In vitro Dissolution Testing of Rifampicin Powder Formulations For Prediction of Plasma Concentration-Time Profiles After Inhaled Delivery.

PURPOSE: The purpose of this study was to evaluate the in vitro lung dissolution of amorphous and crystalline powder formulations of rifampicin in polyethylene oxide (PEO) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), and to predict the in vivo plasma concentration-time profiles using the in vitro data. METHODS: The in vitro dissolution and permeation profiles of respirable rifampicin particles were studied using a custom-made dissolution apparatus. Data from the in vitro dissolution test were used to estimate the parameters to be used as the input for the simulation of in vivo plasma concentration-time profiles using STELLA® software. For prediction of in vivo profiles, a one-compartment model either with a first order elimination or with a Michaelis-Menten kinetics-based elimination was used. RESULTS: Compared to the crystalline formulation, the amorphous formulation showed rapid in vitro dissolution suggesting their possible faster in vivo absorption and higher plasma concentrations of rifampicin following lung delivery. However, the simulations suggested that both powder formulations would result in similar plasma-concentration time profiles of rifampicin. CONCLUSIONS: Use of an in vitro dissolution test coupled with a simulation model for prediction of plasma-concentration time profiles of an inhaled drug was demonstrated in this work. These models can also be used in the design of inhaled formulations by controlling their release and dissolution properties to achieve desired lung retention or systemic absorption following delivery to the lungs.

The influence of storage relative humidity on aerosolization of co-spray dried powders of hygroscopic kanamycin with the hydrophobic drug rifampicin.

The purpose of this study was to investigate the influence of storage humidity on in vitro aerosolization and physicochemical properties of co-spray dried powders of kanamycin with rifampicin. The powders were stored for one-month in an open Petri dish at different relative humidities (RHs) (15%, 43%, and 75%) and 25 ± 2 °C. The in vitro aerosolization (fine particle fraction, FPF) of the powders was determined by a next generation impactor (NGI). The moisture content, particle morphology and crystallinity of the powders were determined by Karl Fischer titration, scanning electron microscopy, and X-ray powder diffractometry, respectively. At all RH, the FPF of hydrophobic rifampicin-only powder was unaffected but the FPF of hygroscopic kanamycin-only powder significantly decreased even at 43% RH. The kanamycin-only particles fused together, crystallized and formed hard cakes at 75% RH. The aerosolization of kanamycin and rifampicin in the combination powders remained unaffected at 15% and 43% RH, but aerosolization significantly decreased at 75% RH. Enrichment of the surface of the particles with hydrophobic rifampicin did not protect the combination powders from moisture uptake but it prevented particle agglomeration up to 43% RH. At 75% RH, the moisture uptake led to agglomeration of the particles of the combination powder particles and consequently an increase in aerodynamic diameter. Further studies are required to investigate how rifampicin enrichment prevents particle agglomeration, the possible mechanisms (e.g. particle interactions due to capillary forces or electrostatic forces) for the changes in the aerosolization and changes in surface composition during storage.

Carrier-free combination dry powder inhaler formulation of ethionamide and moxifloxacin for treating drug-resistant tuberculosis.

This study aimed to develop a combination dry powder formulation of ethionamide and moxifloxacin HCl as this combination is synergistic against drug-resistant Mycobacterium tuberculosis (Mtb). L-leucine (20% w/w) was added in the formulations to maximize the process yield. Moxifloxacin HCl and/or ethionamide powders with/without L-leucine were produced using a Buchi Mini Spray-dryer. A next generation impactor was used to determine the in vitro aerosolization efficiency. The powders were also characterized for other physicochemical properties and cytotoxicity. All the spray-dried powders were within the aerodynamic size range of <5.0 µm except ethionamide-only powder (6.0 µm). The combination powders with L-leucine aerosolized better (% fine particle fraction (FPF): 61.3 and 61.1 for ethionamide and moxifloxacin, respectively) than ethionamide-only (%FPF: 9.0) and moxifloxacin-only (%FPF: 30.8) powders. The combination powder particles were collapsed with wrinkled surfaces whereas moxifloxacin-only powders were spherical and smooth and ethionamide-only powders were angular-shaped flakes. The combination powders had low water content (<2.0%). All the powders were physically stable at 15% RH and 25 ± 2 °C during 1-month storage and tolerated by bronchial epithelial cell-lines up to 100 µg/ml. The improved aerosolization of the combination formulation may be helpful for the effective treatment of drug-resistant tuberculosis. Further studies are required to understand the mechanisms for improved aerosolization and test the synergistic activity of the combination powder.

