Exploratory translational modeling approach in drug development to predict human brain pharmacokinetics and pharmacologically relevant clinical doses.

The central nervous system (CNS) pharmacokinetics (PK) of drugs that have pharmacological targets in the brain are not often understood during drug development, and this gap in knowledge is a limitation in providing a quantitative framework for translating nonclinical pharmacologic data to the clinical patient population. A focus of translational sciences is to improve the efficiency of clinical trial design via a more judicious selection of clinical doses on the basis of nonclinical data. We hypothesize that this can be achieved for CNS-acting drugs based on knowledge of CNS PK and brain target engagement obtained in nonclinical studies. Translating CNS PK models from rat to human can allow for the prediction of human brain PK and the human dose-brain exposure relationship, which can provide insight on the clinical dose(s) having potential brain activity and target engagement. In this study, we explored the potential utility of this translational approach using rat brain microdialysis and PK modeling techniques to predict human brain extracellular fluid PK of atomoxetine and duloxetine. The results show that this translational approach merits consideration as a means to support the clinical development of CNS-mediated drug candidates by enhancing the ability to predict pharmacologically relevant doses in humans in the absence of or in association with other biomarker approaches.

Peptide transporter function and prolidase activities in Caco-2 cells: a lack of coordinated expression.

Peptide transport and prolidase activities were measured to determine whether the expression of these two components of protein nutrition are coordinately regulated; i.e., whether an increase in the peptide transporter function will necessarily lead to a higher prolidase activity, or vice versa. The results indicated that peptide transporter function and prolidase activity respond differently to cell differentiation and feeding schedules. The results also indicated that peptide transport and prolidase activities were different in two Caco-2 cell "clones", with S-K cells transported peptides at higher rates but had lower total prolidase activities, when compared to ATCC cells. These results suggest that the expression of the peptide transporter function and prolidase activity is not coordinated. In addition, both the transporter and the prolidase activities affected the overall transport of Phe when given as the dipeptide Phe-Pro, supporting the notion that intestinal absorption of peptides is an essential component of amino acid absorption. In conclusion, the evidence suggests that the peptide transporter function and prolidase activity are not coordinately expressed by the human intestinal Caco-2 cells.

Aminopeptidase activity in albino rabbit extraocular tissues relative to the small intestine.

Aminopeptidase activity in the corneal epithelium and the conjunctiva of the albino rabbit eye was determined using L-leucine-, L-alanine-, and L-arginine-4-methoxy-beta-naphthylamide as substrates. This was compared with the aminopeptidase activity in the duodenum, jejunum, and ileum. The two ocular tissues were found to be 5-15% as active as the ileum in aminopeptidase activity. There was indirect evidence that the dominant aminopeptidase in the corneal epithelium is different from that in the conjunctiva, suggesting that further studies are warranted to investigate the possibility of delivering certain peptides systemically via the conjunctiva without simultaneously exposing the intraocular tissues to these peptides.

Systemic absorption of ocularly administered enkephalinamide and inulin in the albino rabbit: extent, pathways, and vehicle effects.

The systemic absorption of ocularly applied tritiated [D-Ala2]metenkephalinamide (YAGFM) and inulin was studied in the albino rabbit with respect to rate, extent, pathways, and vehicle effects and compared with epinephrine. Peak concentration was achieved within 20 min except for inulin, for which absorption was still ongoing at 120 min. For YAGFM, the apparent absorption rate was slower than the elimination rate, thus obeying "flip-flop" pharmacokinetics. Based on the area under the plasma concentration curve from zero to 120 min, the percent of dose systemically absorbed was 36.1 +/- 4.4% for YAGFM, at least 3.3 +/- 0.2% for inulin, and 58.5 +/- 4.4% for epinephrine. This suggests that loss of drug to the systemic circulation is a more important factor in reducing the ocular absorption of YAGFM than for inulin. The conjunctival mucosa played as important a role as the nasal mucosa in the systemic absorption of YAGFM, while playing a secondary role in the case of inulin. Unlike nonpeptide drugs, the systemic absorption of ocularly administered YAGFM and inulin was not adversely affected by incorporation in 5% polyvinyl alcohol. Overall, the contact time of the instilled dose with the conjunctival and the nasal mucosae, their intrinsic permeability, and the extent of dilution of the instilled dose are key factors determining the vehicle effects on the extent of systemic absorption of ocularly applied peptides.

Uptake characteristics of loracarbef and cephalexin in the Caco-2 cell culture model: effects of the proton gradient and possible presence of a distinctive second component.

