Stability-Indicating Analytical Approach for Stability Evaluation of Lactoferrin.

Lactoferrin is a multifunctional iron-binding glycoprotein in milk. Due to its potential for the treatment of various diseases, interest in products containing lactoferrin is increasing. However, as a protein, it is prone to degradation, which critically affects the quality of products. Therefore, the main purpose of our work was to develop a stability-indicating analytical approach for stability evaluation of lactoferrin. We were focused on two complementary methods: reversed-phase and size-exclusion chromatography. The stability-indicating nature of the selected methods was confirmed. They were successfully validated by following the ICH guidelines and applied to preliminary lactoferrin stability studies. Up to three degradation products, as well as aggregates and fragments of lactoferrin, were detected in various samples using complementary reversed-phase and size-exclusion chromatographic methods. The analytical approach was additionally extended with three spectroscopic techniques (absorbance, intrinsic fluorescence, and bicinchoninic acid method), which may provide valuable complementary information in some cases. The presented analytical approach allows the stability evaluation of lactoferrin in various samples, including the ability to detect differences in its degradation mechanisms. Furthermore, it has the potential to be used for the quality control of products containing lactoferrin.

Comprehensive Stability Study of Vitamin D3 in Aqueous Solutions and Liquid Commercial Products.

Vitamin D3 has numerous beneficial effects, such as musculoskeletal, immunomodulatory, and neuroprotective. However, its instability is the main obstacle to formulating quality products. Despite increased attention and growing use, data on vitamin D3 stability is scarce because data from individual studies is inconclusive and mostly qualitative. Therefore, we have systematically investigated the influence of various factors (temperature, light, oxygen, pH, concentration, and metal ions) on its stability in aqueous media using a stability-indicating HPLC-UV method. First-order kinetics fitted its degradation under all tested conditions except light and oxygen. In both cases, the established models in chemical kinetics were inappropriate and upgraded with the Weibull model. Metal ions and acidic conditions had the main destabilizing effect on vitamin D3 in aqueous media, but these solutions were successfully stabilized after the addition of ethylenediaminetetraacetic acid (EDTA), ascorbic acid, and citric acid, individually and in combination. EDTA showed the most significant stabilizing effect. Synergism among antioxidants was not observed. Our findings on vitamin D3 instability in aqueous media also correlated with its instability in commercial products. Vitamin D3 aqueous products require proper stabilization, thereby signifying the importance and contribution of the obtained results to the formulation of stable and quality products.

Stability of Reduced and Oxidized Coenzyme Q10 in Finished Products.

The efficiency of coenzyme Q10 (CoQ10) supplements is closely associated with its content and stability in finished products. This study aimed to provide evidence-based information on the quality and stability of CoQ10 in dietary supplements and medicines. Therefore, ubiquinol, ubiquinone, and total CoQ10 contents were determined by a validated HPLC-UV method in 11 commercial products with defined or undefined CoQ10 form. Both forms were detected in almost all tested products, resulting in a total of CoQ10 content between 82% and 166% of the declared. Ubiquinol, ubiquinone, and total CoQ10 stability in these products were evaluated within three months of accelerated stability testing. Ubiquinol, which is recognized as the less stable form, was properly stabilized. Contrarily, ubiquinone degradation and/or reduction were observed during storage in almost all tested products. These reactions were also detected at ambient temperature within the products' shelf-lives and confirmed in ubiquinone standard solutions. Ubiquinol, generated by ubiquinone reduction with vitamin C during soft-shell capsules' storage, may lead to higher bioavailability and health outcomes. However, such conversion and inappropriate content in products, which specify ubiquinone, are unacceptable in terms of regulation. Therefore, proper CoQ10 stabilization through final formulations regardless of the used CoQ10 form is needed.

Quantification of reduced and oxidized coenzyme Q10 in supplements and medicines by HPLC-UV.

