Supercritical fluid extraction-supercritical fluid chromatography of saliva: Single-quadrupole mass spectrometry monitoring of caffeine for gastric emptying studies†.

Saliva is an attractive sampling matrix for measuring various endogenous and exogeneous substances but requires sample treatment prior to chromatographic analysis. Exploiting supercritical CO2 for both extraction and chromatography simplifies sample preparation, reduces organic solvent consumption, and minimizes exposure to potentially infectious samples, but has not yet been applied to oral fluid. Here, we demonstrate the feasibility and benefits of online supercritical fluid extraction coupled to supercritical fluid chromatography and single-quadrupole mass spectrometry for monitoring the model salivary tracer caffeine. A comparison of 13 C- and 32 S-labeled internal standards with external standard calibration confirmed the superiority of stable isotope-labeled caffeine over nonanalogous internal standards. As proof of concept, the validated method was applied to saliva from a magnetic resonance imaging study of gastric emptying. After administration of 35 mg caffeine via ice capsule, salivary levels correlated with magnetic resonance imaging data, corroborating caffeine's usefulness as tracer of gastric emptying (R2  = 0.945). In contrast to off-line methods, online quantification required only minute amounts of organic solvents and a single manual operation prior to online bioanalysis of saliva, thus demonstrating the usefulness of CO2 -based extraction and separation techniques for potentially infective biomatrices.

Design and Optimization of a Novel Strategy for the Local Treatment of Helicobacter pylori Infections.

Infections with Helicobacter pylori are a global challenge. Currently, H. pylori infections are treated systemically, but the eradication rates of the different therapy regimens are declining due to the growing number of bacterial strains resistant to major antibiotics. Here, we present a strategy for the local eradication of H. pylori by the use of Penicillin G sodium (PGS). In vitro experiments revealed that PGS shows high antibiotic activity against resistant strains of Helicobacter pylori with a minimum inhibitory concentration (MIC) of 0.125 µg/ml. In order to provide luminal concentrations above the MIC for longer periods of time, an extended release tablet was developed. Alkalizers were included to prevent acidic degradation of PGS within the tablet matrix. Out of the tested alkalizers MgO, l-Lysine, NaHCO3, and Na2CO3 NaHCO3 provided the strongest rise in pH inside the hydrated matrix when tested in simulated gastric fluid. Better PGS stability can mainly reasoned from that, addition of MgO resulted in high pH values within the matrix, causing basic degradation of PGS. This work is a first step towards the use of extended release tablets containing PGS for the local treatment of H. pylori as a safe and cost-effective alternative to common systemic treatment regimens.

Application of the GastroDuo to study the interplay of drug release and gastric emptying in case of immediate release Aspirin formulations.

The process of gastric emptying is of major importance for the in vivo performance of immediate release dosage forms. In the fed state, this process consists of two phases: the rapid emptying of water along the "Magenstrasse" and the continuous emptying of the chyme. The relevance of these phases for the pharmacokinetic (PK) profile of a drug depends on the release behavior from its dosage form. It was the aim of this study to investigate the role of gastric emptying for the pharmacokinetics of a fast disintegrating and dissolving Aspirin® tablet (FDDT). For this purpose, a three way pharmacokinetic study with 30 healthy volunteers was performed to investigate the performance of the FDDT under fasted and fed conditions and compare it to a regular Aspirin® tablet (RT) administered in the fed state. Plasma samples were taken at predetermined time points and analyzed by LC MS/MS. In the second part of this work, both products were tested in a biorelevant dissolution test device - the GastroDuo. To simulate the occurrence of the Magenstrasse at different time points, two test programs have been applied. The results of the PK study clearly demonstrated the superiority of the FDDT over the RT. We observed an earlier tmax (0.39 h vs. 2.00 h) and a higher Cmax (6.33 ± 2.37 µg/mL vs. 3.23 ± 1.28 µg/mL), whereas the AUC was only slightly different between both formulations. The administration of the FDDT together with food had no marked effect on tmax (0.34 h vs. 0.39 h), but caused a decrease in Cmax compared to fasted intake (14.76 ± 4.81 µg/mL vs. 6.33 ± 2.37 µg/mL). This effect could be explained by the in vitro data collected with the GastroDuo. The FDDT showed a faster drug release and improved emptying kinetics in the GastroDuo. In contrast, the RT showed incomplete emptying in both test programs. Thus, the early tmax observed for the FDDT under fed conditions could be related to the presence of the Magenstrasse. In contrast, drug release from the RT was insufficient to allow gastric emptying via the Magenstrasse, which resulted in later tmax. This study highlighted the importance of gastric emptying for immediate release dosage forms and illustrated that the application of suitable formulation techniques provides a strategy to generate a fast and reliable onset of drug plasma concentrations even in the fed state.

