Citrulline: from metabolism to therapeutic use.

Citrulline possesses a highly specific metabolism that bypasses splanchnic extraction because it is not used by the intestine or taken up by the liver. The administration of citrulline may be used to deliver available nitrogen for protein homeostasis in peripheral tissues and as an arginine precursor synthesized de novo in the kidneys and endothelial and immune cells. Fresh research has shown that citrulline is efficiently transported across the intestinal luminal membrane by a set of transporters belonging to the B°,⁺, L, and b°,⁺ systems. Several pharmacokinetic studies have confirmed that citrulline is efficiently absorbed when administered orally. Oral citrulline could be used to deliver arginine to the systemic circulation or as a protein anabolic agent in specific clinical situations, because recent data have suggested that citrulline, although not a component of proteins, stimulates protein synthesis in skeletal muscle through the mammalian target of rapamycin signaling pathway. Hence, citrulline could play a pivotal role in maintaining protein homeostasis and is a promising pharmaconutrient in nutritional support strategies for malnourished patients, especially in aging and sarcopenia.

Endotoxemia affects citrulline, arginine and glutamine bioavailability.

BACKGROUND: Sepsis considerably alters the intestinal barrier functions, which in turn modify the absorption and bioavailability of nutrients. However, the effects of septic shock on aminoacid (AAs) bioavailability are poorly documented. The aim of this study was to compare the bioavailability of citrulline, arginine and glutamine during endotoxemia. MATERIALS AND METHODS: Thirty-six rats were randomised into two groups: control and lipopolysaccharides (LPS). The LPS group received an intraperitoneal injection of endotoxins (7·5 mg/kg). After 12 h, each group was again randomised into three subgroups, each of which received an oral bolus of citrulline, arginine or glutamine (5·7 mmol/kg). Blood samples were collected at various times from 0 to 600 min after AA administration. The concentrations of citrulline, arginine, glutamine and their metabolites arginine and ornithine were measured to determine pharmacokinetic parameters Area Under Curve (AUC), C(max) and T(max). RESULTS: The AUC values of citrulline decreased in LPS rats [citrulline, control: 761 ± 67 and LPS: 508 ± 72 µmol min/mL (P = 0·02)]. Maximum concentrations of citrulline were also significantly decreased by endotoxemia (P = 0·01). The pharmacokinetic parameters of arginine and glutamine were not significantly modified by endotoxemia. The AUC value of arginine from citrulline conversion was diminished in endotoxemic rats. The other pharmacokinetic parameters of arginine were not significantly modified after arginine or citrulline supply in either group (control or LPS). CONCLUSION: Endotoxemia affects the bioavailability of AAs differently according to the amino acid considered. This feature may be important for nutritional strategy in ICU patients.

Mechanisms and kinetics of citrulline uptake in a model of human intestinal epithelial cells.

BACKGROUND & AIMS: Citrulline is a major precursor of arginine by de novo synthesis in the kidneys. Oral citrulline supplementation may be beneficial in some clinical conditions. However, citrulline bioavailability depends on its intestinal absorption. Since the mechanism of citrulline transport across the intestine has not been established yet, this study was designed to characterize L-[(14)C]-citrulline uptake by Caco-2 cells. METHODS: Caco-2 cells were cultured in a bicameral insert system. Inhibition studies were conducted in the presence of neutral, cationic, acidic and non-metabolized amino acids. We performed control inhibition studies for arginine uptake. RESULTS: Citrulline uptake was pH-independent whereas the uptake rate was reduced in the absence of Na(+). Kinetic analysis indicated the involvement of Na(+)-dependent and Na(+)-independent saturable transport components. For competition studies, both the transport components were markedly inhibited by large, small neutral and cationic amino acids. It was also noticed that specific inhibitor of system lBCH inhibited uptake. The inhibition profile of arginine transport was different from that of citrulline transport as arginine uptake was insensitive to BCH. CONCLUSIONS: These characteristics suggest that system B(0,+) might be responsible for the Na(+)-dependent uptake of citrulline, whereas Na(+)-independent uptake may include systems L and b(0,+). Our results show that systems involved in citrulline transport are partly different from those involved in arginine transport.

Dissolution and permeation properties of naproxen from solid-state systems with chitosan.

