Permeability of Chemical Delivery Systems Across Rabbit Corneal (SIRC) Cell Line and Isolated Corneas: A Comparative Study

The purpose of this study was to investigate the corneal permeability of phenylephrone chemical delivery systems (CDS) across isolated cornea and to evaluate the utility of the SIRC cell line (epithelial cells originating from rabbit cornea) as an in vitro model for predicting the ocular permeability. The effect of benzalkonium chloride (BAC) on the drug permeability through SIRC cell layers was also studied. The transport of phenylephrone CDS across the isolated cornea of the albino rabbit was measured at various pH values using a two-chamber glass diffusion cell, and the results were compared with the reported permeability values across SIRC cells of rabbit origin. Corneal membranes showed lower flux values for compounds, especially for hydrophilic compounds, than the SIRC cell line. A significant correlation was observed between the permeability coefficients through corneal membranes and SIRC cells. When the pH of the transport medium was increased, the permeability coefficients increased and lag times decreased in both in vitro models. Furthermore, both in vitro models showed significant correlation between permeability coefficients and lipophilicities of the drugs. The three esters, having higher lipophilic characteristics, showed higher permeability than phenylephrine HCl. The phenylacetyl ester of phenylephrone showed a three-fold increase in penetration across SIRC cell layers in the presence of 0.01% BAC. These results suggest that the use of SIRC cell layers can reasonably predict the permeability of ophthalmic drugs across corneal membranes.     

Permeability of chemical delivery systems across rabbit corneal (SIRC) cell line and isolated corneas: a comparative study

The purpose of this study was to investigate the corneal permeability of phenylephrone chemical delivery systems (CDS) across isolated cornea and to evaluate the utility of the SIRC cell line (epithelial cells originating from rabbit cornea) as an in vitro model for predicting the ocular permeability. The effect of benzalkonium chloride (BAC) on the drug permeability through SIRC cell layers was also studied. The transport of phenylephrone CDS across the isolated cornea of the albino rabbit was measured at various pH values using a two-chamber glass diffusion cell, and the results were compared with the reported permeability values across SIRC cells of rabbit origin. Corneal membranes showed lower flux values for compounds, especially for hydrophilic compounds, than the SIRC cell line. A significant correlation was observed between the permeability coefficients through corneal membranes and SIRC cells. When the pH of the transport medium was increased, the permeability coefficients increased and lag times decreased in both in vitro models. Furthermore, both in vitro models showed significant correlation between permeability coefficients and lipophilicities of the drugs. The three esters, having higher lipophilic characteristics, showed higher permeability than phenylephrine HCl. The phenylacetyl ester of phenylephrone showed a three-fold increase in penetration across SIRC cell layers in the presence of 0.01% BAC. These results suggest that the use of SIRC cell layers can reasonably predict the permeability of ophthalmic drugs across corneal membranes.     

Drug delivery studies in Caco-2 monolayers. III. Intestinal transport of various vasopressin analogues in the presence of lysophosphatidylcholine

The transport of a series of vasopressin and oxytocin analogues with varying lipophilicities was studied in Caco-2 monolayers. Transport was studied across the bare monolayer and after treatment with a phospholipid absorption enhancer, palmitoyl lysophosphatidylcholine. The range in lipophilicity of the analogues, estimated as the capacity factor, was found to be from 0.19 to 3.43. The intrinsic transport of the peptides across Caco-2 monolayers was found to be low. The apparent permeability coefficients, P_app, were in the range of 2 × 10⁻⁸–6 × 10⁷ cm/s. However, peptide transport was significantly greater (P_app in the range of 5 × 10⁻⁶–2 × 10⁻⁵ cm/s) when facilitated by addition of palmitoyllysophosphatidylcholine. The results suggest that polypeptide transport across Caco-2 monolayers does not depend on lipophilicity, but that the facilitated transport does depend on the lipophilicity.     

