Tissue-direct PCR, a rapid and extraction-free method for barcoding of ferns.

Fern gametophytes and young sporophytes often provide too little material for DNA extraction and are particularly difficult to identify to genus. Here we developed an efficient procedure called 'Tissue-direct PCR', in which a slice of fern tissue is mixed with PCR reagents and primers, allowing certain genomic regions to be amplified directly in the thermal cycler. For these diminutive and featureless stages of ferns, Tissue-direct PCR combined with amplifying plant barcodes promises to make the identification of immature ferns easy and rapid. Tissue-direct PCR would also be very helpful for large-scale ecological studies surveying distribution and population structure.

Role of P-glycoprotein in the hepatic metabolism of tacrolimus.

The main objective was to determine the potential effect of P-glycoprotein (P-gp) modulation on hepatic metabolism of tacrolimus, a P-gp and cytochrome P450(CYP)3A4 substrate, and to investigate various potential factors that may contribute to the interaction between P-gp and CYP. An isolated perfused rat liver system was used to study the hepatic disposition of tacrolimus in the presence of a P-gp inhibitor, GF120918, and a comprehensive pharmacokinetic analysis was conducted. GF120918 significantly decreased mean intrinsic metabolic clearance (by 86 and 41% based on the well-stirred and tube models, respectively) as well as hepatic clearance (from 47.3 to 44.2 ml min(-1)). Potential factors that might contribute to these observations, such as the effects of GF120918 on hepatic metabolism or on distribution of tacrolimus, were investigated and found to be negligible. In conclusion, it was shown that P-gp inhibition by GF120918 reduces hepatic clearances of its substrate drugs although the mechanism is yet to be determined.

Causes and consequences of high osmotic potentials in epiphytic higher plants.

Past reports of the water relations of epiphytes, particularly bromeliads, indicate that tissue osmotic potentials in these tropical and subtropical plants are very high (close to zero) and are similar to values for aquatic plants. This is puzzling because several ecophysiological studies have revealed a high degree of drought stress tolerance in some of these epiphytes. The goal of this study was two-fold: (1) to increase the number of epiphytic taxa sampled for tissue osmotic potentials; and (2) to explain the apparent discrepancy in the significance of the tissue water relations and tolerance of drought stress in epiphytes. Tissue osmotic potentials of 30 species of epiphytic ferns, lycophytes, and orchids were measured in a subtropical rain forest in northeastern Taiwan. Nearly all values were less negative than -1.0 MPa, in line with all previous data for epiphytes. It is argued that such high osmotic potentials, indicative of low solute concentrations, are the result of environmental constraints of the epiphytic habitat on productivity of these plants, and that low rates of photosynthesis and transpiration delay the onset of turgor loss in the tissues of epiphytes such that they appear to be very drought-stress tolerant. Maintenance of photosynthetic activity long into drought periods is ascribed to low rates of transpiration and, hence, delayed tissue desiccation, and hydration of the photosynthetic tissue at the expense of water from the water-storage parenchyma.

A comprehensive account on the role of efflux transporters in the gastrointestinal absorption of 13 commonly used substrate drugs in humans.

BACKGROUND: The potential absorption-limiting effect of intestinal efflux transporters such as P-glycoprotein (P-gp) has been well recognized, primarily based on results of numerous Caco-2 cell studies showing that flux, permeability, or transport clearance of drugs from the basolateral to the apical (B --> A) compartment is greater than that from the apical to the basolateral (A --> B) compartment. Except for very limited examples such as celiprolol, talinolol, pafenolol and paclitaxel, the potential clinical impact of these transporters on oral absorption of the vast number of commonly prescribed drug substrates in humans has not been closely examined to date. OBJECTIVE: To evaluate whether these efflux transporters may play a significant role in limiting oral absorption of 13 commonly used drugs (digoxin, etoposide, felodipine, fexofenadine, furosemide, indinavir, losartan, nadolol, propranolol, ritonavir, saquinavir, tacrolimus, and verapamil) in humans. METHODS: Drug absorption properties such as the rate (as judged by the Cmax and tmax) and extent (as judged by AUC or urinary excretion of drugs) of absorption as a function of dose, as well as the completeness of oral absorption were obtained from the literature. RESULTS: The absorption properties of these 13 drugs are not consistent with absorption-retarding expectations from in vitro studies because they all show apparent dose-independent kinetics in absorption or bioavailability and completeness of oral absorption is shown for most of the drugs evaluated. CONCLUSIONS: In spite of being substrates of intestinal efflux transporters such as P-gp, the in vivo oral absorption of 13 drugs examined apparently is not significantly impeded by efflux transporters. Thus, there may exist an apparent discrepancy between in vitro "expectations" and in vivo results; potential reasons for this are discussed. The present findings, however, do not de-emphasize potential in vivo importance of efflux transporters in affecting (increasing or decreasing) oral absorption of certain substrate drugs, especially those with low to moderate intestinal permeability and with low therapeutic index, or the importance of efflux transporters in the study of mechanisms of drug absorption and some potentially clinically significant drug-drug and drug-food interactions.

