Guus Duchateau

Bile salts and intranasal drug absorption

Abstract The nasal route has been proven to be very effective for drug absorption. Bioavailability of intranasally administered drugs depends on the structure of the drug and can be as high as 100%. The absorption of drugs with a lower bioavailability can be improved with the aid of absorption promotors such as some surfactants, e.g. bile salts. The intranasal absorption of gentamicin, as a model drug, was studied in rabbits with six different bile salts as absorption promotors (cholate, taurocholate, glycocholate, deoxycholate, taurodeoxycholate and glycodeoxycholate). Without a surfactant, gentamicin is not absorbed by the nasal mucosa. The serum concentrations of gentamicin after intranasal administration were compared with those obtained after an intravenous injection. Concentrations were measured with EMIT. The bioavailability of the intranasal gentamicin in rabbits was related to the hydrophobicity and the pK a s of the bile salts. Furthermore the effects on ciliated epithelium of the six bile salts, used in the absorption experiment, were studied in an in vitro model. Ciliary activity was studied with a photo-electric method. Bioavailability increased with the increase of the hydrophobicity of the trihydroxy bile salts (cholate, taurocholate and glycocholate). Sodium cholate and sodium taurodeoxycholate were the most active absorption promotors (F = 41 ± 16%, respectively, 34 ± 13%). The dihydroxy bile salts (deoxycholate, taurodeoxycholate and glycodeoxycholate) showed a decreasing activity in the promotion of gentamicin absorption as the hydrophobicity increases. Depending on the pH/pK a relation, increasing hydrophobicity results in lower solubility and therefore decreasing activity. Ciliotoxicity of the bile salts increased with increasing hydrophobicity. Dihydroxy bile salts are more toxic than trihydroxy bile salts. At the used concentrations ciliary beat arrested within 30 min. Deoxycholate is extremely ciliotoxic, ciliary arrest occurred within 1 min at a concentration of 5 mmol/l.

Correlation between nasal ciliary beat frequency and mucus transport rate in volunteers

The correlation between mucus transport time (MTT) and nasal ciliary beat frequency (CBF) in human volunteers was investigated. Mucus transport was measured with the indigo carmine/saccharin sodium test. The test can be performed easily, with no need for sophisticated equipment. CBF was measured photometrically in biopsies from the ciliated epithelium of the nose.

Postprandial evolution in composition and characteristics of human duodenal fluids in different nutritional states.

The purpose of this study was to assess the changes in duodenal composition in three nutritional states: fasted, fed, and fat-enriched fed state. Two isocaloric meals were administered to healthy subjects on nonconsecutive days. Subsequently, duodenal samples were collected every 30 min after which they were characterized with respect to pH, lipolytic products, bile salts, phospholipids, osmolality, and surface tension. The resulting time profiles displayed fluctuating patterns, which reflect high inter- and intrasubject variability. Duodenal composition was not altered by the higher fat percentage of the fat-enriched liquid meal. Monoglycerides, amounting from 5% to 88% of total lipids, were the dominant lipolytic species, followed by free fatty acids. Within 30 min after meal administration, individual intraduodenal concentrations of lipid products were 0.0-5.5, 1.0-14.9, and 3.1-22.4 mg/mL in fasted, fed, and fat-enriched fed state, respectively. The corresponding values for bile salts were 2.0-9.0, 6.9-9.3, and 4.4-30.3 mM and for phospholipids 0.06-2.4, 2.6-5.7, and 1.4-9.3 mM, respectively. Specific trends though, were not detected. This study illustrates the variable intraluminal conditions that can result after food intake. As intraduodenal events (e.g., intraduodenal dissolution) affect absorption of poorly water soluble and/or highly lipophilic drugs, this variability may possibly contribute to the highly variable drug plasma-time profiles often observed.

The angiotensin converting enzyme inhibitory tripeptides Ile-Pro-Pro and Val-Pro-Pro show increasing permeabilities with increasing physiological relevance of absorption models

Transepithelial transport of the ACE inhibitory peptides Ile-Pro-Pro and Val-Pro-Pro was studied in different models of absorption. Apparent permeability (P(app)) values for absorptive transport across Caco-2 monolayers were 1.0+/-0.9 x 10(-8) (Ile-Pro-Pro) and 0.5+/-0.1 x 10(-8)cms(-1) (Val-Pro-Pro). Ex vivo transport across jejunal segments in the Ussing chamber was 5-times (Ile-Pro-Pro) to 10-times (Val-Pro-Pro) higher with no significant differences (p>0.05) observed between both peptides. The peptidase inhibitor bestatin increased permeability for the absorptive direction for Ile-Pro-Pro by twofold. Neither a transepithelial pH gradient nor increased apical tripeptide concentration nor longitudinal localization of the intestinal segment influenced P(app) in the ex vivo experiments. Val-Pro-Pro transport across Peyers patches, however, was 4-times higher (P(app)=21.0+/-9.3 x10(-8)cms(-1)) as compared to duodenum (P(app)=4.8+/-1.4 x 10(-8)cms(-1)). In the in situ perfusion experiments P(app) values varied greatly among different animals ranging from 0.5 to 24.0 x10(-8)cms(-1) (Ile-Pro-Pro) and from 1.0 to 15.6 x 10(-8)cms(-1) (Val-Pro-Pro). In summary, Caco-2 and ex vivo absorption models differ considerably regarding their peptide permeability. The in situ model seems to be less appropriate because of the observed large variability in peptide permeability. The results of this study demonstrate that the ACE inhibitory peptides Ile-Pro-Pro and Val-Pro-Pro are absorbed partially undegraded.

