatographic analyses revealed the presence of eight substances related to RTG, four of which were already described and were qualified impurities (impurities B, C, K and E) and four new degradation products (DP-1 - DP-4), whose structures were characterized by high-resolution mass spectrometry through Q-Orbitrap and electrospray ionization. In the stress testing of the active pharmaceutical ingredient in solid form, significant RTG degradation was observed in the presence of the oxidative matrix. The results corroborate the literature that confirm the high susceptibility of RTG to oxidation and the importance of using different detectors to detect degradation products in forced degradation studies.

Serum Stabilities and Antiviral Activities of Chemically Modified Peptides Against Dengue Serotypes 1-4.

Dengue presents a major public health concern in over 100 countries due to the absence of an effective vaccine and antiviral therapy against all four dengue virus (DENV) serotypes. Several antiviral peptides were previously reported to inhibit at least three or all four DENV serotypes. Chemical modifications such as d-amino acid substitutions, polyethylene glycol (PEG)ylation, and cyclization could be applied to peptides to improve their biological activities and stability in serum. The PEGylated peptide 3 (PEG-P3) was identified to be the most promising antiviral candidate as it demonstrated good inhibitory effects against all four DENV serotypes during the pre- and post-infection stages, Based on the RP-HPLC and LC/MS analysis, peptide 4 was identified to be more stable in human serum than peptide 3, with 78.9 % and 41.6 % of the peptides remaining after 72 h of incubation in human serum, respectively. Both peptides were also able to retain their antiviral activities against specific DENV serotypes after 72 h incubation in human serum. PEG-P3 was found to be more stable than the unmodified peptide 3 with 89.4 % of PEG-P3 remaining in the human serum after 72 h of incubation. PEG-P3 was able to retain its inhibitory effects against DENV-1 to 4 after 72 h of incubation in human serum. This study provided insights into the antiviral activities and stabilities of the unmodified and chemically modified peptides in human serum.

Impact of Trisulfide on the Structure and Function of Different Antibody Constructs.

Trisulfide is a post-translational modification (PTM) commonly found in recombinant antibodies. It has been demonstrated that trisulfide had no impact on the bioactivity of mono-specific antibodies (MsAbs). However, the impact of trisulfide on multi-specific antibodies has not been evaluated. In this study, two mass spectrometric methods were developed for comprehensive trisulfide characterization. The non-reduced peptide mapping method combined with the unique electron activated dissociation (EAD) provided signature fragments for confident trisulfide identification as well as trisulfide quantitation at individual sites. A higher throughput method using Fab mass analysis was also developed and qualified to support routine monitoring of trisulfide during process development. Fab mass analysis features simpler sample preparation and shorter analysis time but provides comparable results to the non-reduced peptide mapping method. In this study, a bi-specific (BsAb) and a tri-specific antibody (TsAb) were compared side-by-side with a MsAb to evaluate the impact of trisulfide on the structure and function of multi-specific antibodies. Results indicated that trisulfide dominantly formed at similar locations across different antibody constructs and had no impact on the size heterogeneity, charge heterogeneity, or bioactivities of any assessed antibodies. Together with the in vitro stability under heat stress (25 °C and 40 °C for up to four weeks) and rapid conversion from trisulfide to disulfide during in vivo circulation, trisulfide could be categorized as a non-critical quality attribute (non-CQA) for antibody products.

Investigation of High Molecular Weight Size Variant Formation in Antibody-Drug Conjugates: Microbial Transglutaminase-Mediated Crosslinking.