Coenzyme Q10 (CoQ10) supplements are widely used because of their antioxidant and anti-inflammatory effects, especially in the management of cardiovascular diseases. The latest pharmaceutical approach to increase CoQ10 bioavailability and efficiency is the formulation of its reduced form. Regardless of the growing number and usage of CoQ10 preparations, their analytics and quality control is inadequate, neglecting interconversion between the two CoQ10 forms. Therefore, this study proposes a HPLC-UV method for the simultaneous quantification of both reduced and oxidized coenzyme Q10, as well as total CoQ10, as a sum of its individual forms. The suitability of the developed method was confirmed by two additional approaches for total CoQ10 determination - its total reduction and oxidation, differing from the proposed procedure only in the final stage of sample preparation. The results for total CoQ10 content were consistent between the three procedures and also with the official USP method for total CoQ10 determination. The proposed method was applied to 13 dietary supplements and medicines in the form of soft- and hard-shell capsules, revealing the co-existence of both CoQ10 forms in 85% of the tested preparations. CoQ10 forms that were undeclared accounted for up to 75% of the CoQ10 content, which is overlooked by current official methods that evaluate only the total CoQ10 content. This validated HPLC-UV method for the unequivocal quantification of reduced and oxidized CoQ10 is therefore appropriate for the routine analysis of CoQ10 preparations in quality control laboratories, as well as for stability studies, and is suggested to be adopted as an official method.

A self-microemulsifying drug delivery system to overcome intestinal resveratrol toxicity and presystemic metabolism.

A mixed lipid-mixed surfactant self-microemulsifying drug delivery system (SMEDDS) was developed to exploit the health benefits of resveratrol, a Biopharmaceutical Classification System Class 2 natural polyphenol, subject to extensive intestinal presystemic metabolism. SMEDDS with a mixed lipid phase (castor oil/Capmul MCM 1:1) and a mixed surfactant phase (Kolliphor EL/Kolliphor RH 40 1:1) was developed and evaluated for its self-emulsifying properties and in vitro dispersion. The impact of SMEDDS on the permeability properties of resveratrol and its metabolite fluxes through the rat intestine and Caco-2 cells was monitored. The inhibitory effect of selected SMEDDS components on the efflux transporters multidrug resistance-associated protein and P-gp as well as cytotoxicity was assessed on Caco-2 cells. The formulation allowed for high resveratrol loading (122.5 mg/g SMEDDS), excellent self-emulsifying properties, and very rapid release. When formulated in SMEDDS, resveratrol metabolite efflux significantly declined. The formulation (SMEDDS without incorporated resveratrol) and its individual components did not compromise in vitro cell vitality and integrity. Mixed lipid-mixed surfactant SMEDDS is a prospective formulation to improve resveratrol biopharmaceutical, pharmacokinetic, and toxicological properties, leading the way to resveratrol use not only as a supplement but also as a pharmacological drug.

The mechanisms responsible for garlic - drug interactions and their in vivo relevance.

Garlic phytochemicals and garlic supplements influence the pharmacokinetic and pharmacodynamic behavior of concomitantly ingested drugs. In this paper we have summarized the mechanisms responsible for first-pass intestinal pharmacokinetic interactions by investigating the intestinal permeability of some cardiovascular, antiviral drugs, their transport with hepatic transporters and CYP3A4 metabolism. Transporter-enzyme interplay was studied with several in vitro models of varying complexity: rat small intestine and Caco-2 cell monolayers were used in studies of intestinal processes, and hepatic pharmacokinetics was monitored in HepG2 cells, isolated rat hepatocytes and rat liver slices. Garlic phytochemicals from aged garlic extract modified the activities of secretory and absorptive transporters in both intestine and liver and competitively inhibited CYP3A4 enzyme. The increased activities of the most important intestinal efflux (P-glycoprotein - Pgp, Multidrug Resistance Associated Protein 2 - MRP-2, Breast Cancer Resistance Protein - BCRP) and uptake (MonoCarboxylate Transporter 1 - MCT1, Organic Anion Transporting Polypeptide - OATP, Peptide transporter 1 - PepT1) transporters were caused by changes in electrophysiological membrane properties and by allosteric modifications. Because clinical studies investigating interactions between garlic and human immunodeficiency virus protease inhibitors saquinavir and ritonavir have already been performed, we used these in vivo data to evaluate the in vitro results and the reliability of the models employed as screening tools for forecasting the potential of first-pass intestinal metabolism changes. We also assessed the probability of pharmacokinetic interactions with garlic of the novel drug darunavir and other cardiovascular drugs. Finally, selected garlic phytochemicals were tested for their ability to influence P-glycoprotein and CYP3A4 activities.