The rate and extent of oral bioavailability versus the rate and extent of oral absorption: clarification and recommendation of terminology.

In the literature, the meanings of the terms oral absorption and oral bioavailability of drugs vary greatly. Absorption has been considered to take place at the mucosal membrane of the gastrointestinal (GI) tract. It has also been defined as the process from the site of drug administration to the site of measurement. In the latter definition, the extent of oral absorption depends on the extent of first-pass elimination in the gut wall and liver even though a drug may be completely absorbed from the GI tract. Moreover, these two terms have also been used interchangeably. Inconsistency in the definition of these two terms has led to varying interpretations of these terms among students, researchers and laymen, and such an inconsistency seems undesirable. Apparently because of these inconsistencies, improper correlations between the Caco-2 permeability or intestinal permeability and the oral bioavailability of drugs subject to extensive first-pass effect may have occurred. It is suggested that absorption be defined as movement of drug across the outer mucosal membranes of the GI tract, while bioavailability be defined as availability of drug to the general circulation or site of pharmacological actions. Since transit times (this may range from about 1 min to several hours) across enterocytes, liver, lungs, and the peripheral venous sampling tissue are virtually unknown for all drugs, this factor alone would favor the use of "oral bioavailability rate" rather than "oral absorption rate" in all routine studies.

Similarity in the linear and non-linear oral absorption of drugs between human and rat.

OBJECTIVE: The main aim of this study was to provide a simple, pharmacokinetic rationale for great similarity in the extent (Fab) of gastrointestinal absorption of about 100 different, diverse compounds between human and the rat in linear dosing range, and to test the general applicability of a novel empirical method to correlate the non-linear Fab between the human and the rat by normalizing doses by body surface area (BSA) or body weight 0.67. METHOD: The mean small intestinal transit time (t) of 36 rats was estimated from the reported study, and this was used to compare with that in humans. The reported great similarity in apparent first-order absorption rate constants (k) of seven structurally diverse compounds between the two species were obtained. Extensive computer search was made and non-linear Fab data for the two species were obtained for chlorothiazide, acyclovir, miglitol and pafenolol. RESULTS: The mean t for rats was estimated to be 3.32 h which is almost identical to that reported in humans. The great similarity in Fab between human and rat in linear absorption range can be rationalized by similar t and k between the two species. The markedly different Fab vs dose/kg of body weight profiles between human and rat for the four drugs showing dose-dependent Fab were found to collapse when doses were normalized by BW0.67. CONCLUSION: For Fab not limited by the solubility problem, the great similarity in Fab between human and rat in linear absorption range can be rationalized by the similar t and k. For non-linear Fab drugs, great similarity in Fab can also be obtained between human and rat when doses are normalized by BSA or BW0.67. Regardless of absorption properties (active, passive or facilitated), similar Fab between the two species may be generally obtained when doses used in humans are about 5 to 7 times lower than that in rats. The above findings may be valuable in drug development.

Evaluation of using dog as an animal model to study the fraction of oral dose absorbed of 43 drugs in humans.