Microbial transglutaminase (mTG) has become a powerful tool for manufacturing antibody-drug conjugates (ADCs). It enables site-specific conjugation by catalyzing formation of stable isopeptide bond between glutamine (Q) side chain and primary amine. However, the downstream impact of mTG-mediated conjugation on ADC product quality, especially on high molecular weight (HMW) size variant formation has not been studied in a systematic manner. This study investigates the mechanisms underlying the formation of HMW size variants in mTG-mediated ADCs using size exclusion chromatography (SEC) and liquid chromatography-mass spectrometry (LC-MS). Our findings revealed that the mTG-mediated glutamine and lysine (K) crosslinking is the primary source of the increased level of HMW size variants in the ADCs. In the study, two monoclonal antibodies (mAbs) with glutamine engineered for site-specific conjugation were used as model systems. Based on the LC-MS analysis, a single lysine (K56) in the heavy chain (HC) was identified as the major Q-K crosslinking site in one of the two mAbs. The HC C-terminal K was observed to crosslink to the target Q in both mAbs. Quantitative correlation was established between the percentage of HMW size variants determined by SEC and the percentage of crosslinked peptides quantified by MS peptide mapping. Importantly, it was demonstrated that the level of HMW size variants in the second ADC was substantially reduced by the complete removal of HC C-terminal K before conjugation. The current work demonstrates that crosslinking and other side reactions during mTG-mediated conjugation needs to be carefully monitored and controlled to ensure process consistency and high product quality of the final ADC drug product.

The effect of Gamma and Ethylene Oxide Sterilization on a Selection of Active Pharmaceutical Ingredients for Ophthalmics.

Ensuring the sterility of life science products plays a pivotal role in the healthcare sector. Gamma irradiation and ethylene oxide sterilization are two commonly applied methods for the sterilization of medical devices, packaging components and Active Pharmaceutical Ingredients (API) for medicinal products. Focussed studies on the effects of sterilization processes on APIs remain limited. In this research study, five APIs, frequently used in sterile ophthalmic preparations were subjected to both gamma irradiation and ethylene oxide under different process conditions. The following APIs of GMP quality were selected: dexamethasone, aciclovir, tetracycline hydrochloride, triamcinolone and methylprednisolone. Analyses were performed using High Performance Liquid Chromatography equipped with UV detection and the effect of sterilization conditions on the APIs was evaluated by the assay and related substances test prescribed by the European Pharmacopoeia (Ph. Eur.). It was concluded that exposure to ethylene oxide resulted in compliance with Ph. Eur. for all APIs. While dexamethasone and methylprednisolone did not meet the requirement for the Ph. Eur. after exposure to gamma irradiation, the other three APIs did meet the requirement under the specified irradiation conditions. Subsequent optimization of sterilization parameters positively influenced the compliance to the Ph. Eur. requirements.

Development of Protein-Specific Analytical Methodologies to Evaluate Compatibility of Recombinant Human (rh)IGF-1/rhIGFBP-3 with Intravenous Medications Co-Administered to Neonates.

The protein complex of recombinant human insulin-like growth factor-1 and insulin­like growth factor binding protein­3 (rhIGF-1/rhIGFBP-3; mecasermin rinfabate), is an investigational product for the prevention of complications of prematurity. Delivery of rhIGF-1/rhIGFBP-3 is by continuous central line intravenous infusion in preterm infants until endogenous IGF-1 production begins. Protein-specific analytical methodologies were developed to evaluate the compatibility of rhIGF-1/rhIGFBP-3 at low protein concentrations (∼2.5-10 µg/mL) expected when co-administered with other required medications in the NICU. Highly sensitive detection of the biologic potential degradants (fragments) and/or molecular modifications (oxidized species, aggregates) required the use of reversed-phase high-performance liquid chromatography and size-exclusion ultra-performance liquid chromatography coupled with mass spectrometric detection. We report on the quantification of rhIGF-1/rhIGFBP-3, its components and degradants, to a limit of quantitation of 3.1 µg/mL upon mixing with 24 commonly administered neonatal medications. Methods developed for the rhIGF-1/rhIGFBP-3 admixtures, optimized in studies with furosemide, caffeine citrate and ampicillin, demonstrated good reproducibility, linearity, and limit of detection/quantitation. Using these methods, no increase in degradation of rhIGF-1/rhIGFBP-3 components and no increase in oxidation or aggregation level was observed with caffeine citrate, while admixtures of rhIGF-1/rhIGFBP-3 with ampicillin yielded lower mass recovery of rhIGF-1/rhIGFBP-3 components, which likely resulted from adduct formation. Furosemide was found to be physically incompatible with rhIGF-1/rhIGFBP-3. Our findings support the use of these methodologies for detection of protein modifications under various clinical administration conditions, and additionally supplement physical compatibility data studies of ultra-low concentrations of rhIGF-1/rhIGFBP-3 post co-administration to preterm infants with other medications (manuscript in-preparation).