Bio-relevant media to assess drug permeability: sodium taurocholate and lecithin combination or crude bile?

The assessment of in vivo drug absorption with in vitro permeability models demands the use of transport media with surface acting compounds. With the aim to establish their influence on in vitro permeability of 30 drugs through Caco-2 monolayers, cell vitality/integrity and micellar drug entrapment, taurocholate/lecithin (NaTC/Leci) and pig crude bile were applied. Drug permeabilities were correlated to fraction of drugs absorbed and appropriate NaTC/Leci and bile concentrations were proposed to simulate fasted/fed conditions in vitro (bile in the concentration range 1-5 v/v% or 0.2/0.05mM NaTC/Leci for fasted; 10 v/v% bile or 3/0.75mM NaTC/Leci for fed conditions) without detrimental effects on monolayer integrity/vitality (NaTC/Leci was more toxic than bile). Surfactants exerted different affinities for drugs; free drug concentration (c(free)) of some was significantly lowered only by bile, while for the others NaTC/Leci and bile significantly diminished c(free). For some substances NaTC/Leci and bile significantly increased their permeabilities (i.e. more than 3-times) in spite of profound c(free) decrease indicating the existence of an alternative absorption mechanism. Based on these data, the impact of bile on in vitro drug permeability and micellar drug entrapment cannot be adequately simulated by NaTC/Leci, because their effects on drug absorption differ.

The effect of garlic supplements and phytochemicals on the ADMET properties of drugs.

INTRODUCTION: Garlic supplements have received wide public attention because of their health-beneficial effects. Although these products are considered as innocuous, several case reports and studies have shown the capacity of individual garlic phytochemicals/supplements to interfere with drug pharmacokinetics. AREAS COVERED: This review covers recently published literature on garlic chemistry and composition, and provides a thorough review of published studies evaluating drug-garlic interactions. The authors illustrate the mechanisms underlying pharmacokinetic interactions, which could serve as important highlights in further research to explain results for drugs with narrow therapeutic indices or for drugs, utilizing multiple absorption, distribution and metabolism pathways. EXPERT OPINION: To increase the relevance of further research on safety and efficacy of garlic supplements and phytochemicals, their composition should be addressed before conducting in vitro or in vivo research. It is also strongly recommended to characterize in vitro formulation performance to assess the rate and extent of garlic phytochemical release in order to anticipate the in vivo impact on the pharmacokinetics of concomitantly consumed drugs. The main conclusion of this review is that the impact of garlic on different stages of pharmacokinetics, especially on drug absorption and metabolism, is drug specific and dependent on the type/quality of utilized supplement.

Transwell-grown HepG2 cell monolayers as in vitro permeability model to study drug-drug or drug-food interactions.

HepG2 cell monolayers, formed during cell growth on collagen-coated Transwell® (Corning® Inc., Corning, NY, USA) inserts, can be used for the evaluation of interactions between food supplements and drugs that are substrates for P-glycoprotein (Pgp) and/or multidrug resistance-associated protein 2 (MRP-2). Samples obtained during such permeability studies were relatively free of intracellular proteins or cell debris compared to usually performed uptake experiments with HepG2 cells; therefore no special preparation protocol prior to the analysis was needed. In the presence of aged garlic extract the activities of hepatic efflux transporters (Pgp, MRP-2) changed, which was observed as significant permeability changes of tested human immunodeficiency virus (HIV) protease inhibitors. Darunavir efflux significantly increased, whereas that of saquinavir significantly decreased. Because of the observed in vitro interactions between aged garlic extract and HIV protease inhibitors (darunavir, saquinavir), any alterations of in vivo liver transport in the presence of garlic phytochemicals could also significantly influence darunavir/saquinavir hepatocyte intracellular concentrations and hence their bioavailabilities. Therefore this aspect needs further in vivo animal evaluation.

HIV protease inhibitors: garlic supplements and first-pass intestinal metabolism impact on the therapeutic efficacy.