Pharmacokinetics of Abamectin/Levamisole Combination in a Medium Chain Mono and Diglyceride-Based Vehicle and an In Vitro Release and In Vitro In Vivo Correlation Study for Levamisole.

A combination of lipophilic and hydrophilic drugs in a single solution is a challenge due to their different physicochemical properties. In vitro and in vivo release studies are useful to optimize this solution. The in vitro (Franz diffusion cell) release rate of levamisole phosphate from an isotropic vehicle of medium chain mono and diglycerides (MCMDG) was significantly slower than the release from water. The injectable solution of the isotropic MCMDG-based system was prepared with 13.65% of levamisole phosphate and 0.5% of abamectin. Two milliliters/50 kg (0.04 ml/kg) was injected subcutaneously into five healthy adult sheep. None of the animals showed the signs of inflammation at injection site. Both drugs were assayed using validated HPLC methods. The absorption rates for levamisole (0.71 ± 0.32 h-1) and abamectin (0.24 ± 0.08 day-1) from the MCMDG-based formulation were considerably slower than those of other studies conducted on the commercial products. The tmax was delayed for levamisole (2.20 ± 0.45 h) and abamectin (4.20 ± 1.64 days) compared with those in published studies. Longer MRT values for levamisole (6.14 ± 1.14 h) and abamectin (8.80 ± 1.39 days) were found in this study compared to those reported. A correlation was observed between in vivo fraction absorbed and in vitro fraction released for levamisole phosphate in the MCMDG-based formulation. The injection vehicle of isotropic MCMDG-based system delayed the subcutaneous absorption of levamisole phosphate and abamectin compared to the commercial subcutaneous injection products for levamisole and abamectin. Notably, this isotropic MCMDG-based vehicle system is prepared with a combination of two drugs with different physicochemical properties.

The influence of bile salts on the response of liposomes to ultrasound.

CONTEXT: Triggering drug release from delivery vehicles with ultrasound has potential applications in targeted drug delivery. It was hypothesized that the addition of bile salts would increase the sensitivity of liposomes to ultrasound through creation of defects. OBJECTIVE: The aim of this study was to investigate whether incorporating bile salts into liposomes would lead to differential effects on their response to low and high frequency ultrasound. MATERIALS AND METHODS: Cholate, chenodeoxycholate, ursodeoxycholate, glycocholate and taurocholate were the selected bile salts. Response to ultrasound was characterized by measuring the release of carboxyfluorescein (CF). RESULTS: At 30 kHz ultrasound, taurocholate containing liposomes were most responsive and released 70% (± 2) CF after 30 seconds of sonication. Compared to this, liposomes that did not contain bile salts released just 7% (± 2). At 1.1 MHz ultrasound, all liposome formulations were unresponsive. To increase the response of liposomes at 1.1 MHz ultrasound, a combination of membrane destabilizers were added to DSPC liposomes. DOPE, a hexagonal phase lipid was used in combination with taurocholate. Surprisingly, liposomes containing DOPE and taurocholate were more resistant to 1.1 MHz ultrasound than ones containing only DOPE. DISCUSSION: This suggests that the sensitivity of liposomes towards ultrasound may not simply be defined by a single membrane component but instead depends on the interaction between constituting lipid components. Furthermore, strategies other than membrane destabilization may be required to sensitize liposomes towards high frequency ultrasound. CONCLUSION: Bile salts may be used to increase or decrease the sensitivity of liposomes to low frequency ultrasound.

Signaled drug delivery and transport across the blood-brain barrier.