The purpose of this study was to investigate the influence of different types of chitosan and of the preparation technique of the drug-polymer combination in improving the dissolution and permeation abilities of naproxen, a very poorly water-soluble anti-inflammatory drug. Drug-chitosan systems were prepared by simple physical mixing, kneading, cogrinding, or coevaporation using five types of chitosan (base and glutamate or hydrochloride salts, both at two different molecular weights). The products were tested for drug-dissolution behavior and for permeation properties through both Caco-2 cell monolayers and artificial lipophilic membranes. All combinations with chitosan base were significantly (p < .01) more effective in enhancing drug-dissolution rate than those with both its salts, probably in virtue of its higher amorphizing effect toward the drug, as observed in solid-state studies. A different rank order was found in permeation experiments in which chitosan glutamate was the most powerful partner in improving the drug-apparent permeability (p < .01), followed by the hydrochloride salt (p < .05), whereas no significant effect was obtained with chitosan base. Cogrinding was the most powerful technique in promoting both dissolution and permeation properties of the drug, thus pointing out the importance of the preparation method in obtaining efficacious drug-carrier systems. Finally, the good correspondence between permeation experiments with Caco-2 cells and those with the artificial lipophilic membrane indicated the suitability of this latter in preformulation studies for a rapid screening of the best carrier and the most efficient drug-carrier preparation method for improving the biopharmaceutical properties of drugs.

Effect of chitosan-coated alginate microspheres on the permeability of Caco-2 cell monolayers.

Alginate microspheres were prepared by emulsification/internal gelation and coated with chitosan. The ability of chitosan-coated alginate microspheres to increase the paracellular transport across Caco-2 cell monolayers was evaluated in comparison to uncoated microspheres and chitosan solutions. Transport studies were performed by using a permeability marker, Lucifer Yellow (LY), and by measuring the transepithelial electric resistance (TEER) variations. Furthermore, the occurrence of cytotoxic effects was assessed by evaluating neutral red uptake in viable cells and lactate dehydrogenase (LDH) release from damaged cells. A 3-fold increase on LY permeability was obtained for coated microspheres when compared to chitosan solutions. TEER variations were in agreement with permeability results. Chitosan solutions exhibited a dose-dependent toxicity, but coated microspheres did not decrease the viability of cells. Chitosan-coated alginate microspheres have potential to be used as carriers of poorly absorbable hydrophilic drugs to the intestinal epithelia and possibly increase their oral bioavailability.

Development and evaluation of an in vitro method for prediction of human drug absorption II. Demonstration of the method suitability.

The aim of the present work was to assess the actual suitability and general applicability of a new in vitro permeation method based on an appositely developed artificial membrane to evaluate and predict drug absorption potential. The proposed method was employed to evaluate the apparent permeability of a set of 21 structurally diverse drugs having very different solubility and permeability properties, covering the whole range of fraction absorbed in humans (F(a) from 13 to 100%); 13 of the drugs in this study were part of the list suggested by FDA for validation of in vitro permeation methods. An excellent linear correlation (R(2)=0.957) was obtained between artificial membrane apparent permeability and human absorption data in the whole range of F(a) values examined (including all the drugs belonging to the above FDA list), indicating the good predictive ability of the proposed method not only for highly absorbed hydrophobic compounds but also, differently from other in vitro permeation methods, for poorly or middling permeable drugs. The predictive ability of the new method was greater than those obtained for the same set of drugs with PAMPA and Caco-2 permeability literature data, probably due to the poor sensitivity of these methods towards hydrophilic drugs. The better performance of our artificial membrane was attributed to the hydrophilic nature of the support that, differently from the commonly used hydrophobic supports, offers less resistance to permeation of hydrophilic compounds. A comparison of permeation data of theophylline, ketoprofen, aciclovir and furosemide (selected, respectively, as models of I-IV BCS classes) obtained using a Caco-2 cell based dynamic method and the developed artificial membrane and the corresponding F(a) values in humans further confirmed the suitability of the proposed permeation method as predictor of the oral absorption of passively absorbed drugs.

Characterization of the absorption of theophylline from immediate- and controlled-release dosage forms with a numerical approach using the in vitro dissolution-permeation process using caco-2 cells.

After oral administration, drug absorption rate is recognized to be dependent on two major factors: dissolution and intestinal cells permeability. Caco-2 monolayer cells have been largely used as a permeation study model. In this study, a numerical approach funded on an exponential first-order time relationship was tested to compare immediate- and controlled-release tablets of theophylline using a dissolution-permeation system. The dissolution performance using USP II paddle apparatus was coupled to the permeability studies investigated in Caco-2 cell monolayers. The dissolved samples were taken at different times; their pH and osmolarity were adjusted to render them suitable to Caco-2 permeability studies (osmolarity = 300 mosm, pH = 7.4). The experimental data show that the dissolution fits the exponential first-order relationship rate. The permeability values were in a range of 4.45 10(- 6)-5.28 10(- 6) cm/s, and percentages of absorbed drug dose were dependent on the fraction initially present in the donor compartment, indicating that absorption of theophylline was dissolution rate limited. Plotting experimental absorbed fractions (F(a)) against experimental dissolved fractions (F(d)) show that permeation is the rate-limiting step in drug absorption process in the extended release form of theophylline. Our results demonstrate a general agreement between observed F(a)/F(d) relationships and theoretical F(a)/F(d) relationships obtained with our approach funded on dissolution and permeation behavior. We concluded that the couple dissolution-caco-2 system could be a useful tool to characterize intestinal permeation for a new formulation of a drug compared with the conventional one.