BACKGROUND/AIMS: The aim of this study was to elucidate the impact of first-pass intestinal metabolism to therapeutic efficacy of antiretrovirals and to ascertain interaction mechanisms between garlic supplements (aged garlic extract) and HIV-protease inhibitors. METHODS: In vitro permeability through rat jejunum, Caco-2 cell monolayers was determined and CYP3A4 metabolism studies were performed. RESULTS: Saquinavir and darunavir efflux from enterocytes into gastrointestinal lumen significantly increased in the presence of aged garlic extract, whereas their CYP3A4 metabolism was inhibited. In the case of saquinavir a significant increase of its efflux was observed also in the presence of lower ritonavir doses. Because both drugs bound to different binding sites on P-glycoprotein and/or multidrug resistance associated protein 2 than garlic phytochemicals or ritonavir, lower amounts of antiretrovirals were absorbed. CONCLUSIONS: The fractions of tested anti-HIV drugs absorbed could decrease significantly during self-medication with garlic supplements or ritonavir dose adjustments. Due to distinct saquinavir and darunavir preferences for binding sites on efflux transporters, the presence of other compounds (garlic phytochemicals, ritonavir), capable of influencing intestinal transporter-enzyme interplay, might lead to pharmacokinetic interactions observed in clinical studies and case reports with anti-HIV drugs.

Garlic flavonoids and organosulfur compounds: impact on the hepatic pharmacokinetics of saquinavir and darunavir.

The therapeutic efficacy of saquinavir and darunavir is affected by the presence of xenobiotics (such as garlic compounds) capable of modifying transporter-enzyme interplay. To ascertain the mechanism of interactions between antiretroviral drugs and garlic supplements and to identify the garlic constituents responsible, the hepatic pharmacokinetics of two antiretroviral drugs was investigated in the presence of garlic phytochemicals and aged garlic extract. For this purpose, rat liver slices and isolated rat hepatocytes were used. Aged garlic extract significantly inhibited saquinavir efflux from rat hepatocytes, while the efflux of darunavir significantly increased. Phytochemicals inducing distribution changes of saquinavir and darunavir were most probably flavonoids and lipophilic organosulfur compounds, respectively. All tested phytochemicals (except S-allyl L-cysteine) and aged garlic extract inhibited CYP3A4 metabolism of both drugs and modulated hepatic distribution of the corresponding saquinavir and darunavir metabolites. The competition between saquinavir and garlic constituent(s) for the same binding site on the efflux transporter and the positive cooperative effect between darunavir and garlic phytochemical(s), which bind to separate binding places on transporter, are the most probable mechanisms explaining the plasma profile changes, which could occur in vivo during concomitant consumption of antiretrovirals and garlic supplements.

The influence of aged garlic extract on the uptake of saquinavir and darunavir into HepG2 cells and rat liver slices.

Bioavailability and therapeutic outcome of treatment with HIV-protease inhibitors depends on intestinal and hepatic transporter-enzyme interplay. Liver transport of HIV protease inhibitors (saquinavir, darunavir) was assessed in the presence of aged garlic extract, because the HIV-infected often consume garlic supplements together with prescribed therapy. The in vitro uptake of both drugs into HepG2 cells and precision cut rat liver slices significantly increased in the presence of Pgp and MRP-2 inhibitor ritonavir. The incubation medium containing aged garlic extract caused significant inhibition of saquinavir efflux from HepG2 cells and precision cut liver slices, while the activity of darunavir efflux transporters in both liver models significantly increased. Due to opposite in vitro interactions observed between aged garlic extract and HIV protease inhibitors, darunavir and saquinavir most probably bind to different binding sites on one or both efflux transporters. Based on this study, coadministration of investigated compounds with garlic supplements could result in significant in vivo modification of hepatic transport-enzyme interplay, possibly leading to further bioavailability change.

In vitro interactions between aged garlic extract and drugs used for the treatment of cardiovascular and diabetic patients.