Comparison of In Vitro and In Vivo Results Using the GastroDuo and the Salivary Tracer Technique: Immediate Release Dosage Forms under Fasting Conditions.

The fasted state administration of immediate release (IR) dosage forms is often regarded as uncritical since physiological aspects seem to play a minor role for disintegration and drug release. However, recent in vivo studies in humans have highlighted that fasted state conditions are in fact highly dynamic. It was therefore the aim of this study to investigate the disintegration and drug release behavior of four different IR formulations of the probe drug caffeine under physiologically relevant conditions with the aid of the GastroDuo. One film-coated tablet and three different capsule formulations based on capsule shells either made from hard gelatin or hydroxypropylmethyl cellulose (HPMC) were tested in six different test programs. To evaluate the relevance of the data generated, the four IR formulations were also studied in a four-way cross-over study in 14 healthy volunteers by using the salivary tracer technique (STT). It could be shown that the IR formulations behaved differently in the in vitro test programs. Thereby, the simulated parameters affected the disintegration and dissolution behavior of the four IR formulations in different ways. Whereas drug release from the tablet started early and was barely affected by temperature, pH or motility, the different capsule formulations showed a longer lag time and were sensitive to specific parameters. However, once drug release was initiated, it typically progressed with a higher rate for the capsules compared to the tablet. Interestingly, the results obtained with the STT were not always in line with the in vitro data. This observation was due to the fact that the probability of the different test programs was not equal and that certain scenarios were rather unlikely to occur under the controlled and standardized conditions of clinical studies. Nonetheless, the in vitro data are still valuable as they allowed to discriminate between different formulations.

Application of the GastroDuo as an in Vitro Dissolution Tool To Simulate the Gastric Emptying of the Postprandial Stomach.

In the postprandial stomach, processes such as secretion, digestion, and gastric emptying all occur simultaneously. Therefore, the system is highly heterogeneous and dynamically changing, for instance, in terms of various physicochemical parameters such as pH value or viscosity. Thus, the administration of a drug together with food can result in highly variable drug plasma concentrations, which may affect the efficacy and safety of the pharmacotherapy. In this work, the pharmacokinetic (PK) data obtained from two fed-state bioequivalence studies with the immediate release (IR) drug products Viagra (sildenafil) and Adenuric (febuxostat) have been analyzed. This evaluation revealed that basically three characteristic types of onset behaviors of drug plasma concentration can be distinguished. It was hypothesized that the different types of onset behaviors were mainly caused by the interplay between gastric drug dissolution and gastric emptying. To study this interplay in vitro, a biopredictive dissolution tool-GastroDuo-was developed and used for both drug products. Therefore, three different test programs have been applied to simulate certain aspects of the postprandial human stomach, which included dynamic pH changes, gastric peristalsis, and the kinetics of gastric emptying. Specifically, the behavior of noncaloric fluids by the so-called "Magenstrasse" was taken into deeper consideration. The experiments revealed that the dissolution and emptying behavior of the two drug products were affected in different ways by the three test programs. The in vitro data nicely explained the tendencies of the drug products for certain types of onset behaviors observed in the PK data. While Viagra was strongly affected by simulated peristalsis, Adenuric was more sensitive to the simulated emptying kinetics. This work clearly demonstrated the important role of gastric fluid emptying for the onset of drug plasma concentration after oral administration of IR formulations in the fed state. Moreover, this was the first study in which GastroDuo was applied as a biopredictive in vitro model which is able to simulate crucial parameters of the human stomach (e.g., pH profiles and gastric emptying) in a realistic manner.

In vivo characterization of enTRinsic™ drug delivery technology capsule after intake in fed state: A cross-validation approach using salivary tracer technique in comparison to MRI.