PURPOSE: To conduct a retrospective evaluation of using dog as an animal model to study the fraction of oral dose absorbed (F) of 43 drugs in humans and to briefly discuss potential factors that might have contributed to the observed differences in absorption. METHODS: Mean human and dog absorption data obtained under fasted state of 43 drugs with markedly different physicochemical and pharmacological properties and with mean F values ranging from 0.015 to 1.0 were obtained from the literature. Correlation of F values between humans and dogs was studied. Based on the same references, additional F data for humans and rats were also obtained for 18 drugs. RESULTS: Among the 43 drugs studied, 22 drugs were virtually completely absorbed in both dogs and humans. However, the overall correlation was relatively poor (r2 = 0.5123) as compared to the earlier rat vs. human study on 64 drugs (r2 = 0.975). Several drugs showed much better absorption in dogs than in humans. Marked differences in the nonliner absorption profiles between the two species were found for some drugs. Also, some drugs had much longer Tmax values and prolonged absorption in humans than in dogs that might be theoretically predicted. Data on 18 drugs further support great similarity in F between humans and rats reported earlier from our laboratory. CONCLUSIONS: Although dog has been commonly employed as an animal model for studying oral absorption in drug discovery and development, the present study suggests that one may need to exercise caution in the interpretation of data obtained. Exact reasons for the observed interspecies differences in oral absorption remain to be explored.

Apparent lack of effect of P-glycoprotein on the gastrointestinal absorption of a substrate, tacrolimus, in normal mice.

PURPOSE: To study the contribution of P-glycoprotein (P-gp) to the oral absorption of a substrate, tacrolimus, by comparing the extent and rate of bioavailability in normal and mdr1a knockout mice. Methods. Intravenous and oral (2 mg/kg) blood concentration data of tacrolimus in normal and knockout mice were obtained from a study by K. Yokogawa et al. in Pharm. Res. 16:1213-1218 (1999). Mean bioavailability (F), mean hepatic first-pass extraction ratio (Fh), mean bioavailability rates, mean oral clearance, and mean total hepatic intrinsic clearance were calculated using standard pharmacokinetic methods. RESULTS: The mean F of tacrolimus (an apparently highly permeable compound) was increased from 0.22 in normal mice to 0.72 in knockout mice. These values were consistent with mean predicted Eh (based on intravenous data) of 0.77 and 0.27 in normal and knockout mice, respectively. Great similarity in the relative bioavailability profile (such as short Tmax) between normal and knockout mice was also found. Mean oral clearance and mean total or unbound hepatic intrinsic clearance of tacrolimus in knockout mice were found to be about 10 times lower compared to those in normal mice. CONCLUSIONS: The above results suggest an apparent lack of effect of P-gp on the gastrointestinal absorption of tacrolimus in normal mice under the study condition. It is postulated that the effect of P-gp on the rate and extent of oral absorption should be more pronounced for those more slowly or incompletely absorbed drugs (i.e., drugs with relatively low permeabilities) as illustrated by talinolol in humans. The clearance data also suggest a very dominant role of P-glycoprotein in controlling the rate of hepatic metabolism of tacrolimus in normal mice, and P-glycoprotein may serve as an effective efflux pump for direct transport of metabolites formed in hepatocytes into the blood circulation.

Elucidation of human amphotericin B pharmacokinetics: identification of a new potential factor affecting interspecies pharmacokinetic scaling.

PURPOSE: To elucidate the pharmacokinetics of amphotericin B in rats, mice and humans, and to perform interspecies scaling to humans using allometry. METHODS: Plasma concentrations following intravenous bolus administration in rats, and mice were determined by HPLC. Human pharmacokinetic parameters elucidated from literature data were validated in a preliminary study involving a patient receiving daily infusion dose for 27 days. A critical literature review was conducted to identify appropriate pharmacokinetic parameter values in other species for interspecies scale-up. Interspecies allometric scale-up was performed across mice, rats, rabbits and dogs and the resulting predictions in humans were compared to observed values. RESULTS: A triexponential decline in rat, mouse and human plasma concentrations were observed. No gender differences in rat pharmacokinetics were observed. In contrast to allometry, mouse CL was smaller (82 vs 116 ml/h/kg) and T0.5 (33 vs 20 h) was longer compared to rat. In the preliminary human study, Cpeak and Cmin values remained relatively constant over the duration of therapy, and a CL, MRT, T0.5, Vss and Vdarea of 26 ml/h/kg, 10 and 23 days, 6.2 and 20 L/kg, respectively, were estimated. The relative contributions of the terminal phase area in rat, mouse and human were 75%, 92% and 31%, respectively. Interspecies allometric scale-up predictions of human CL (41 ml/h/kg), CLu (467 ml/h/kg) and Vss (3.3 L/kg) were similar to reported values, whereas poor predictions of human Vuss (33 L/kg), Vdarea (4.1 L/kg) and T0.5 (3 days) were obtained. CONCLUSIONS: Insignificant accumulation in humans inspite of the long terminal T0.5 was rationalized to be due to the small terminal-phase area contribution. While human CL and Vss were successfully predicted in the interspecies scaling, poor predictions of human Vdarea and T0.5 were obtained, which was attributed to disposition pattern differences between humans and other species, a potential new critical factor affecting interspecies scale-up.