Best Practices for Aggregate Quantitation of Antibody Therapeutics by Sedimentation Velocity Analytical Ultracentrifugation.

Analytical ultracentrifugation (AUC) is a critical analytical tool supporting the development and manufacture of protein therapeutics. AUC is routinely used as an assay orthogonal to size exclusion chromatography for aggregate quantitation. This article distills the experimental and analysis procedures used by the authors for sedimentation velocity AUC into a series of best-practices considerations. The goal of this distillation is to help harmonize aggregate quantitation approaches across the biopharmaceutical industry. We review key considerations for sample and instrument suitability, experimental design, and data analysis best practices and conversely, highlight potential pitfalls to accurate aggregate analysis. Our goal is to provide experienced users benchmarks against which they can standardize their analyses and to provide guidance for new AUC analysts that will aid them to become proficient in this fundamental technique.

Dosing Recommendations Based on Population Pharmacokinetics of Lamotrigine in Mexican Adult Patients With Epilepsy.

Dose individualization is essential in epilepsy treatment, especially in antiepileptic drugs that present high interindividual variability such as lamotrigine. We aimed an observational study to develop a population pharmacokinetic model for quantitative evaluation of the factors that influence lamotrigine pharmacokinetics in Mexican adults with epilepsy. Patients on stable treatment with lamotrigine therapy were included, plasma concentrations were analyzed by a high-performance liquid chromatography method and UGT2B7-161C > T polymorphism was determined. The data were analyzed by NONMEM® 7.3, model validation was performed using bootstrap approach and visual predictive check. Finally, stochastic simulations were carried out to propose dosage regimens. A total of 73 lamotrigine plasma concentrations from 2 h after last dose and up to 0.5 h prior to next administration were fitted to a one-compartment open model. The final population pharmacokinetic model for lamotrigine indicates that concomitant treatment with valproic acid and carbamazepine should be considered to individualize epilepsy treatment with this drug. Based on this model, we proposed dosage regimens to achieve trough lamotrigine concentrations within reference interval (2.5-15 mg/L). These results provide clinical useful data to give more rational anticonvulsant therapy in our population.

Controlled polysorbate 20 hydrolysis - A new approach to assess the impact of polysorbate 20 degradation on biopharmaceutical product quality in shortened time.

Hydrolysis of polysorbate in biopharmaceutical liquid formulations upon long-term storage represents a risk factor, since reduction of the intact surfactant concentration may compromise protein stability. Moreover, accumulation of polysorbate degradation products is associated with the formation of particulates potentially affecting drug product stability and quality. These effects are conventionally assessed by real-time end-of-shelf life studies constituting an integral yet lengthy process of formulation development. To accelerate this procedure, we describe here a powerful tool to conduct shake stress studies based on the controlled hydrolysis of polysorbate 20 by beads-immobilized lipases. For this purpose, the production of stable, partially degraded material characterized by a representative presence of non-emulsifying degradants such as ethoxylated sorbitan and free fatty acids was monitored by state-of-the-art chromatographic methods ensuring realistic pharmaceutical conditions. Freeze-thaw, shaking and shipping stress studies of a mAb formulation did not only demonstrate that this approach is useful to determine the critical degradation level impairing drug product quality, but furthermore revealed significant differences in protective effects depending on the hydrolysis pattern. As these results emphasize, the outlined strategy may support formulation scientists to unveil the interrelationship between polysorbate hydrolysis products and stabilization of the active pharmaceutical ingredient in a holistic and time-saving manner.