Functional differences in nucleoside and nucleobase transporters expressed on the rabbit corneal epithelial cell line (SIRC) and isolated rabbit cornea

The purpose of this study was to investigate the expression of nucleoside/nucleobase transporters on the Statens Seruminstitut rabbit corneal (SIRC) epithelial cell line and to evaluate SIRC as an in vitro screening tool for delineating the mechanism of corneal permeation of nucleoside analogs. SIRC cells (passages 410–425) were used to study uptake of [³H]thymidine, [³H]adenine, and [³H]ganciclovir. Transport of [³H]adenine and [³H]ganciclovir was studied across isolated rabbit cornea. Uptake and transport studies were performed for 2 minutes and 120 minutes, respectively, at 34°C. Thymidine uptake by SIRC displayed saturable kinetics (K _m=595.9±80.4μM, and V _max=289.5±17.2 pmol/min/mg protein). Uptake was inhibited by both purine and pyrimidine nucleosides but not by nucleobases. [³H]thymidine uptake was sodium and energy independent but was inhibited by nitrobenzylthioinosine at nanomolar concentrations. Adenine uptake by SIRC consisted of a saturable component (K _m=14.4±2.3μM, V _max=0.4±0.04 nmol/min/mg protein) and a nonsaturable component. Uptake of adenine was inhibited by purine nucleobases but not by the nucleosides or pyrimidine nucleobases and was independent of sodium, energy, and nitrobenzylthioinosine. [³H]ganciclovir uptake involved a carrier-mediated component and was inhibited by the purine nucleobases but not by the nucleosides or pyrimidine nucleobases. However, transport of [³H]adenine across the isolated rabbit cornea was not inhibited by unlabeled adenine. Further, corneal permeability of ganciclovir across a 100-fold concentration range remained constant, indicating that ganciclovir permeates the cornea primarily by passive diffusion. Nucleoside and nucleobase transporters on rabbit cornea and corneal epithelial cell line, SIRC, are functionally different, undermining the utility of the SIRC cell line as an in vitro screening tool for elucidating the corneal permeation mechanism of nucleoside analogs.     

MDCK (Madin-Darby Canine Kidney) Cells: A Tool for Membrane Permeability Screening

The goal of this work was to investigate the use of MDCK (Madin–Darby canine kidney) cells as a possible tool for assessing the membrane permeability properties of early drug discovery compounds. Apparent permeability (P_app) values of 55 compounds with known human absorption values were determined using MDCK cell monolayers. For comparison, P_app values of the same compounds were also determined using Caco‐2 cells, a well‐characterized in vitro model of intestinal drug absorption. Monolayers were grown on 0.4‐µm Transwell‐COL membrane culture inserts. MDCK cells were seeded at high density and cultured for 3 days, and Caco‐2 cells were cultured under standard conditions for 21 to 25 days. Compounds were tested using 100 µM donor solutions in transport medium (pH 7.4) containing 1% DMSO. The P_app values in MDCK cells correlated well with those in Caco‐2 cells (r² = 0.79). Spearman's rank correlation coefficient for MDCK P_app and human absorption was 0.58 compared with 0.54 for Caco‐2 P_app and human absorption. These results indicate that MDCK cells may be a useful tool for rapid membrane permeability screening.     

Design of potent lipophilic-peptide inhibitors of human neutrophil elastase: In vitro and in vivo studies

In keeping with the presence of an extended hydrophobic binding site in human neutrophil elastase (HNE), a series of lipophilic peptide derivatives were synthesized. In vitro studies, using an oligopeptide substrate for HNE, showed that lipidic amino acid derivatives 1b, 1c and 1d were potent HNE inhibitors; the lipophilic amino acid trimer conjugate ld (IC₅₀ = 1.8 × 10⁻¹⁰) was a more powerful inhibitor than the dimer conjugate lc (IC₅₀ = 2.8 × 10⁻¹⁰) which was more potent than the monomer conjugate lb (IC₅₀ = 2.9 × 10⁻⁹). When injected intradermally to rabbit dermis, compound ld protected skin elastic fibres against degradation by HNE.     