The mechanisms of apical (AP) uptake of cephalexin (CEPH) and loracarbef (LOR) in the absence or presence of an (extemally imposed) proton gradient were determined using well-stirred diffusion chambers that minimize the effects of the unstirred water layer. The results indicated that, compared to AP uptake in the presence of an imposed proton gradient, AP uptake in the absence of an imposed proton gradient had higher K(m) values and lower Jmax values. Furthermore, when inhibition studies were performed in the absence of a proton gradient, only natural peptides were effective, whereas the peptide analogs (e.g., enalapril) were not. In addition to the effects of concentration and competitive inhibitors, the results also indicated that (1) the AP uptake of both drugs was decreased more than 60% by FCCP, regardless of whether the proton gradient was present or absent; (2) effects of protein kinase C promoter were dependent upon the presence of a proton gradient; and (3) AP uptake in the presence of an imposed proton gradient was not affected by feeding restriction, whereas AP uptake in the absence of an imposed proton gradient was. These results showed for the first time that two substrates with similar AP uptake characteristics in the presence of an imposed proton gradient may not share those characteristics in the absence of an imposed proton gradient. Taken together, these results suggest that the AP uptake component that functions in the absence of an imposed proton gradient is distinctly different from the one that functions in the presence of an imposed proton gradient. Data generated from the present study and those in the literature lend support to the hypothesis that this distinctive component represents the second binding site on the AP peptide transporter. However, an alternative hypothesis that there are two AP peptide transporters remains to be disapproved.

Regional-dependent intestinal absorption and meal composition effects on systemic availability of LY303366, a lipopeptide antifungal agent, in dogs.

Low oral bioavailability and a negative meal effect on drug plasma levels motivated studies on formulation and meal composition effects on the absorption of LY303366, a poorly water-soluble, semisynthetic, cyclic peptide antifungal drug. Solid drug particle size and meal composition studies were evaluated in beagle dogs. Canine regional absorption studies were also carried out utilizing surgically implanted intestinal access ports, and Caco-2 studies were performed to evaluate drug candidate intestinal permeability. Particle size and Caco-2 data indicate that drug permeability limitations to absorption are more important than dissolution rate limits. Caco-2 cell-associated LY303366 approached 10% of incubation concentration that is in the range of the oral bioavailability of the drug. Canine regional absorption studies showed that the extent of LY303366 absorption following duodenal administration was similar to that following oral administration. Significantly lower drug plasma levels were obtained following administration through a colonic access port, a result consistent with poor membrane permeation. Administration of drug with meals of mixed composition, as well as simple fat and protein meals, resulted in significant reductions in AUC(0-48h) compared with results from fasted dogs. In contrast, carbohydrate meals did not reduce drug plasma levels compared to controls. Intravenous pretreatment with devazepide, a cholecystokinin (CCK) antagonist that blocks canine biliary secretion, did not reverse the negative effect of the fat meal on LY303366. Taken together, the results from the present study suggest that membrane-permeability-limited absorption is the cause of the observed regionally dependent absorption of LY303366 in the dog and that the observed negative meal effects depend on composition but are independent of biliary secretion.

Application of oral bioavailability surrogates in the design of orally active inhibitors of rhinovirus replication.

Previous studies in rats and humans demonstrated poor oral bioavailability of potent in vitro 2-aminobenzimidazole inhibitors of rhinovirus replication due to significant first-pass elimination and possibly also to poor aqueous solubility. Estimations of aqueous solubility, as well as measurements of caco-2 permeability and NADPH dependent compound loss in rat liver microsomal incubations were employed alongside traditional in vivo experiments in rats to guide subsequent chemistry efforts. Retention of activity upon replacement of the metabolically labile vinyl oxime in the lead molecule with a vinyl carboxamide was a major breakthrough; however, oral bioavailability among the latter compounds was variable. Based on the ability to independently measure solubility, permeability, and metabolic stability of new compounds, variable solubility across the series (ranging from approximately 1 to 10 microg/mL) was identified as the cause of the inconsistent performance. Subsequent efforts to improve solubility led to the discovery of highly soluble (>10 mg/mL) and potent dessulfonyl vinyl carboxamide benzimidazoles. Determination of the metabolic stability of these compounds as a surrogate of the extent of their first-pass elimination supported a prediction of excellent oral bioavailability. In comparison to the sulfonyl-containing vinyl carboxamides, caco-2 permeabilities were reduced 5 to 10-fold; however, these were considered to be in the range of well-absorbed compounds based on comparison to a series of reference compounds of known percentage absorption in humans. Subsequent experiments in the rat verified the oral bioavailability of these N-alkyl compounds, with one compound (368177) having an absolute oral bioavailability of 89.4%. The application of solubility and caco-2 permeability as surrogates for oral absorption potential, in conjunction with the use of microsomal incubations as a surrogate for first-pass metabolism, was shown to augment a rational chemistry approach to discover orally bioavailable inhibitors of rhinovirus replication. Future expanded use of these surrogates is planned.