The instability of various small molecules, vaccines and peptides in the human stomach is a complex challenge for oral drug delivery. Recently, a novel gastro-resistant capsule - the enTRinsic™ Drug Delivery Technology capsule - has been developed. In this work, the salivary tracer technique based on caffeine has been applied to study the in vivo disintegration of enTRinsic™ capsules in 16 healthy volunteers. In addition, magnetic resonance imaging (MRI) was used to visualize GI transit and to verify the disintegration times determined by using the salivary tracer technique. The enTRinsic™ capsules filled with 50mg of caffeine and 5mg of black iron oxide were administered in the fed state, i.e. 30min after a light meal (500kcal). In the first hour after capsule intake, the subjects were placed in supine position in the MRI scanner and scans were performed in short time intervals. After 1h, the subjects could leave the MRI scanner in between the MRI measurements, which were performed every 15min until disintegration of the capsule was confirmed (maximum observation time: 8h). Saliva samples were obtained simultaneously with MR imaging. Caffeine concentrations in saliva were determined by LC/MS-MS. The starting point of capsule disintegration was determined visually by inspection of the MR images as well as by the onset of salivary caffeine concentrations. In 14 out of 16 subjects, the capsule disintegrated in the small intestine. In one subject, the enTRinsic™ capsule was not emptied from the stomach within the observation time. In another subject, disintegration occurred during gastric emptying in the antropyloric region. In this study, we demonstrated that the enTRinsic™ capsules are also gastro resistant when taken under fed state conditions. Furthermore, we demonstrated the feasibility of using low dose caffeine as a salivary tracer for the determination of the disintegration of an enteric formulation.

Combined Application of MRI and the Salivary Tracer Technique to Determine the in Vivo Disintegration Time of Immediate Release Formulation Administered to Healthy, Fasted Subjects.

The process of disintegration is a crucial step in oral drug delivery with immediate release dosage forms. In this work, the salivary tracer technique was applied as a simple and inexpensive method for the investigation of the in vivo disintegration time of hard gelatin capsules filled with caffeine. The disintegration times observed with the salivary tracer technique were verified by magnetic resonance imaging (MRI). After an overnight fast of at least 10 h and caffeine abstinence of minimum 72 h, conventional hard gelatin capsules containing 50 mg caffeine and 5 mg iron oxide were administered to 8 healthy volunteers. For the period of 1 h after capsule intake, subjects were placed in supine position in the MRI scanner, and scans were performed in short time intervals. Each MRI measurement was directly followed by saliva sampling by drooling. Salivary caffeine concentrations were determined by high performance liquid chromatography followed by mass spectrometric detection (LC/MS-MS). The time point of capsule disintegration was determined by visual inspection of the MR images as well as by an increase in the salivary caffeine concentration. The results indicated that the difference in mean disintegration times of the capsules as determined by the two in vivo methods was around 4 min (8.8 min for MRI vs 12.5 min for saliva). All disintegration times determined by the salivary tracer technique were slightly higher. This delay could be explained by the fact that the appearance of caffeine in saliva required drug absorption in the small intestine. Because capsule disintegration happened mainly in the stomach, the exact site of disintegration as well as the processes of gastric mixing and gastric emptying contributed to the delay between the two methods. This work demonstrated the feasibility of the salivary tracer technique to investigate the in vivo disintegration of immediate release dosage forms in a simple and reliable manner.

Low dose caffeine as a salivary tracer for the determination of gastric water emptying in fed and fasted state: A MRI validation study.