Simultaneous Retrodialysis by Drug for Cefuroxime Using Meropenem as an Internal Standard-A Microdialysis Validation Study.

Microdialysis is a valuable pharmacokinetic tool for obtaining samples of drug concentrations from tissues of interest. When an absolute tissue concentration is needed, a calibration of the microdialysis catheter is required. The use of an internal standard offers a number of advantages compared to standard calibration methods. However, meticulous validation both in vitro and in vivo is needed, as this method requires an internal standard with physiochemical similarities to the analyte of interest with no interference. A series of in vitro and in vivo setups were conducted to determine the relative recovery by gain and by loss for cefuroxime, with and without a constant meropenem concentration. The cefuroxime and meropenem concentrations were determined using ultra-HPLC. The main finding was that cefuroxime and meropenem relative recovery behaved similarly both in vitro and in vivo, signifying that meropenem is a representative internal standard for cefuroxime. Furthermore, cefuroxime relative recovery in vitro was not affected by either the cefuroxime concentration or the presence of meropenem, and the in vivo meropenem relative recovery was constant over 6 h.

Characterization of MB-102, a New Fluorescent Tracer Agent for Point-of-Care Renal Function Monitoring.

MB-102 is a fluorescent tracer agent designed for measurement of point-of-care glomerular filtration rate (GFR) and is currently in clinical studies. MB-102 possesses a strong UV absorbance at 266 nm and 435 nm, and broad fluorescent emission at ~560 nm when excited at ~440 nm. The MB-102 formulation is stable at 2°C-8°C for >3 years. The pKa's of the 2 acid groups are 2.71 and 3.40. Both X-ray crystallography and HPLC confirmed the D, D chirality of MB-102 in solid, in solution, and in the drug formulation. Initial safety and toxicity was published previously [Bugaj and Dorshow, 2015], which enabled the commencement of clinical studies. In vitro studies showed that 4.1% of MB-102 is bound to human plasma proteins, compared to 6.0% for the accepted standard GFR agent iohexol. The blood-to-plasma ratio for MB-102 was 0.590, illustrating minimal distribution of MB-102 into red blood cells. The manufacture of MB-102 under good manufacturing practice yields the designed molecular structure at high purity (>95% wt/wt).

Bioavailability Enhancement of Polymyxin B With Novel Drug Delivery: Development and Optimization Using Quality-by-Design Approach.

Polymyxin-B (Poly-B) is an effective antibiotic used to treat infections mainly caused due to sensitive gram-negative bacteria. They belong to the group of cyclic peptide antibiotics and are minimally absorbed from the gastrointestinal tract. This arises the need for bioavailability enhancement and is achieved in the present case using niosomes as carrier system. The Poly-B niosomes had been developed using Span 60 and cholesterol while optimization is achieved with quality-by-design (QBD) approach. In this QBD approach, 3 independent variables (Span 60:cholesterol, volume of phosphate-buffered saline [%], and amount of drug [mg]) each at 3 levels were studied. A total of 17 runs were suggested by the model which was further analyzed by optimizing 3 different responses (particle size, zeta potential, and entrapment efficiency [EE%]). The results had clearly shown that the optimum formulation selected by QBD was based on the criteria of attaining the maximum value of EE% and low value of size and zeta potential. Poly-B niosomes were further examined by in vitro antifungal, rat creatinine, and cytotoxicity assay. The pharmacokinetics and scintigraphy studies were also performed for in vivo behavior of Poly-B.

Factors Influencing Polysorbate's Sensitivity Against Enzymatic Hydrolysis and Oxidative Degradation.