Ocular aminopeptidase activity and distribution in the albino rabbit.

Ocular aminopeptidase activity in the albino rabbit was determined using L-leucine-, L-alanine-, and L-arginine-4-methoxy-2-naphthylamide as substrates. The corneal epithelium and the iris-ciliary body were found to be the most active, followed by, in turn, conjunctiva and corneal stroma, lens and aqueous humor, and lastly tears. The pattern of substrate hydrolysis suggests that a common dominant aminopeptidase is present in these tissues except the conjunctiva. While the role of these peptidases in ocular physiology is unknown, they may play a role in limiting the entry of topically applied peptides into the eye.

Ocular distribution of liposome-encapsulated epinephrine and inulin in the albino rabbit.

A methodology has been developed to evaluate the disposition of liposome-encapsulated epinephrine and inulin in the conjunctival sac and selected ocular tissues. Relative to inulin, epinephrine effluxed much more rapidly from liposomes and disappeared more rapidly from the tear pool. As a result, liposomes were found to exert opposite effects on the ocular uptake of epinephrine and inulin. Over a period of 45 minutes, the concentration of epinephrine in the eye was 1.5 - 3 times lower when presented in liposomes than in aqueous solutions. This contrasted with the 3 to 15 fold increase in inulin concentration in the eye from liposomal preparations of the compound. Specifically, much of the increase in inulin concentration in the cornea can be attributed to an increase seen in the epithelium. Despite elevated inulin concentrations in the ocular tissues it bathes, the aqueous humor was devoid of inulin unless the corneal epithelium was physically removed prior to topical dosing. Collectively, these findings suggest that both the manner in which an entrapped compound interacts with the constituents of liposomes and the manner in which liposomes interact with the absorptive surfaces of the eye can significantly influence its pharmacokinetics in the eye.

Disposition of topically applied sodium cromoglycate in the albino rabbit eye.

The influence of pH, tonicity, preservatives, polymers and instilled drop size on the disposition of sodium cromoglycate, an agent used in the prophylaxis of vernal keratoconjunctivitis, in the tear chamber of the albino rabbit eye has been examined. Radiotracer techniques were used throughout. The initial decline in concentration in the tear chamber was found to be unaffected by the presence of preservatives, pH, and tonicity over the ranges studied. However, significant increases in the residence time of sodium cromoglycate in the precorneal area were noted when a smaller instilled drop size was used and when 5% polyvinyl alcohol (PVA) was added to the aqueous vehicle. Tissue uptake was found to be greatest in the conjunctiva, followed by the cornea, the iris-ciliary body and the aqueous humor. In both the conjunctiva and the cornea, the addition of 5% PVA produced an elevation in the peak concentration of sodium cromoglycate and an increase in the time at which the peak concentration was achieved, indicating improved drug delivery to these sites.

Development of a plasma high-performance liquid chromatographic assay for LY303366, a lipopeptide antifungal agent, and its application in a dog pharmacokinetic study.

An HPLC assay for plasma analysis of LY303366 (I), a semi-synthetic lipopeptide antifungal related to echinocandin B (ECB), was developed to support the selection and subsequent preclinical development of I. The method involved extraction of I from plasma with the aid of solid-phase extraction (SPE) cartidges followed by reversed-phase HPLC with UV detection at 300 nm. The method is simple, selective and is applicable to dog, rat, mouse and rabbit plasma. Validation studies using dog plasma showed that the values obtained for parameters of linearity, precision and accuracy were within acceptable limits. Based on analysis of 0.3 ml of plasma, the lower limit of quantitation was 20 ng/ml. The method has been successfully applied to determine the pharmacokinetic parameters of I in the dog following intravenous (i.v.) and oral administration. Compared to first generation ECB antifungal agents, the results of the i.v. dog study indicated a 50% reduction in clearance of the drug from plasma (0.1 l/h/kg) and an 18-fold increase in the volume of distribution at steady state (1.8 l/kg). When administered orally, compound I had an absolute bioavailability of 9%; however, plasma levels remained above the MIC for C. albicans (0.005 microg/ml) through 48 h. Given the excellent potency of I and its broad spectrum of activity relative to first generation ECB antifungal agents, the assay results for I indicate the potential for its use as a broad spectrum i.v. and oral antifungal agent.