Current In Vitro Testing of Bioactive Peptides Is Not Valuable

Dietary peptides have been suggested to possess biological activity in vivo and could affect cardiovascular disease parameters, based on data derived from in vitro experiments. Isolated peptides are often tested in in vitro cellular assays or on heterologously expressed molecular target proteins. The stimulatory or inhibitory effect on target proteins in vitro has often been used as the justification to test these compounds directly in vivo. Unfortunately, this research approach has an inherent flaw. It neglects the poor absorption, distribution, metabolism, and excretion (ADME) properties of peptides resulting in low peptide bioavailability. Because peptides are prone to extensive hydrolysis in the gastrointestinal tract by stomach, small intestinal, and brush border peptidases, most of them do not reach the absorption stage in the duodenum and jejunum. Therefore, a valid research approach should include the demonstration of stability of the peptide toward luminal and brush border peptidases and evaluate its ADME properties. Surprisingly, only very few animal and human studies determined absolute concentrations and kinetics of bioactive peptides. These studies have shown the presence of selected peptides in plasma samples at pico- and nanomolar concentrations with fast elimination kinetics in the minute range. For the correct interpretation of results, it is advised that researchers refer to the data currently available concerning bioavailability and ADME properties in humans. Two mandatory criteria for future in vitro studies investigating potential biological activities of peptides should be using physiologically relevant concentrations and times.

Food-dependent disintegration of immediate release fosamprenavir tablets: In vitro evaluation using magnetic resonance imaging and a dynamic gastrointestinal system

In the present study, we demonstrated the value of two advanced tools, the TNO gastric and small Intestinal Model (TIM-1) and magnetic resonance imaging (MRI), for the in vitro evaluation of food-dependent disintegration of immediate release fosamprenavir tablets. Upon introduction of a tablet with the nutritional drink Scandishake Mix® in the stomach compartment of TIM-1, simulating the fed state, disintegration and fosamprenavir dissolution were significantly postponed compared to the fasted state (lag time 80 ± 23 min). This resulted in a lag in the appearance of bioaccessible fosamprenavir (<5% during the first 2h), even though the nutritional state did not significantly alter the cumulative bioaccessibility after 5h. These results were in agreement with the previously observed postprandial delay in gastric fosamprenavir tablet disintegration and subsequent amprenavir absorption in healthy volunteers. Therefore, TIM-1 can be used in tablet development to identify food-induced disintegration issues causing unexpected clinical behavior. From a mechanistic perspective, we applied MRI to illustrate impaired water ingress in fosamprenavir tablets immersed in the nutritional drink compared to simulated gastric fluid. This effect may be attributed to both competition between nutritional components and the tablet for the available water (indicated by reduced rotational and translational diffusion) as well as the possible formation of a food-dependent precipitation layer on the HPMC-coated tablet.

Release and absorption rate aspects of intramuscularly injected pharmaceuticals

Abstract Injection depth is an important parameter influencing absorption rate after intramuscular injection. A too shallow injection will, especially in the gluteal region, only reach the subcutaneous fat layer. This fat layer appears to exert a retarding effect on lipophilic drugs, which is moreover dependent on formulation factors. A cohesive mechanistic description of the variables and their interrelationships does not exist. From a review study on the relevant information it appears that the mean absorption times of drugs in aqueous or oily suspension i.m. injected is longer (weeks to months) than of drugs in solution (minutes to hours, incidentally weeks, dependent on the lipophilicity of the drug). The injection depth is an important variable since the mean absorption times are considerably longer when the drug is shallowly injected in the adipose layer. In intramuscular injection kinetics the following steps are considered to be important (in hierachical sequence): dissolution rate, solvent supply (vascularisation or perfusion), phase transfer and diffusion to the vascular system, all of them more or less susceptible to formulation, physiological variables and injection depth (in fat or muscular tissue). Dissolution and solvent supply are associated with zero order-, phase transfer and diffusion processes with first order release and absorption kinetics. In general a “mixed” order appearance in the circulation will result from a more complicated situation. After application to mucosal membranes such as buccal and vaginal walls many lipophilic drugs appear to show a depot effect resulting in larger mean absorption times (the order of magnitude is hours) than hydrophilic drugs. A similar phenomenon is seen in the intramuscular absorption. The differences can be explained by the nature of the retaining bonds and differences in histological structure. It can be concluded that the subcutaneous adipose layer has important retarding effects on the absorption of drugs which are intentionally or otherwise injected in the adipose layer.