Improving our knowledge about human gastrointestinal physiology and its impact on oral drug delivery is crucial for the development of new therapies and effective drug delivery systems. The aim of this study was to develop an in vivo tool to determine gastric emptying of water by administration of a caffeine as a tracer substance followed by subsequent saliva caffeine analysis. For this purpose, 35 mg of caffeine were given to six healthy volunteers after a 10 h overnight together with 240 mL of tap water either on a fasted stomach or 30 min after the high-caloric, high-fat breakfast recommended for bioavailability/bioequivalence (BA/BE) studies. Caffeine was administered in form of an ice capsule in order to omit the contamination of the oral cavity with caffeine. Parallel to saliva sampling, magnetic resonance imaging (MRI) was applied in order to validate this novel approach. After administration of the ice capsule, MRI measurements were performed every 2 min for the first 20 min followed by further measurements after 25, 30, 35, 40, 50 and 60 min. Saliva samples were collected always 1 min after the MRI measurement in supine position in the MRI scanner and continued for further 240 min. The caffeine concentration in saliva was quantified after liquid-liquid extraction by a validated HPLC/MS-MS method. The obtained MRI data revealed a fast emptying of the co-administered water within 10 to 50 min in the fasted state and likewise in the fed state. Salivary caffeine kinetics showed a Cmax from 150 to 400 ng/mL with a tmax from 20 to 90 min. MRI data were normalized by setting the maximum emptied volume to 100% and the salivary caffeine kinetics were normalized by setting Cmax to 100%. In order to compare the results obtained by the MRI and the saliva method, the normalized data for each volunteer was correlated based on a linear regression. In the fasted state the mean slope for six comparisons was 0.9114 ±â€¯0.1500 and the mean correlation coefficient was 0.912 ±â€¯0.055. In the fed state, a mean slope of 0.8326 ±â€¯0.1630 and a mean correlation coefficient of 0.887 ±â€¯0.047 were obtained. Based on these results, we could show that salivary caffeine concentrations are suitable to describe the emptying of water as a non-caloric liquid from the fasted and the fed stomach. The presented technique provides a straight-forward, inexpensive and noninvasive method to assess gastric emptying of hydrophilic liquids, which can be broadly used in oral biopharmaceutics. Possible applications are the characterization of real-life conditions, specific populations (e.g. elderly people) and the better understanding of the contribution of gastric emptying to pharmacokinetic profiles of orally administered drugs.

Effect of Coadministered Water on the In Vivo Performance of Oral Formulations Containing N-Acetylcysteine: An In Vitro Approach Using the Dynamic Open Flow-Through Test Apparatus.

The drug plasma profile after oral administration of immediate release dosage forms can be affected by the human gastrointestinal physiology, the formulation, and the drug itself. In this work, we investigated the in vivo and in vitro performance of two formulations (granules vs. tablet) containing the highly soluble drug N-Acetylcysteine (BCS class I). Thereby, special attention was paid to the effect of the dosage form and the coadministration of water on drug release. Interestingly, the in vivo results from a pharmacokinetic study with 11 healthy volunteers indicated that the drug plasma concentrations were comparable for the tablet given with water as well as for the granules given with and without water. In order to mechanistically understand this outcome, we used a biorelevant dissolution test device, the dynamic open flow-through test apparatus. With the aid of this test apparatus, we were able to simulate biorelevant parameters, such as gastric emptying, hydrodynamic flow as well as physical stress. By this, it was possible to mimic the intake conditions of the clinical trial (i.e., drug intake with and without water). Whereas the experiments in the USP paddle apparatus revealed differences between the two formulations, we could not observe significant differences in the release profiles of the two formulations by using the dynamic open flow-through test apparatus. Even by considering the different intake conditions, drug release was slow and amounted to around 30% until simulated gastric emptying. These results suggest that dissolution was irrespective of coadministered water and the formulation. Despite the high aqueous solubility of N-Acetylcysteine, the limiting factor for drug release was the slow dissolution rate in relation to the gastric emptying rate under simulated gastric conditions. Thus, in case of administration together with water, large amounts of the drug are still present in the stomach even after complete gastric emptying of the water. Consequently, the absorption of the drug is largely controlled by the nature of gastric emptying of the remaining drug. The data of this study indicated that the water emptying kinetics are only determining drug absorption if drug release is rapid enough. If this is not the case, physiological mechanisms, such as the migrating motor complex, play an important role for oral drug delivery.

Navigating the human gastrointestinal tract for oral drug delivery: Uncharted waters and new frontiers.

Many concepts of oral drug delivery are based on our comprehension of human gastrointestinal physiology. Unfortunately, we tend to oversimplify the complex interplay between the various physiological factors in the human gut and, in particular, the dynamics of these transit conditions to which oral dosage forms are exposed. Recent advances in spatial and temporal resolution of medical instrumentation as well as improved access to these technologies have facilitated clinical trials to characterize the dynamic processes within the human gastrointestinal tract. These studies have shown that highly relevant parameters such as fluid volumes, dosage form movement, and pH values in the lumen of the upper GI tract are very dynamic. As a result of these new insights into the human gastrointestinal environment, some common concepts and ideas of oral drug delivery are no longer valid and have to be reviewed in order to ensure efficacy and safety of oral drug therapy.