Esterase distribution in the rabbit cornea and its implications in ocular drug bioavailability.

Being the major pathway by which topically applied ophthalmic drugs enter the eye, the cornea can control the amount of active drug ultimately absorbed by offering resistance to drug permeation and by metabolizing the drug during permeation. This research seeks to determine the esterase activity in the cornea and two of its component layers--epithelium and stroma-endothelium--so as to anticipate the extent to which drugs containing ester linkages will be metabolized during transport. This has been achieved by incubating corneal homogenates of albino and pigmented rabbits of various age groups with alpha-naphthyl acetate, the model substrate, and monitoring the fluorescence intensity due to alpha-naphthol as a function of time. The results indicate that: (1) esterase activity in the epithelium is approximately twice that in the stroma-endothelium; (2) esterase activity in the intact cornea is linearly related to those in the epithelium and the stroma-endothelium; and (3) the esterase activity in the cornea and its component layers varies with rabbit's age and strain. According to these results the bulk of esterase-mediated hydrolysis is expected to take place in the epithelium, so that the residence time of a drug in this tissue can have a significant impact on its ocular bioavailability.

Metabolic and permeation barriers to the ocular absorption of topically applied enkephalins in albino rabbits.

The objective of this study was to test the hypothesis that there existed substantial metabolic and permeation barriers to the ocular absorption of topically applied peptides in the albino rabbit. Leucine enkephalin, methionine enkephalin, and [D-ala2]-met-enkephalinamide were used as model peptides, while radiotracer techniques were used as the analytical methodology. The corneal epithelium was found to offer considerable resistance to the permeation of these peptides from tears into aqueous humor, as evidenced by the 1% or less of topical dose absorbed and by a 14 fold improvement in the amount of peptide absorbed upon removing the corneal epithelium prior to solution instillation. The metabolic barrier, believed to be composed of aminopeptidases and other peptidases, was also substantial in that less than 1% and 13% of the methionine and leucine enkephalins absorbed, respectively, remained intact. Unfortunately, this barrier was not perturbed appreciably by pretreating the eye with bestatin, an aminopeptidase inhibitor. Results obtained with [D-ala2]-met-enkephalinamide suggest that a more practical strategy to circumvent this metabolic barrier is to administer metabolically stable peptide analogs.

Mechanism and kinetics of transcellular transport of a new beta-lactam antibiotic loracarbef across an intestinal epithelial membrane model system (Caco-2).

Various processes involved in the transcellular transport (TT) of loracarbef (LOR) were studied in the Caco-2 cell monolayer, a cell culture model of the small intestinal epithelium. The results provide support for presence of two AP to BL peptide TT pathways in the intestinal epithelial cell monolayer (Caco-2). The H+ gradient-dependent pathway (Km = 0.789 mM, and Jmax = 163 pmol/min per cm2) is relatively "high affinity" and "low capacity" compared to H+ gradient-independent pathway (Km = 8.28 mM, and Jmax = 316 pmol/min per cm2). In addition, TT of LOR in the presence of a H+ gradient was inhibited 77% to 88% (p < 0.05) by 10 mM of cephalexin, enalapril, Gly-Pro and Phe-Pro, while TT of LOR in the absence of a H+ gradient was only inhibited 42% to 48% (p < 0.05) by 10 mM of Gly-Pro and Phe-Pro. Since AP uptake is H+ gradient-dependent and saturable while the BL efflux is mostly nonsaturable and not driven by a H+ gradient, these two transmembrane transport processes must be different, which could be the result of two different peptide carriers. In vivo, these two transport processes must have worked in concert to produce transcellular flux of loracarbef. To explain the differences between kinetic characteristics of AP uptake and TT transport, a cellular pharmacokinetic (PK) model was developed and the results indicate that the PK model appropriately described the kinetics of LOR TT. The use of this PK model may provide an additional advantage to the use of the cell culture model because kinetic parameters at both sides of the intestinal epithelial membrane may be obtained using the same preparation. Taken together, the Caco-2 model system represents an excellent model system for the study of carrier-mediated processes involved in the TT of peptides and peptide-like drugs.

Mechanisms of transport of quinapril in Caco-2 cell monolayers: comparison with cephalexin.