We use a mathematical model to describe the delivery of a drug to a specific region of the brain. The drug is carried by liposomes that can release their cargo by application of focused ultrasound (US). Thereupon, the drug is absorbed through the endothelial cells that line the brain capillaries and form the physiologically important blood-brain barrier (BBB). We present a compartmental model of a capillary that is able to capture the complex binding and transport processes the drug undergoes in the blood plasma and at the BBB. We apply this model to the delivery of levodopa (L-dopa, used to treat Parkinson's disease) and doxorubicin (an anticancer agent). The goal is to optimize the delivery of drug while at the same time minimizing possible side effects of the US.

Kinetic and equilibrium studies of bile salt-liposome interactions.

Research has suggested that exposure to sub-micellar concentrations of bile salts (BS) increases the permeability of lipid bilayers in a time-dependent manner. In this study, incubation of soy phosphatidylcholine small unilamellar vesicles (liposomes) with sub-micellar concentrations of cholate (C), deoxycholate (DC), 12-monoketocholate (MKC) or taurocholate (TC) in pH 7.2 buffer increased membrane fluidity and negative zeta potential in the order of increasing BS liposome-pH 7.2 buffer distribution coefficients (MKC < C ≈ TC < DC). In liposomes labeled with the dithionite-sensitive fluorescent lipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE) in both leaflets and equilibrated with sub-micellar concentrations of BS, fluorescence decline during continuous exposure to dithionite was biphasic involving a rapid initial phase followed by a slower second phase. Membrane permeability to dithionite as measured by the rate of the second phase increased in the order control < MKC < TC ∼ C < DC. In liposomes labeled with NBD-PE in the inner leaflet only and incubated with the same concentrations of C, DC and MKC, membrane permeability to dithionite initially increased very rapidly in the order MKC < C < DC before impermeability to dithionite was restored after which fluorescence decline was consistent with NBD-PE flip-flop. For liposomes incubated with TC, membrane permeability to dithionite was only slightly increased and the decline in fluorescence was mainly the result of NBD-PE flip-flop. These results provide evidence that BS interact with lipid bilayers in a time-dependent manner that is different for conjugated and unconjugated BS. MKC appears to cause least disturbance to liposomal membranes but, when the actual MKC concentration in liposomes is taken into account, MKC is actually the most disruptive.

Stability of penethamate, a benzylpenicillin ester prodrug, in oily vehicles.

Penethamate (PNT) is an ester prodrug of benzylpenicillin which is marketed as dry powder for reconstitution with aqueous vehicle prior to injection. The purpose of this paper was to investigate the chemical stability of PNT in oily formulations to provide a basis for a ready-to-use (RTU) oil-based PNT formulation. The chemical stability of PNT solutions and suspensions in light liquid paraffin (LP), medium chain triglyceride (MIG), ethyl oleate (EO) and sunflower oil (SO) was investigated at 30 °C. Solid state stability of PNT powder and stability of PNT in EO suspensions with different moisture contents were also evaluated. The solubility of PNT in the oils was in order SO > EO > MIG > LP. Degradation of PNT was rapid in oily solutions and less than 10% remained after 7-15 days. Stability of PNT decreased with increase in moisture content in ethyl oleate suspensions. PNT was stable over four weeks in the solid state. Hydrolysis, due to moisture in the oil formulation is not the only degradation mechanism. PNT stability (% drug remaining) in oily suspensions after 3.5 months was in the order LP (96.2%) > MIG (95.4%) > EO (94.1%) > SO (86%). A shelf-life of up to 5.5 years at 30 °C may be achieved for PNT suspension in these oils.

Parameterisation affects identifiability of population models.

Identifiability is an important aspect of model development. In this work, using a simple one compartment population pharmacokinetic model, we show that identifiability of the variances of the random effects parameters are affected by the parameterisation of the fixed effects parameters.

Evaluation of an on-farm culture system for the detection of subclinical mastitis pathogens in dairy cattle.