The aim was to compare the sensitivity of different grades of polysorbate 20 (PS20) and polysorbate 80 (PS80) against enzymatic hydrolysis and oxidative degradation in pharmaceutically relevant buffer systems. For this purpose, a fast liquid chromatography charged aerosol detection method was developed which allows to (1) differentiate between hydrolytic and oxidative PS degradation and (2) to monitor the PS decay over time. Systematic enzymatic and oxidative forced degradation studies were conducted with multicompendial PS20 and PS80, as well as all-laurate PS20 and all-oleate PS80 (with >98% oleic acid, as required by the Chinese Pharmacopoiea since 2015). No differences in the sensitivity toward enzymatic degradation were observed between multicompendial PS and high purity grade PS. However, all-laurate PS20 and all-oleate PS80 have a higher predisposition for oxidative degradation as compared to multicompendial PS20 and PS80. The buffer system used within the study played thereby a key role: histidine showed a protective effect against hydrogen peroxide-induced oxidation, whereas hydrogen peroxide oxidation of PS in acetate buffer was severe under the experimental conditions. Furthermore, ethylenediaminetetraacetic acid protected PS20 and PS80 against oxidative degradation in histidine buffer.

Oxidation-Induced Destabilization of Model Antibody-Drug Conjugates.

Oxidation of biopharmaceutics represents a major degradation pathway, which may impact bioactivity, serum half-life, and colloidal stability. This study focused on the quantification of oxidation and its effects on structure and colloidal stability for a model antibody and its lysine (ADC-L) and cysteine (ADC-C) conjugates. The effects of oxidation were evaluated by a forced degradation study using H2O2 and a shelf-life simulation, which used degrading polysorbate 80 as source for reactive oxygen species. Differential scanning fluorimetry revealed decreasing transition temperatures of the CH2 domain with rising oxidation, resulting in a loss of colloidal stability as assessed by size-exclusion high pressure liquid chromatography. The conjugation technique influences structural changes of the monoclonal antibody (mAb) and subsequently alters the impact of oxidation. ADC-C was most effected by oxidation as the CH2 domain showed the biggest destabilization on conjugation compared to the mAb and ADC-L. Quantification of Fc methionine oxidation by analytical protein A chromatography revealed 4-fold higher oxidation after 8 weeks for the ADC-C compared to the mAb. Payload degradation was observed independently of the conjugation technique used or if free in solution by ultraviolet-visible. In addition, adding antioxidants can be a suitable approach to prevent oxidation and achieve baseline stabilization of the proteins.

Impacting Global Health Through Equipment Repurposing: Measurable Progress Through the Accumulation of Many Tiny Victories.

There is an active and growing effort occurring in laboratories throughout Africa to research the underpinnings of endemic communicable diseases, many of which are considered "neglected tropical diseases" as defined by the World Health Organization. Across the continent, scientists, doctors, health care workers, and students investigate the in vitro activity of pharmacologically active extracts against known pathogens in hope of discovering new treatments for the diseases that affect the local population. During the summer of 2014, I had the opportunity to visit laboratories in 3 different countries engaged in this area of research through participation in the Merck Fellowship for Global Health (Merck is known as Merck, Sharp & Dohme outside of the United States and Canada.), in which Merck sponsors employees on a short-term sabbatical to work with a global health-focused nonprofit organization. This commentary describes the objectives of the fellowship program, the specific project to which my co-fellow and I contributed, and the story of a subsequent equipment donation effort that was inspired by my individual fellowship experience. It also captures a few of the more notable challenges and opportunities for the scientists in the laboratories we visited. Finally, for the reader who may be curious as to how she or he can contribute, I hope to move you to action by highlighting some of the opportunities for researchers to positively and creatively impact global health from their "home" lab benches and hoods.

A stability-study of expired ampoules manufactured more than 40 years ago.