PURPOSE: To determine the transport mechanisms of quinapril and cephalexin in Caco-2 cell monolayers, a cell culture model of the human small intestinal epithelium. METHODS: Uptake, transepithelial transport and intracellular accumulations of these two drugs were measured using Caco-2 cell monolayers grown onto Millicells and magnetically stirred diffusion chambers. RESULTS: Transepithelial transport, apical (AP)4 uptake and intracellular accumulation of both drugs depended on the maintenance of a transepithelial proton gradient and temperature of the medium. However, quinapril transport and accumulation, which did not display a maximum at approximately pH 6, was more sensitive to proton gradient change, whereas cephalexin transport was more sensitive to concentration change (range 0.5-5 mM). In addition, quinapril (1 mM) transport was decreased significantly (p < 0.05) by 10 mM cephalexin, loracarbef, Gly-Pro and Phe-Pro, but not by enalapril; whereas cephalexin (0.1 mM) transport was decreased significantly (p < 0.05) by all four compounds. Similarly, AP quinapril (1 mM) uptake was also decreased by 10 mM loracarbef, Gly-Pro, cephalexin, and enalapril, but these inhibitory effects (20-50%) were quantitatively less than their inhibitory effects on cephalexin uptake (50-90%). Finally, the AP uptake of quinapril was also significantly (p < 0.05) inhibited by FCCP (10 micrograms/ml), amiloride (0.5 mM), DEP (0.5 mM), and staurosporine (5 nM). CONCLUSIONS: The transport of quinapril in the Caco-2 cells is via a combination of the carrier-mediated proton gradient-dependent peptide transporter and passive diffusion.

Effects of reserpinization, surgical denervation and in vitro chemical denervation with 6-hydroxydopamine on the contractile response of isolated rabbit ear artery to propranolol.

The contractile effect of propranolol in isolated rabbit ear artery was assessed in reserpinized and in surgically and chemically denervated blood vessels. Reserpinization and surgical denervation either had no effect on or enhanced the ear artery contractile response to 10(-6) to 10(-4) M propranolol. In contrast, the contractile response to propranolol was nearly abolished after denervation of the ear artery in vitro with 6-hydroxydopamine (6-OHDA). Dose-response curves to norepinephrine were shifted to the left by factors of 4.9 in 6-OHDA denervated ear arteries and 15.6 in untreated arteries in the presence of 10(-7) M desipramine. The diluent for 6-OHDA shifted both the norepinephrine and propranolol dose-response curves to the right. It is proposed that propranolol caused a contractile response in ear artery by an action on the postsynaptic tissues of this vessel. 6-OHDA denervation caused nonspecific desensitization in rabbit ear artery leading to the loss of response of this vessel to propranolol.

Three-dimensional quantitative structure-permeability relationship analysis for a series of inhibitors of rhinovirus replication.

Multiple three-dimensional quantitative structure-activity relationship (3D-QSAR) approaches were applied to predicting passive Caco-2 permeability for a series of 28 inhibitors of rhinovirus replication. Catalyst, genetic function approximation (GFA) with MS-WHIM descriptors, CoMFA, and VolSurf were all used for generating 3D-quantitative structure permeability relationships utilizing a training set of 19 molecules. Each of these approaches was then compared using a test set of nine molecules not present in the training set. Statistical parameters for the test set predictions (r(2) and leave-one-out q(2)) were used to compare the models. It was found that the Catalyst pharmacophore model was the most predictive (test set of predicted versus observed permeability, r(2) = 0.94). This model consisted of a hydrogen bond acceptor, hydrogen bond donor, and ring aromatic feature with a training set correlation of r(2) = 0.83. The CoMFA model consisted of three components with an r(2) value of 0.96 and produced good predictions for the test set (r(2) = 0.84). VolSurf resulted in an r(2) value of 0.76 and good predictions for the test set (r(2) = 0.83). Test set predictions with GFA/WHIM descriptors (r(2) = 0.46) were inferior when compared with the Catalyst, CoMFA, and VolSurf model predictions in this evaluation. In summary it would appear that the 3D techniques have considerable value in predicting passive permeability for a congeneric series of molecules, representing a valuable asset for drug discovery.

Activities of the semisynthetic glycopeptide LY191145 against vancomycin-resistant enterococci and other gram-positive bacteria.

LY191145 is the prototype of a series of compounds with activities against vancomycin-resistant enterococci derived by modification of the glycopeptide antibiotic LY264826. LY191145 had MICs for vancomycin- and teicoplanin-resistant enterococci of < or = 4 micrograms/ml for 50% of isolates and < or = 16 micrograms/ml for 90% of isolates. Its MICs for vancomycin-resistant, teicoplanin-susceptible enterococci were 1 to 8 micrograms/ml. LY191145 retains the potent activities of its parent compound against staphylococci and streptococci. In vivo studies in a mouse infection model confirmed these activities. This compound indicates the potential of semisynthetic glycopeptides as agents against antibiotic-resistant gram-positive bacteria.