The purpose of this observational study was to compare the performance of a novel on-farm culture (OFC) test with the reference method (RM) in identifying pathogens, and in particular Staphylococcus aureus, associated with subclinical mastitis (SCM) in dairy cattle. The OFC test (Mastatest HiSCC; Mastaplex Limited) for SCM uses a cartridge with 2 × 12 wells allowing 1 sample to be analyzed in duplicate (24 wells) or 2 samples analyzed simultaneously, each in 12 wells. Results of the milk analyses are reported hierarchically (Staph. aureus → coagulase-negative staphylococci (CNS) → other gram positive or coliform/gram negative → no bacteria present) and emailed within 24 h. Milk samples (617 quarter level from 158 cows and 70 cow level) were collected from 288 cows [individual cow somatic cell count (ICSCC) ≥150,000 cells/mL] on 9 purposefully selected farms known to have a high prevalence of clinical and subclinical Staph. aureus mastitis in Southland New Zealand. Quarter samples were analyzed individually (617 samples) and after animal-level pooling, providing 228 (158 + 70) cow-level samples. Samples were analyzed by the OFC test (in duplicate) and the RM (culture agar medium and latex test based on the recommendation by the National Mastitis Council) and identifications confirmed with MALDI-TOF mass spectrometry. Sensitivity (Se), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) for detection of Staph. aureus were all ∼90% with tight 95% confidence limits, and Cohen's kappa (κ) for agreement between the OFC test and RM was 0.81. Kappa for agreement between the OFC test duplicates was 0.93. About 35% of cows had only one quarter infected with Staph. aureus and all these animals could still be identified when pooled cow-level milk was analyzed. Although the high prevalence of Staph. aureus in the herds used in this study does not affect the Se and Sp values, it does elevate the PPV value (and decrease the NPV) and therefore use of PPV to extrapolate to a population with lower prevalence is not appropriate. For CNS, Sp, PPV, and NPV were all >0.8, κ was ≥0.6, and Se was >0.7. Kappa for agreement between the OFC test duplicates was 0.83. A result of "no bacteria detected" was reported in 13% of the cows with 93% agreement between OFC test and RM. We conclude that the OFC test provides a reliable method for detecting Staph. aureus in pooled cow-level milk even if only one quarter is infected; in the absence of Staph. aureus in the milk, it reliably identified CNS in pooled cow-level milk; it reliably identified cows with <10 cfu/10 µL of their milk. Compared with the RM, the method was rapid with results returned in 24 h of loading the cartridge.

Mechanistic studies of the effect of bile salts on rhodamine 123 uptake into RBE4 cells.

To examine the ability of bile salts (BS) to act as permeation enhancers at the blood brain barrier, the effect of four BS (cholate, deoxycholate, monoketocholate and taurocholate) on accumulation of rhodamine 123 (R123) in rat brain endothelial (RBE4) cells was investigated. Experiments were performed using BS concentrations shown to be noncytotoxic to RBE4 cells. Uptake and efflux of R123 in the absence and presence of BS were studied by fluorescence spectroscopy and confocal microscopy. Changes in RBE4 cell membrane fluidity in the presence of BS were evaluated using fluorescence anisotropy. The direct interaction between BS and R123 (ion pairing) and the effect of BS on distribution of R123 into liposomes were studied by capillary electrophoresis. All BS influenced R123 uptake in a concentration-dependent manner and increased cell membrane fluidity. Monoketocholate produced the greatest increase in uptake and also significantly reduced R123 efflux probably by inhibition of P-glycoprotein (P-gp). Direct interaction of BS and R123 was weak, but distribution of R123 into liposomes was increased by BS. The results suggest that BS increase R123 uptake by increasing cell membrane fluidity and, in the case of MKC, by inhibiting P-gp.

Characterization of a nanoparticulate drug delivery system using scanning ion occlusion sensing.