Absorption mechanism of oxymatrine in cultured Madin–Darby canine kidney cell monolayers

Context Oxymatrine (OMT) is beneficial to human health by exerting various biological effects. Objective To investigate the absorption mechanism of OMT and discover absorption enhancers using Madin–Darby canine kidney (MDCK) cell monolayers.

Materials and methods Concentration effects on the transport of OMT were measured in the range of 1.0?×?10^?5–1.0?×?10^?3 M in 2?h. Then, the effect of time, direction, temperature and pH on the transport of OMT at 10^?4 M was studied. Moreover, P_app of OMT was determined in the absence/presence of cyclosporine and surfactants at 100?μM to further confirm the relative transport mechanism.

Results The P_{app AP→BL} ranged from (3.040?±?0.23)?×?10^?6 to (3.697?±?0.19)?×?10^?6?cm/s as the concentration varied from 10^?5 to 10^?3 M. OMT showed similar P_app at 4 and 37?°C (p?>?0.05). Increasing the apical pH 7.4 and 8.0 resulted in P_app versus pH 5.0 (p?<?0.01). Furthermore, in the presence of cyclosporine and surfactants including sodium citrate, sodium dodecyl sulphate (SDS) and deoxysodium cholate, P_app was (0.318?±?0.033)?×?10^?5, (0.464?±?0.048)?×?10^?5, (0.897?±?0.115)?×?10^?5 and (1.341?±?0.122)?×?10^?5?cm/s, respectively. In the presence of surfactants, P_app significantly increased up to 1.5–4.3-fold (p?<?0.05).

Discussion and conclusion OMT transport across MDCK cell monolayers was by passive diffusion. Sodium citrate, SDS and deoxysodium cholate serve as excellent absorption enhancers which are useful for the related research improving the oral bioavailability of OMT.     

Facile α-chymotrypsin-catalyzed degradation of the HIV inhibitor [d-Ala1]-Peptide T amide

[d-Ala¹]-Peptide T amide is an octapeptide presently undergoing clinical evaluation as a potential agent for the treatment of AIDS. As a part of studies aiming at obtaining an oral delivery form of the peptide, its stability towards α-chymotrypsin was examined. The peptide was found to be rapidly degraded by this pancreatic enzyme, the degradation being due to cleavage of the Tyr-ThrNH₂ bond. At pH 7.4 and 37°C the peptide showed a K_m value of 1.3 × 10⁴ M, a k_cat value of 560 min⁻¹ and a specificity constant (k_cat/K_m) of 4.3 × 10⁶ M⁻¹ min⁻¹. At physiological concentrations of α-chymotrypsin and at pH 6.0–7.4 the half-life of degradation of the peptide was calculated to be less than 5 s. It is thus concluded that a means to protect [d-Ala¹]-Peptide T amide against cleavage by α-chymotrypsin is an absolute requirement for the development of an orally absorbable product. The peptide proved highly stable toward aminopeptidase as well as in human plasma.     

Design of novel prodrugs for the enhancement of the transdermal penetration of indomethacin

A series of esters of indomethacin containing tertiary amine functional groups and designed for transdermal delivery were synthesized. The rates of chemical hydrolysis of all the esters in pH 7.4 phosphate buffer were determined. For N,N-diethylaminopropyl N-(4-chlorobenzoyl)-5-methoxy-2-methyl-3-indole acetate (ester 4), the rates of chemical hydrolysis in pH 5.5–11.25 buffers were investigated in detail. Ester 4 was chemically stable and had a higher n-octanol/water partition coefficient and a greater solubility in water at pH 7.4 than indomethacin. Furthermore, the rate of transdermal penetration of ester 4 through cadaver skin in Franz cells was found to be more than 10-times faster than that of indomethacin itself. Ester 4 was found to possess surface-active properties (CMC = 0.5 mg/ml). Assuming that micelles cannot penetrate biologic membranes, a corrected permeability coefficient was calculated for ester 4 using only the monomer concentration. This value, 3.6 × 10⁻² cm/h, was 100-times greater than that of indomethacin. These results suggest that prodrugs with structures similar to that of ester 4 may be useful for enhancing transdermal penetration of other carboxylic acid-containing anti-inflammatory agents.     