BACKGROUND: Disease preventing effects gained by garlic consumption have been recognized since early period of history, making commercially available garlic supplements attractive to the general public. Possible pharmacokinetic interactions which could occur between applied drugs and aged garlic extract (AGE) are unknown. AIM: To test in vitro impact of some garlic phytochemicals on P-glycoprotein (Pgp), the most recognized efflux transporter, and the effect of AGE on passive membrane permeability, absorptive and secretory intestinal transporters. METHODS: Rat small intestine and Caco-2 cell monolayers, mounted in side-by-side diffusion chambers were used. RESULTS: Hydrophilic sulphur compounds increased Pgp mediated Rhodamine 123 (Rho123) efflux, whereas the lipophilic ones increased Pgp efflux through rat ileum but not through Caco-2 cell monolayers. Increased activities of secretory (Pgp, multidrug-resistance associated protein 2) and absorptive (monocarboxylate transporter 1, organic anion transporting polypeptide) transporters involved in drug absorption were observed in rat small intestine and Caco-2 cell monolayers in the presence of AGE. Transport of drugs mediated by breast cancer resistance protein and H(+)-oligopeptide transporter 1 was activated in rat intestine but inhibited through Caco-2 cells. Passive membrane permeability of tested compounds remained unaltered through rat small intestine, while significant changes were observed with Caco-2 cell monolayers. CONCLUSIONS: Due to the observed in vitro pharmacokinetic interactions between AGE and investigated cardiovascular, antidiabetic and antiviral drugs, in vivo absorption changes are possible, but the magnitude of change depends on the most profound process involved (influx, efflux, passive diffusion) in compounds permeability.

Physicochemical and preclinical pharmacokinetic and toxicological evaluation of LK-423--a new phthalimido-desmuramyl-dipeptide derivative with immunomodulating activity.

INTRODUCTION: LK-423 is a new phthalimido-desmuramyl-dipeptide derivative with immunomodulating activity. As optimized delivery to the site of action appears crucial for further preclinical development of LK-423, the aim of this study was to perform a physicochemical and preclinical pharmacokinetic and toxicological evaluation. METHODS: The solubility, partition coefficient, permeability, and stability profile were determined. Pharmacokinetics were evaluated in rats following intravenous and oral application of LK-423, and in dogs after intravenous administration and oral administration of microcapsules, designed for colon-specific delivery of LK-423 based on pH-, time-, and enzyme-controlled release mechanisms. Additionally, the acute and subchronic toxicity was examined. RESULTS AND DISCUSSION: LK-423 is hydrophilic, sparingly to slightly soluble, and poorly permeable. Stability profile in aqueous solution is pH dependent. A pharmacokinetic study following intravenous application to rats and dogs revealed that LK-423 is rapidly eliminated with a short terminal phase half-life, and high plasma clearance, as well as a limited distribution to the peripheral tissue. Oral bioavailability of LK-423 is low, presumably due to low permeability. Debris of insoluble microcapsule coating in feces and obtained plasma concentration profiles confirm that LK-423 microcapsules are a promising approach for local treatment of inflammatory diseases of the large intestine. Acute and a subchronic toxicity results indicate that LK-423 is a safe and nontoxic drug under the applied experimental conditions.

Aged garlic extract stimulates p-glycoprotein and multidrug resistance associated protein 2 mediated effluxes.

The growing concomitant consumption of drugs and herbal preparations such as garlic, and the numerous reports about the influence of herbal preparations on intestinal transport, led us to evaluate the influence of aged garlic extract on the transport function and electrophysiological parameters of the small intestinal mucosa. Aged garlic extract induced increase of the absolute value of the transepithelial potential difference and of the short-circuit current in both permeability models tested (rat jejunum, Caco-2 cell monolayers) without affecting transepithelial electrical resistance. It also caused a significant increase of the P-glycoprotein and multidrug resistance associated protein 2 mediated effluxes through rat jejunum of marker substrates Rhodamine 123 and 2,4-dinitrophenyl-S-glutathione, respectively. Rhodamine 123 efflux through the Caco-2 cell monolayers was not altered by aged garlic extract, whereas the efflux of 2,4-dinitrophenyl-S-glutathione increased significantly. So altered activity of the important transport proteins could significantly change the pharmacokinetic properties of conventional medicines taken concomitantly with aged garlic extract.