PURPOSE: To explore the application of scanning ion occlusion sensing (SIOS) as a novel technology for characterization of nanoparticles. METHODS: Liposomes were employed as model nanoparticles. The size distribution of the liposomes was measured by both SIOS and dynamic light scattering (DLS). Particle number concentration was determined based on particle translocation rate. The ability of SIOS and DLS to resolve bimodal samples was evaluated by measuring a mixture of 217 and 355 nm standard nanoparticles. Opsonization of liposomes by plasma was also studied using SIOS. RESULTS: SIOS was shown to measure the size of different liposomes with higher sensitivity than DLS and it requires a smaller sample volume than DLS. With appropriate calibration, SIOS could be used to determine particle number concentrations. In comparison, SIOS analysis of the mixture showed accurate resolution of the population as a bimodal distribution over a wide range of number ratios of the particles. SIOS could detect plasma opsonization of liposomes by demonstrating a increase in particle size and also changes in the particle translocation rate. CONCLUSION: SIOS is a useful technology for nanoparticle characterization. It shows some advantages over DLS and is clearly a useful tool for the study of nanoparticle drug delivery systems.

Pre-formulation and chemical stability studies of penethamate, a benzylpenicillin ester prodrug, in aqueous vehicles.

Penethamate (PNT) is a diethylaminoethyl ester prodrug of benzylpenicillin used to treat bovine mastitis via the intramuscular route. Because of its instability, PNT products must be reconstituted before administration and the reconstituted injection has a short shelf life (7 days at 2-8°C). The purpose of this paper was to investigate whether the stability of PNT can be improved in order to achieve a chemically stable ready-to-use aqueous-based PNT formulation or at least to extend the shelf life of the reconstituted suspension. A chemical stability study of PNT in aqueous-based solutions as a function of pH, buffer strength, solvent mixtures and temperature, supported by studies of its solubility in mixed solvents, allowed predictions of the shelf life of PNT solution and suspension formulations. PNT degraded in aqueous solutions by several pathways over the pH range 2.0-9.3 with a V-shaped pH-rate profile and a minimum pH of around 4.5. The stability of PNT solutions in mixed solvents was greater than in aqueous solutions. For example, in propylene glycol:citrate buffer (60:40, v/v, pH 4.5), the half-life of PNT was 4.3 days compared with 1.8 days in aqueous buffer. However, solubility of PNT in the mixed solvent was higher than that in aqueous solution and this had an adverse effect on the stability of suspensions. By judicious choosing of pH and mixed solvent, it is possible to achieve a storage life of a PNT suspension of 5.5 months at 5°C, not sufficient for a ready-to-use product but a dramatic improvement in the storage life of the reconstituted product.

Delivery of drugs to the brain via the blood brain barrier using colloidal carriers.

Delivering drugs to the brain is challenging given the selective permeability of the blood brain barrier (BBB). Targeted colloidal carriers containing drug payloads offer some promise for enhanced and perhaps selective delivery to brain. This review examines the recent literature and identifies issues to be addressed if these systems are to be rationally designed. These include opsonization of nanoparticles and off-target clearance; the cerebral microvasculature, flow of nanoparticles in capillaries and binding to the capillary wall; and transcytosis. Capillary architecture, blood flow and BBB permeability are affected by disease and age and there are species differences. These complexities caution against making extravagant claims for a particular nanosystem but they also highlight the rich opportunities and need for critical research in this field.

Modeling the interaction of polymeric nanoparticles functionalized with cell penetrating peptides at the nano-bio interface.

The interaction of nanoparticles with Caco-2 monolayers in cell culture underpins our predictions of the uptake of nanoformulations in vivo for drug delivery. Cell-penetrating peptides (CPP), such as oligoarginine, are currently of interest to enhance cellular uptake of bioactives and nanoparticles. This paper assesses the cellular association of poly(ethyl-cyanoacrylate) nanoparticles functionalized with di-arginine-histidine (RRH) in a Caco-2 cell model. We applied a computational model of particokinetics, In vitro Sedimentation, Diffusion and Dosimetry (ISDD) to predict the accumulation of nanoparticles on the cell surface. An important finding is that the proportion of nanoparticles associated with cells was less than 5 %. This has important implications for interpreting nanoparticle uptake in vitro. RRH-decoration does not appear to alter nanoparticle deposition, but increases association of nanoparticles with Caco-2 cells. Immediate deposition of nanoparticles on the cell surface was apparent and similar between formulations, but underestimated by the ISDD model. Key to understanding the nano-bio interface for drug delivery, nanoparticles that reach the cells were not necessarily absorbed by them, but can become detached. This variable of nanoparticle release from cells was incorporated into a new mathematical model presented here.