Myocardial and vascular effects of efonidipine in vitro as compared with nifedipine,verapamil and diltiazem

• 1.1. Effects of efonidipine on isolated myocardial and aortic preparations were compared with those of nifedipine, verapamil and diltiazem.
• 2.2. All drugs produced concentration-dependent negative chronotropic effects on isolated guinea-pig atrial preparations. The potency order was efonidipine⩾nifedipine>diltiazem⩾verapamil, EC₃₀ values being 3.08×10⁻⁸M, 3.48× 10⁻⁸M, 1.27×10⁻⁷M and 1.47×10⁻⁷M, respectively.
• 3.3. Nifedipine, verapamil and diltiazem produced concentration-dependent negative inotropic effects on isolated guinea-pig left atrial preparations. The potency order was nifedipine>verapamil>diltiazem, EC₃₀ values being 4.94× 10⁻⁸M, 1.49×10⁻⁷M and 8.03×10⁻⁷M, respectively. Efonidipine, even at 1 μM produced no inotropic effect: 10 μM efonidipine decreased the contractile force by about 20%.
• 4.4. All drugs concentration-dependently attenuated the KCl-induced contraction of isolated rat aortic ring preparation. The potency order was nifedipine>efonidipine>verapamil>diltiazem, EC₃₀ values being 2.98× 10⁻⁹M, 1.24×10⁻⁸M, 3.96×10⁻⁸M and 2.13×10⁻⁷M, respectively.
• 5.5. Thus, efonidipine was demonstrated to be a potent vasodilator with negative chronotropic but minimal negative inotropic activity, which may be of benefit in the treatment of cardiovascular disorders.

     

A fluorimetric,potentiometric and conductimetric study of the aqueous solutions of naproxen and its association with hydroxypropyl-β-cyclodextrin

The dissociation constant, K_a, of naproxen, a nonsteroidal antiinflammatory drug of the family of arylpropionic acids, has been determined in aqueous solutions at 25°C by using a potentiometric and a conductimetric techniques. The solubility limit of the drug in water, a controversial point in the literature, has been found to be less than 3×10⁻⁵ M. The interaction of naproxen with hydroxypropyl-β-cyclodextrin (HPBCD), in terms of the binding constants of the complexes formed by the CD and the nonionic (HNAP) and ionic (NAP⁻) species of the drug, has been evaluated at 25°C as well by means of steady-state fluorescence enhancement studies. A discussion of the results, K_HPBCD:HNAP=6500±400 M⁻¹ and K_HPBCD:NAP−=1400±80 M⁻¹, emphasizing the crucial importance of the choice of the pH at a value that pH≥pK_a +2 or pH≤pK_a −2, is also included.     

Water-soluble salts of aminoacid esters of the anaesthetic agent Propofol

The glycinates 4, 5, acetates 6, 7, 10, propionate 8, butyrate 9 and carbonate 11 were synthesized and evaluated as potential water-soluble prodrugs of Propofol (2,6-diisopropylphenol) 1 suitable for parenteral administration. The 4–9 · HCl salts were also prepared and some of them (i.e. 4 · HCl and 6 · HCl) were found sufficiently soluble in aqueous solutions. The kinetics of hydrolysis of the esters 4–11 and 4–9 · HCl salts were studied in 0.05 M phosphate buffer pH 7.4, and a number of derivatives (4, 6, 7, and corresponding HCl salts) were examined for their stability in human plasma and brain homogenate. Our results indicated that the salts 4 · HCl and 6 · HCl, sufficiently soluble in water, are relatively stable in physiological media. Most of the examined compounds, in particular compound 6, were found to inhibit the binding of [³⁵S]-tert-butylbicyclophosphorothionate ([³⁵S]TBPS) demonstrating to possess affinity for the Propofol recognition site on GABA_A receptors.     