Pharmaceutical manufacturers have to study the stability of drug products before marketing according to ICH guideline Q1A(R2); data of those investigations aim to set expiry dates. The expiry date on the container of a remedy assures the physician and the patient a stability of the drug in its formulation i.e. within a specification of 95-105%. Only few studies show that shelf-lives of pharmaceutical products are often longer than expiration dates. The objective of the study presented here was determining the content of nine expired ampoules manufactured in the last century and identifying the impurity profile by means of HPLC-UV and HPLC-MS, respectively. The ampoules are part of the "PEAK-collection" of long expired finished pharmaceutical products at IBMP, Nürnberg-Heroldsberg, and consists among others of epinephrine (Suprarenin and Adrenalin in Oil), etilefrine (Effortil®), synephrine (Sympatol®), caffeine and procaine (Impletol), caffeine and sodium salicylate (Caffeinum Salicylicum), dipyridamole (Persantin®), furosemide (Lasix®), and metamizole (Novalgin®). For chromatographic investigations methods of the European Pharmacopoeia for related substances were used; for determining the content, they were validated for linearity, precision, and accuracy. The results were compared to current reference ampoules. Five out of nine ampoules were still within the specified content limits. In Suprarenin and Adrenalin in Oil, both containing epinephrine, Impletol (procaine), and Persantin® (dipyridamole) contents were decreased to 70%, 74%, 79%, and 86%, respectively, and therefore out of specification.

Impact of Experimental Variables on the Protein Binding of Tigecycline in Human Plasma as Determined by Ultrafiltration.

Tigecycline, a tetracycline derivative, shows atypical plasma protein binding behavior. The unbound fraction decreases with increasing concentration at therapeutic concentrations. Moreover, uncertainty exists about the magnitude of tigecyline's protein binding in man. Unbound fractions between 2.5% and 35% have been reported in plasma from healthy volunteers, and between 25% and 100% in patients, respectively. In the present study, the protein binding of tigecycline has been investigated by ultrafiltration using different experimental conditions. Whereas temperature had only a marginal influence, the unbound fraction at 0.3/3.0 mg/L was low at pH 8.2 (9.4%/1.9%) or in unbuffered pooled plasma (6.3%/1.2%), compared with plasma buffered with HEPES to pH 7.4 (65.9%/39.7%). In experiments with phosphate buffer and EDTA, the concentration dependency was markedly attenuated or abolished, which is compatible with a cooperative binding mechanism involving divalent cations such as calcium. The unbound fraction in clinical plasma samples from patients treated with tigecycline was determined to 66.3 ± 13.7% at concentrations <0.3 mg/L compared with 41.3 ± 16.0% at >1 to <5 mg/L. To summarize, tigecycline appears to be only moderately bound to plasma proteins as determined by ultrafiltration, when a physiological pH is maintained.

In Vitro Evaluation of Nasogastric Tube Delivery Performance of Esomeprazole Magnesium Delayed-Release Capsules.

Enteral feeding tubes are used to deliver food or drugs to patients who cannot swallow. To deliver delayed-release drugs that are formulated as enteric coated granules to these patients via feeding tubes requires that they be suspended in water before administration. Importantly, the suspension of enteric granules in water of varying pH can cause damage to the enteric coating and affect the bioavailability of the drug. Here, analytical methods for testing acid resistance stability and particle size distribution (PSD) of esomeprazole granules were used to monitor the integrity of the granule enteric coating after water pretreatment and delivery through an oral syringe and nasogastric (NG) tube. Granules from esomeprazole magnesium delayed-release capsules were transferred to an oral syringe, suspended in water, and delivered on the bench through an NG tube. Subsequently, acid resistance stability (i.e., the amount of drug released after 2-h acid dissolution) was determined via high-performance liquid chromatography, and the PSD were measured with a laser diffraction system. All the granules demonstrated acid resistance stability when the granules were delivered immediately (0 min incubation) through the oral syringe and NG tube. In contrast, some granules demonstrated significant drug release during acid exposure after a 15-min incubation period which mimics a possible delay in delivery of the drug from the syringe by the caregiver. A bimodal PSD was observed with these granules, which was attributed to debris from damaged enteric coating and particle agglomeration. The methods developed in this study could be used to distinguish batches with suboptimal product quality for delivery using NG tubes and to confirm the substitutability of generic drug products for this alternative route of administration.