Studies on the safety and the tissue distribution of inhaled high-dose amorphous and crystalline rifampicin in a rat model.

Inhaled delivery of rifampicin has the potential to achieve high drug concentrations in the lung and the blood for efficient treatment of tuberculosis (TB). Due to its existence as polymorphs, in vivo evaluation of the respiratory tract safety of inhalable amorphous and crystalline rifampicin particles, at clinically relevant high-dose, is necessary. This study investigates the lung and liver safety and the tissue distribution of rifampicin after intra-tracheal administration of high (≥25 mg/kg) doses of amorphous and crystalline powder formulations to Sprague Dawley rats. Powder formulations were administered by intra-tracheal insufflation to rats. Lung and liver safety were evaluated by histopathology. Serum alanine transaminase (ALT) and aspartate aminotransferase (AST) assays were performed to study the hepatic effects. Rifampicin was quantified in the tissues using LC-MS/MS. Intra-tracheal administration of rifampicin decreased the drug burden on the liver compared to oral administration based on its lower serum ALT activity. Repeated-dose intra-tracheal rifampicin was well tolerated by rats, confirmed by the absence of drug or delivery induced complexities. The histopathological evaluation of rat lungs, after both single and repeated drug administration for seven days, suggested the absence of drug-induced toxicity. Following single intra-tracheal delivery of 50 mg/kg doses, comparable rifampicin concentrations to that from same oral dose were observed in lung, liver, heart and brain. Inhaled delivery of high-dose rifampicin was safe to rat lungs and liver suggesting its potential for localized as well as systemic drug delivery without toxicity concerns.

Pharmacokinetics of rifampicin after repeated intra-tracheal administration of amorphous and crystalline powder formulations to Sprague Dawley rats.

Rifampicin is one of the key drugs used to treat tuberculosis and is currently used orally. The use of higher oral doses of rifampicin is desired for better therapeutic efficacy, but this is accompanied by increased risk of systemic toxicity thus limiting its recommended oral dose to 10 mg/kg per day. Inhaled delivery of rifampicin is a potential alternative mode of delivery, to achieve high drug concentrations in both the lung and potentially the systemic circulation. In addition, rifampicin exists either as amorphous or crystalline particles, which may show different pharmacokinetic behaviour. However, disposition behaviour of amorphous and crystalline rifampicin formulations after inhaled high-dose delivery is unknown. In this study, rifampicin pharmacokinetics after intra-tracheal administration of carrier-free, amorphous and crystalline powder formulations to Sprague Dawley rats were evaluated. The formulations were administered once daily for seven days by oral, intra-tracheal and oral plus intra-tracheal delivery, and the pharmacokinetics were studied on day 0 and day 6. Intra-tracheal administration of the amorphous formulation resulted in a higher area under the plasma concentration curve (AUC) compared to the crystalline formulation. For both formulations, the intra-tracheal delivery led to significantly higher AUC compared to the oral delivery at the same dose suggesting higher rifampicin bioavailability from the inhaled route. Increasing the intra-tracheal dose resulted in a more than dose proportional AUC suggesting non-linear pharmacokinetics of rifampicin from the inhaled route. Upon repeated administration for seven days, no significant decrease in the AUCs were observed suggesting the absence of rifampicin induced enzyme auto-induction in this study. The present study suggests an advantage of inhaled delivery of rifampicin in achieving higher drug bioavailability compared to the oral route.

Cell-based, animal and H1 receptor binding studies relative to the sedative effects of ketotifen and norketotifen atropisomers.