Chitosan for enhanced intestinal permeability: Prospects for derivatives soluble in neutral and basic environments

In this study the effects of two chitosan salts, namely chitosan hydrochloride and chitosan glutamate (0.5 and 1.5% w/v), on the transepithelial electrical resistance (TEER) and permeability of Caco-2 cell monolayers, using the radioactive marker [¹⁴C]-mannitol, were investigated in a slightly acidic (pH 6.2) and neutral (pH 7.4) environment. Both salts are soluble in acidic conditions up to a concentration of 1.5% w/v and solutions of this strength, at a pH of 6.2, caused a pronounced lowering in the TEER of Caco-2 cell monolayers in the order of 70±1% (chitosan glutamate) and 77±3% (chitosan hydrochloride), 20 min after incubation started. In agreement with the TEER results the transport of the radioactive marker, [¹⁴C]-mannitol, was increased 25-fold (chitosan glutamate) and 36-fold (chitosan hydrochloride), respectively, at this pH. However, at a pH of 7.4 both salts are insoluble and prove to be ineffective since no reduction in the TEER values or increase in the transport of [¹⁴C]-mannitol were found. The results show that these chitosan salts are potent absorption enhancers in acidic environments. We conclude that there is a need for chitosan derivatives with increased solubility, especially at neutral and basic pH values, for use as absorption enhancers aimed at the delivery of therapeutic compounds in the more basic environment of the large intestine and colon.     

Effect of naringin and naringenin on contractions induced by noradrenaline in rat vas deferens—I. Evidence for postsynaptic alpha-2 adrenergic receptor

• 1.1. Naringin at all doses (2 × 10⁻⁶, 5 × 10⁻⁷, and 1 × 10⁻⁷ M) significantly increased contractions induced by noradrenaline in rat vas deferens but the increments of maximal contraction were not concentration-dependent.
• 2.2. In a medium containing 1 × 10⁻⁶ M yohimbine (a selective blocker of α₂-adrenoceptor) and naringin, the curve constructed with noradrenaline decreased below the control curve.
• 3.3. Naringenin (aglycone of naringin) (2 × 10⁻⁶ and 1 × 10⁻⁷ M) increased the contractile effect of noradrenaline and the maximal effect evoked was related to the maximal dose of naringenin.
• 4.4. The α₂ antagonism produced by yohimbine in the naringenin-noradrenaline association were retained at two doses of naringenin tested and we noticed a similar behaviour when we used clonidine-noradrenaline.

     

Use of SIRC Rabbit Corneal Cell Lines Grown on Polycarbonate- or Polyester-Based Filters to Assess the In Vitro Corneal Transport/Toxicity Screening Using Pilocarpine With or Without Benzalkonium Chloride

The objective of this study was to assess and compare the morphologic and permeability characteristics of SIRC rabbit corneal cells grown on two filter types, polycarbonate-based Costar Transwell and polyester-based Costar Transwell Clear using pilocarpine in presence and absence of benzalkonium chloride. Technically, the microwell inserts were seeded with SIRC rabbit corneal cells and suspended in growth medium. The inserts were covered and kept in a humidified incubator for 10 days. The inserts were rinsed with Dulbecco's phosphate buffered saline at pH 7.3 and placed into microwell plates containing buffer at the same pH. Formulations containing pilocarpine with and without benzalkonium chloride were inoculated into each insert. The inserts were covered and incubated for the predetermined time period. The cell lines were examined with a light microscope using hematoxylin and eosin-stained cross-sections. The permeabilities of pilocarpine across the SIRC cell layers grown on the two filters were quantitatively determined. The results of these experiments show no significant differences in the morphological characteristics between the two filters exposed to either pilocarpine alone or to pilocarpine with benzalkonium chloride. The apparent permeability coefficient values for pilocarpine from both formulations across Costar Transwell-grown SIRC cell layers were relatively higher than that with Costar Transwell Clear-grown layers, and the flux enhancement ratios in the presence of benzalkonium chloride across the two filter types were comparable. These results revealed that the morphologic and the permeability data of SIRC rabbit corneal cells grown on the two filters are comparable for the compounds tested, suggesting their interchangeable use in assessment of the in vitro corneal drug transport and toxicity screening studies for ophthalmic drugs and additives.     