Characterization of Human Duodenal Fluids in Fasted and Fed State Conditions

This work provides an elaborate characterization of human intestinal fluids (HIF) collected in fasted- and fed-state conditions. HIF from 20 healthy volunteers (10 M/F) were aspirated by intubation near the ligament of Treitz in a time-dependent manner (10-min intervals) and characterized for pH, bile salts, phospholipids, cholesterol, triacylglycerides (TAG), diacylglycerides (DAG), monoacylglycerides (MAG), free fatty acids (FFA), pancreatic lipase, phospholipase A2, and nonspecific esterase activity. For almost all parameters, a food-induced effect was observed. Results were characterized by a high variability, as illustrated by the broad ranges observed for each parameter: pH (fasted: 3.4-8.3; fed: 4.7-7.1), bile salts (fasted: 0.03-36.18 mM; fed: 0.74-86.14 mM), phospholipids (fasted: 0.01-6.33 mM; fed: 0.16-14.39 mM), cholesterol (fasted: 0-0.48 mM; fed: 0-3.29 mM), TAG (fed: 0-6.76 mg/mL), DAG (fed: 0-3.64 mg/mL), MAG (fasted: 0-1.09 mg/mL; fed: 0-11.36 mg/mL), FFA (fasted: 0-3.86 mg/mL; fed: 0.53-15.0 mg/mL), pancreatic lipase (fasted: 26-86 g/mL; fed: 146-415 g/mL), phospholipase A2 (fasted: 3-6 ng/mL; fed: 4.3-27.7 ng/mL), and nonspecific esterase activity (fasted: 270-4900 U/mL; fed: 430-4655 U/mL). This comprehensive overview may serve as reference data for physiologically based pharmacokinetic modeling and the optimization of biorelevant simulated intestinal fluids for the use in in vitro dissolution, solubility, and permeability profiling.

Bioavailability of propranolol after oral, sublingual, and intranasal administration.

The bioavailability of propranolol was compared after oral, sublingual, and intranasal administration in eight healthy male volunteers. Relative to the bioavailability after intranasal (in) administration, which was previously shown to be nearly complete (Frelin = 100%), the sublingual (sl) administration of a standard 10-mg tablet gave a bioavailability of Frelsl = 63 ± 22%, while the oral (or) administration yielded only Frelor = 25 ± 8%. The serum concentration–time curves of propranolol after sublingual administration resembled those of a sustained-release preparation. This sustained-release phenomenon after the sublingual route is reflected in the mean residence times (MRTs) of propranolol in the body (MRTor = 5.7 ± 1.3 hr, MRTsl - 6.4 ± 1.3 hr, MRTin = 4.6 ± 1.0 hr; mean ± SD; N = 8). MRTs after sublingual administration were significantly longer than after the oral and the intranasal doses (P < 0.05 and P < 0.002, respectively).

Modeling of the Relationship between Dipeptide Structure and Dipeptide Stability, Permeability, and ACE Inhibitory Activity

Selected di- and tripeptides exhibit angiotensin-I converting enzyme (ACE) inhibitory activity in vitro. However, the efficacy in vivo is most likely limited for most peptides due to low bioavailability. The purpose of this study was to identify descriptors of intestinal stability, permeability, and ACE inhibitory activity of dipeptides. A total of 228 dipeptides were synthesized; intestinal stability was obtained by in vitro digestion, intestinal permeability using Caco-2 cells and ACE inhibitory activity by an in vitro assay. Databases were constructed to study the relationship between structure and activity, permeability, and stability. Quantitative structure-activity relationship (QSAR) modeling was performed based on computed models using partial least squares regression based on 400 molecular descriptors. QSAR modeling of dipeptide stability revealed high correlation coefficients (R > 0.65) for models based on Z and X scales. However, amino acid (AA) clustering showed the best results in describing stability of dipeptides. The N-terminal AA residues Asp, Gly, and Pro as well as the C-terminal residues Pro, Ser, Thr, and Asp stabilize dipeptides toward luminal enzymatic peptide hydrolysis. QSAR modeling did not reveal significant correlation models for intestinal permeability. 2D-fingerprint models were identified describing ACE inhibitory activity of dipeptides. The intestinal stability of 12 peptides was predicted. Peptides were synthesized and stability was confirmed in simulated digestion experiments. Based on the results, specific dipeptides can be designed to meet both stability and activity criteria. However, postabsorptive ACE inhibitory activities of dipeptides in vivo are most likely limited due to the very low intestinal permeability of dipeptides.