OBJECTIVES: Ketotifen (K) and its active metabolite norketotifen (N) exist as optically active atropisomers. They both have antihistaminic and anti-inflammatory properties but the S-atropisomer of N (SN) causes less sedation than K and RN in rodents. This study investigated whether this could be related to a lower concentration of SN in brain or a lower affinity of SN for rat brain H1 receptors. METHODS: Ketotifen and norketotifen atropisomers were quantified using a validated chiral HPLC assay. RBE4 and Caco-2 cell monolayers were used in uptake and permeability studies, respectively. Free and total brain-to-plasma (B/P) ratios were determined after injecting racemic K and N into rat tail veins. Affinity for rat brain H1 receptors (KI ) was determined using the [3 H]mepyramine binding assay. KEY FINDINGS: Uptake and permeation studies indicate no stereoselective transport for K or N. B/P ratios reveal the brain concentration of N is lower than K with no stereoselective transport into brain. Finally, the [3 H]mepyramine binding assay shows SN has the lowest affinity for rat brain H1 receptors. CONCLUSION: The lower sedative effect of SN in rodents is probably due to a combination of a lower uptake of N than K into the brain and less affinity of SN for CNS H1 receptors.

Monoketocholate can decrease transcellular permeation of methotrexate across Caco-2 cell monolayers and reduce its intestinal absorption in rat.

OBJECTIVES: Bile salts have been shown to decrease the absorption of methotrexate in the rat intestine by an unknown mechanism. We aimed to examine this effect. METHODS: We assessed apical-to-basolateral (AP-BL) permeation of methotrexate (5 muM) across Caco-2 cell monolayers pretreated with various concentrations (0, 0.25, 0.5, 1, 3 and 5 mM) of sodium cholate or its semisynthetic analogue, sodium 12-monoketocholate. We also determined the effect of orally administered 12-monoketocholate on the intestinal absorption of methotrexate in rats to evaluate a possible in-vitro-in-vivo correlation. KEY FINDINGS: It was found that sodium cholate and sodium 12-monoketocholate decreased the AP-BL permeation of methotrexate at low concentrations (maximal inhibition at 0.25 and 1 mM, respectively) and increased it at higher concentrations. Determination of [(14)C] mannitol permeation and electrical resistance of monolayers during experiments showed that membrane integrity was not compromised at low concentrations of bile salts but was disrupted at higher concentrations. Subsequently, we examined the effect of the simultaneous oral administration of sodium 12-monoketocholate (4, 20, 40 and 80 mg/kg) on the intestinal absorption of methotrexate in rats after an oral dose (5 mg/kg). The pharmacokinetic study showed that 12-monoketocholate at 4 and 20 mg/kg did not change the methotrexate area under the serum concentration-time curve whereas sodium 12-monoketocholate at 40 and 80 mg/kg significantly reduced it. CONCLUSIONS: Sodium 12-monoketocholate appears to decrease the intestinal absorption of methotrexate in rats by inhibition of transcellular active transport.

An improved HPLC method for the investigation of L-selenomethionine metabolism in rat gut contents.

Selenomethionine (SeMet) is a widely used nutritional supplement that has potential benefit for people living in selenium-deficient areas. Previous research has shown that selenium administered as SeMet undergoes significant enterohepatic recycling which may involve the gut microflora. In order to investigate this we have developed a simple method for the quantitation of l-SeMet in rat gut content suspensions prepared from jejunum, ileum, caecum and colon. After incubation of l-SeMet with gut content suspensions, samples were deproteinized with sulfosalicylic acid and derivatized with o-phthaldialdehyde (OPA) and N-acetyl-l-cysteine (NAC). Mass spectrometry confirmed the formation of a 1:1:1 derivative of l-SeMet with OPA and NAC. Samples were analysed by reversed-phase high-performance liquid chromatography with fluorescence detection. The assay was linear in the concentration range 0.5-100 microg/mL (r(2) = 0.9992) with a limit of detection of 0.025 microg/mL (signal-to-noise ratio of 5). Intra-day and inter-day accuracies were 91.1-92.8 and 91.7-95.5%, respectively with corresponding precisions as relative standard deviation of <5%. Incubation of l-SeMet with gut content suspensions from different parts of the rat intestine showed that l-SeMet metabolism occurs mainly in the caecum.