Independence of substituent contributions to the transport of small-molecule permeants in lipid bilayer

Purpose: To explore the independence of functional group contributions to permeability of nonelectrolytes across egg lecithin bilayers. Methods. The transport rates were measured of a series of α-substituted p-methylhippuric acids (-H,-Cl,-OCH₃,-CN,-OH,-COOH, and-CONH₂) across egg lecithin lipid bilayers, in the form of large unilamellar vesicles (LUVs) at 25EC. Intrinsic permeability coefficients (P_HA) were calculated from apparent permeability coefficients (P_app) measured as a function of pH. Group contributions to the free energy of transfer from water into the barrier domain, Δ(ΔGE)_P,X, were calculated for the substituents and compared to the contributions of these groups when attached to p-toluic acid measured earlier. The Δ(ΔGE)_P,X values from permeability data were also correlated with Δ(ΔGE)_PC,X values of partitioning from water into organic solvents to determine the physicochemical selectivity of the barrier domain. Results. P_app values in LUVs were found to vary approximately linearly with the fraction of neutral permeant over a pH range of 5.5 to 10.5, suggesting that the transport of the ionized species is negligible over this pH range. The Δ(ΔGE)_P,X values from the 2 series of compounds appear to be the same, indicating that the functional group contributions are independent. 1,9-Decadiene was found to be the most similar to the chemical environment of the barrier domain. Conclusions. Functional group contributions to transport across egg lecithin bilayers appear to be independent of the compound to which they are attached, even though the thickness of the barrier domain in lipid bilayers is approximately the same as the extended length of the permeant.     

In Vitro and In Situ Evaluation of pH-Dependence of Atazanavir Intestinal Permeability and Interactions with Acid-Reducing Agents

Purpose

The objectives of this study were to evaluate the effects of intestinal lumen pH, food intake, and acid-reducing agents on the intestinal permeability of atazanavir, an HIV-1 protease inhibitor.

Methods

Atazanavir permeability across Caco-2 cell monolayers (P _app) and in situ steady-state permeability across rat jejunum and ileum (P _eff) were evaluated in buffers of varied pH (4.5–8.5), in fasted- or fed-state simulated intestinal fluid, or in presence of acid-reducing drugs (e.g., omeprazole).

Results

In vitro accumulation and apical-to-basolateral P _app of atazanavir increased with decreasing pH. This effect appeared to be associated with lower atazanavir efflux by P-glycoprotein at acidic pH (5.5) compared to neutral pH. In situ atazanavir P _eff across rat jejunum and ileum also decreased 2.7 and 2.3-fold, respectively, when pH was increased from 4.5 to 8.5. Several acid-reducing agents (e.g., omeprazole) moderately inhibited atazanavir efflux in Caco-2 monolayers; however, this effect was not observed in situ. Fed-state buffer significantly increased atazanavir apical-to-basolateral P _app (p?in situ P _eff (p?Conclusions Atazanavir permeability is sensitive to changes in intestinal lumen pH. This pH-sensitivity may contribute to atazanavir clinical interactions with acid-reducing agents and variable oral bioavailability.     