Short term Caco-2/TC7 cell culture: comparison between conventional 21-d and a commercially available 3-d system.

The Caco-2 cell model is a valuable tool for studying intestinal biotransformation of xenobiotics and to evaluate the potential of human intestinal absorption of new compounds. These properties were evaluated with Caco-2/TC7 cells in accelerated conditions to reduce maturation lag time from 21-d to 3-d in order to increase time and labor efficiency. Transmission electron and fluorescent microscopy were used for morphological characterization. Alkaline phosphatase and lactate dehydrogenase activities were assessed within time. Cytochrome P450 expression was studied by RT-PCR. Apparent permeabilities of a set of passively absorbed molecules across Caco-2/TC7 cell monolayers were determined to evaluate potential of both systems for prediction of human intestinal absorption. Microscopic images revealed that cells under both conditions differentiated as enterocyte-like cells but did so heterogeneously in the 3-d model. TEER values have shown that the 3-d model is a leakier cell system with higher mannitol Papp (cm/s). Biochemical characterization (hydrolase activities, CYP450 expression) suggested that the 3-d model was at a lower maturation level than the 21-d model. Carrier-mediated uptake of L-Phe was lower in the 3-d model suggesting that this model has limited application for mechanistic studies. Reasonable correlation was obtained between the two models (r2=0.88, p>0.01) for 11 passively absorbed compounds with high potential of rank ordering of compounds. Although results suggested that the 3-d cells are under-differentiated, they could be usable to estimate the oral absorption of passively absorbed compounds.

Mucoadhesive and penetration enhancement properties of three grades of hyaluronic acid using porcine buccal and vaginal tissue, Caco-2 cell lines, and rat jejunum.

The influence of the molecular weight on mucoadhesive and penetration enhancement properties of three grades of hyaluronic acid (1878, 693 and 202 kDa) has been evaluated. The mucoadhesive properties were investigated using buccal and vaginal porcine mucosa by means of a tensile stress method and using rat jejunum by means of an inclined plane method. The mucoadhesive performances observed using animal tissues were compared with the mucoadhesive properties observed using submaxillary or gastric mucin dispersions. The penetration enhancement properties were investigated using porcine buccal epithelium membrane or vaginal tissue and a cell monolayer (Caco-2 cell line). Chitosan hydrochloride, already described as a penetration enhancer towards buccal and vaginal mucosae and Caco-2 cell monolayers, was used as reference. Aciclovir (acyclovir), a poorly soluble and absorbable drug, commonly used in the treatment of Herpes simplex virus (type I and II), was used as the model drug. Unlike chitosan hydrochloride, which does not show any mucoadhesive potential at pH close to neutrality (buccal and intestinal), all hyaluronic acid grades show mucoadhesive properties in all the environments considered (buccal, vaginal and intestinal). In all cases, a decrease in molecular weight of hyaluronic acid produced an increase in the mucoadhesive performance. The hyaluronic acid with the lowest molecular weight (202 kD) exhibited the best penetration enhancement properties, that, depending on the substrate under consideration, was either comparable with or even better than chitosan hydrochloride. Therefore, this grade would be the most promising for buccal, vaginal and intestinal delivery of aciclovir.

Comparison of the effect of chitosan and polyvinylpyrrolidone on dissolution properties and analgesic effect of naproxen.

The solubilizing and absorption enhancer properties towards naproxen of chitosan and polyvinylpyrrolidone (PVP) have been investigated. Solid binary systems prepared at various drug-polymer ratios by mixing, cogrinding or kneading, were characterized by differential scanning calorimetry, X-ray diffractometry, Fourier transform infrared spectroscopy, and scanning electron microscopy, and tested for dissolution behavior. Both carriers improved drug dissolution and their performance depended on the drug-polymer ratio and the system preparation method. Chitosan was more effective than PVP, despite the greater amorphizing power of PVP as revealed by solid state analyses. The 3/7 (w/w) drug-carrier coground systems with chitosan and PVP were the best products enabling, respectively, an improvement of 4.8 and 3.6 times of drug dissolution efficiency. In vivo experiments in mice demonstrated that administration of 45 mg/kg of drug coground with PVP or chitosan resulted, respectively, in a 25 and 60% reduction of acetic acid-induced writhings in comparison to pure drug, which, instead, was statistically ineffective as compared to the control group. Moreover, the 3/7 (w/w) drug-chitosan coground product demonstrated an antiwrithing potency 2.4 times higher than the coground with PVP. Thus, the direct-compression properties and antiulcerogenic activity, combined with the demonstrated solubilizing power and analgesic effect enhancer ability towards the drug, make chitosan particularly suitable for developing a reduced-dose fast-release solid oral dosage form of naproxen.