Effects of S-adenosyl-L-methionine on platelet thromboxane and vascular prostacyclin

We therefore designed the present study to evaluate the effect of S-adenosyl-L-methionine (SAMe) on the synthesis of platelet thromboxane and vascular prostacyclin. The experimental materials were human blood and aortic rings from untreated Wistar rats; and platelets and aortic rings from Wistar rats treated for 7 days with SAMe at 5 or 10 mg/kg/day s.c. The administration of 10 mg/Kg/day of SAMe to rats significantly increased vascular production of 6-keto-PGF_1α. In vitro vascular production of 6-keto-PGF_1α increased in a concentration-dependent manner when SAMe was incubated in the range of 10⁻⁷ to 10⁻⁴ M. The greatest increase was 167 ± 15%, obtained in samples incubated with 5 × 10⁻⁵M SAMe. In aortic rings, lipid peroxidase production was inhibited in a concentration-dependent manner in the SAMe range of 10⁻⁷ to 10⁻⁵ M. Maximum inhibition (75.3 ± 6.2%) was obtained with SAMe at 1.5 × 10⁻⁵M. Vascular 6-keto-PGF_1α production showed a significant inverse linear correlation with vascular lipid peroxide production (Y = −0.04 × + 18.1, r = 0.7309, P < 0.0001).     

The Transport and Efflux of Glycosylated Luteinising Hormone-Releasing Hormone Analogues in Caco-2 Cell Model: Contributions of Glucose Transporters and Efflux Systems

Luteinising hormone-releasing hormone (LHRH) analogues have wide therapeutic applications in the treatment of prostate cancers and endocrine disorders. The structure of LHRH was modified using a glycosylation strategy to increase the permeability of the peptide across biological membranes. Lactose, galactose and glucose units were coupled to LHRH peptide, and the impact of glucose transporters, GLUT2 and SGLT1, was investigated in the transport of the analogues. Results showed the contribution of both transporters in the transport of all LHRH analogues. In the presence of glucose transporter inhibitors, reduction in the apparent permeability (P_app) was greatest for compound 6, which contains a glucose unit in the middle of the sequence (P_app = 58.54 ± 4.72 cm/s decreased to P_app = 1.6 ± 0.345 cm/s). The basolateral to apical flux of the glycosylated derivatives and the impact of two efflux pumps was also examined in Caco-2 cell monolayers. The efflux ratios (ERs) of all LHRH analogues in Caco-2 cells were in the range of 0.06–0.2 except for compound 4 (galactose modified, ER = 8.03). We demonstrated that the transport of the glycosylated peptides was facilitated through glucose transporters. The proportion of glucose and lactose derivatives pumped out by efflux pumps did not affect the P_app values of the analogues.     

Kinetics and Mechanism of the Base-Catalyzed Rearrangement and Hydrolysis of Ezetimibe

The pH-rate profile of the pseudo-first-order rate constants for the rearrangement and hydrolysis of Ezetimibe giving (2R,3R,6S)-N,6-bis(4-fluorophenyl)-2-(4-hydroxyphenyl)-3,4,5,6-tetrahydro-2H-pyran-3-carboxamide (2) as the main product at pH of less than 12.5 and the mixture of 2 and 5-(4-fluorophenyl)-5-hydroxy-2-[(4-fluorophenylamino)-(4-hydroxyphenyl)methyl]-pentanoic acid (3) at pH of more than 12.5 in aqueous tertiary amine buffers and in sodium hydroxide solutions at ionic strength I = 0.1 mol L⁻¹ (KCl) and at 39°C is reported. No buffer catalysis was observed and only specific base catalysis is involved. The pH-rate profile is more complex than the pH-rate profiles for the hydrolysis of simple β-lactams and it contains several breaks. Up to pH 9, the log k_obs linearly increases with pH, but between pH 9 and 11 a distinct break downwards occurs and the values of log k_obs slightly decrease with increasing pH of the medium. At pH of approximately 13, another break upwards occurs that corresponds to the formation of compound 3 that is slowly converted to (2R,3R,6S)-6-(4-fluorophenyl)-2-(4-hydroxyphenyl)-3,4,5,6-tetrahydro-2H-pyran-3-carboxylic acid (4). The kinetics of base-catalyzed hydrolysis of structurally similar azetidinone is also discussed. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:2240–2247, 2014