Development and characterization of naproxen-chitosan solid systems with improved drug dissolution properties.

The solubilizing and amorphizing properties toward naproxen (a poorly water-soluble antiinflammatory drug) of chitosan, an emerging pharmaceutical biopolymer, have been investigated. Solid binary systems at different drug/polymer ratios have been prepared according to different techniques (mixing, cogrinding, kneading, coevaporation) using chitosan at low (CS-L(w)) and medium (CS-M(w)) molecular weight, and tested for dissolution properties. Drug-carrier interactions were investigated in both the liquid and solid state, by phase solubility analysis, differential scanning calorimetry, X-ray powder diffractometry, FT-IR spectroscopy, and scanning electron microscopy. Drug dissolution parameters improved with increasing the polymer amount in the mixture, reaching the highest values at the 1:9 (w/w) drug/polymer ratio, and CS-L(w) was more efficacious than CS-M(w). Cogrinding was the most effective technique, showing the strongest amorphizing effect toward the drug and enabling an increase of more than ten times its relative dissolution rate. Coground mixtures at 3:7 (w/w) drug/polymer ratio were able to give directly compressed tablets which maintained unchanged the improved drug dissolution properties. Enhancer dissolution properties combined with its direct compression feasibility and antiulcerogenic action make CS-L(w) an optimal carrier for developing fast-release oral solid dosage forms of naproxen.

Evaluation of the cytotoxicity effect of dimethyl sulfoxide (DMSO) on Caco2/TC7 colon tumor cell cultures.

Dimethyl sulfoxide (DMSO) is usually used to solubilize poorly soluble drugs in permeation assays such as that using Caco2 enterocyte-like cells. The objective of this study was to evaluate the toxicity of DMSO on Caco2/TC7 cells and determinate the maximal concentration usable in permeation experiments. Caco2/TC7 cells were cultured for 21 d on 96-well plates for evaluation of toxicity. The determination of lactate dehydrogenase (LDH) release in cell supernatant and the measurement of Neutral Red (NR) uptake are used for cytotoxicity assays. DMSO solutions (0-100%) in Hank's balanced salt solution containing HEPES (25 mM), pH 7.4, were incubated with Caco-2/TC7 cells on 96 well plates. Caco2/TC7 cells were cultured on Transwell-Clear inserts to evaluate the influence of DMSO on the apparent permeability of the paracellular marker mannitol. DMSO 10% did not induce any significant increase in LDH release whereas a significant increase in LDH activity (ANOVA, p<0.05) occurred at a DMSO concentration of 20 to 50%. NR incorporation in viable cells was statistically reduced by 27 to 36% at DMSO concentration of 20% up to 100% (ANOVA, p>0.05). No statistical difference (p<0.05) in apparent mannitol permeability was observed between the control and 10% DMSO groups. In conclusion, at concentrations of up to 10%, DMSO did not produce any significant alteration in apical membrane permeability or on cell-to-cell tight junctional complexes.

Comparative study of ibuproxam complexation with amorphous beta-cyclodextrin derivatives in solution and in the solid state.

The complexing, solubilizing and amorphizing abilities toward ibuproxam (a poorly water-soluble anti-inflammatory agent) of some randomly substituted amorphous beta-cyclodextrin derivatives (i.e. methyl- (MebetaCd), hydroxyethyl- (HEbetaCd), and hydroxypropyl- (HPbetaCd) beta-cyclodextrins) were investigated and compared with those of the parent beta-cyclodextrin. Equimolar drug-cyclodextrin solid systems were prepared by blending, cogrinding, coevaporation, and colyophilization. Drug-carrier interactions were studied in both the liquid and solid state by phase solubility analysis, supported by molecular modelling, differential scanning calorimetry, X-ray powder diffractometry, Fourier transform infrared spectroscopy and scanning electron microscopy. All the betaCd derivatives showed greater solubilizing efficacies toward ibuproxam than the parent one, due to their higher water solubility. On the contrary, a clear reduction of complexing ability was observed, indicative of some steric interferences to drug inclusion due to the presence of substituents, as confirmed by molecular modelling studies. However, this negative effect was not reflected in the dissolution behaviour (evaluated according to the dispersed amount method) of their solid binary systems, probably thanks to the greater amorphizing properties shown (DSC and X-ray analyses) by betaCd derivatives. In fact their dissolution efficiencies were not significantly different (MebetaCd) or only slightly lower (HEbetaCd and HPbetaCd) than those of the corresponding products with beta-cyclodextrin. Colyophilized products were in all cases the most effective, followed by coground and coevaporated systems, whose dissolution efficiencies were over four times higher than the corresponding physical mixtures and about 15 times higher than the pure drug.