Correlation of plasma clearance of 54 extensively metabolized drugs between humans and rats: mean allometric coefficient of 0.66.

PURPOSE: To evaluate the distribution of allometric exponents for relationship of total plasma clearance of 54 extensively metabolized drugs, with wide-ranging linear clearance values, between humans and rats, to provide a rationale for the observed data, and to discuss potential significance of the findings. METHODS: Human and rat plasma clearance values of 54 drugs with markedly different physicochemical properties were obtained from the literature. Standard allometric analysis was performed for each drug using both rat and human data. Unbound vs. total plasma clearances were obtained for 15 out of 54 drugs and their correlations between humans and rats were compared. RESULTS: The mean+/-SD of the allometric exponent for the 54 drugs studied is 0.660+/-0.190. The median clearance ratio based on unit body weight is 7.41 and the median exponent is 0.645. Excluding two outliers the correlation coefficient of plasma clearance between humans and rats was 0.745 (p < 0.0001). For the 15 drugs, use of unbound plasma clearance approach seems to significantly improve the correlation coefficient compared to total plasma clearance (0.940 vs. 0.841). CONCLUSIONS: The present study indicates that on average, humans and rats may eliminate extensively metabolized drugs at a rate similar to that expected from the allometric or body surface area relationship of basal metabolic rate between the two species. A simple statistical distribution hypothesis is used to rationalize the species difference in plasma drug clearance. Rat may serve as an useful animal model to predict (unbound) plasma clearance of drugs in humans.

Mechanism of ascorbic acid enhancement of the bioavailability and diuretic effect of furosemide.

The following possible explanations for the significant increases in the oral bioavailability and the diuretic and natriuretic effects of orally administered furosemide observed when ascorbic acid was coadministered to dogs were investigated: ascorbic acid might enhance the gastrointestinal (GI) absorption of furosemide, might inhibit GI wall metabolism of furosemide, might enhance the reabsorption of furosemide from the renal tubules, and might increase the unionized fraction of furosemide at the receptor sites. The significant increase in the oral bioavailability with coadministration of ascorbic acid seemed to result from reduced gastric first-pass metabolism of furosemide and not enhanced GI absorption of furosemide. This might be supported by rat studies; the percentages of the oral doses of furosemide recovered from the GI tract at 8 hr after oral administration were similar (p < 0.583) without (39.5%) and with (44.7%) coadministration of ascorbic acid, and the amounts of furosemide remaining per gram of stomach after 30-min incubations of 50 micrograms of furosemide with 9000g supernatant fractions of stomach homogenates were increased significantly (48.5 vs. 42.4 micrograms) by the addition of 100 micrograms of ascorbic acid. The significant increases in the diuretic and natriuretic effects of furosemide with ascorbic acid could be the result of increases in the reabsorption of furosemide from renal tubules and increases in the unionized fraction of furosemide at the renal tubular receptor sites. This was supported by 1.5-4.2-fold increases in urine output and approximately 20% decreases in the time-averaged renal clearance of furosemide when the urine pH was decreased by 1.5-2.5 units by oral administration of ammonium chloride.

Similarity or discrepancy in pharmacokinetic parameter estimation between bolus and infusion studies.

A recent study by Heatherington and Rowland showing discrepancies in steady-state volume of distribution (Vss) estimation of two barbiturates between bolus and infusion studies in rat hindlimb preparations was reviewed. Their rationale is that increasing the duration of administration may increase the accessibility for tissue distribution and thus increase Vss for compounds showing slow tissue uptake. Such a dosing-duration-dependent distribution concept is, however, inconsistent with the principle in linear kinetics that the fate or disposition function of any drug molecules is independent of time of administration and presence of other molecules. When their well-designed bolus studies were reanalyzed by including extrapolated outflow data from the last sampling time to infinity, the Vss values for the two barbiturates were found to be very similar to those obtained by the infusion method. Our analysis seems to validate a theoretical concept that parameter estimation is independent of the duration of administration in linear kinetics. A potential complication of using the bolus method to study Vss is presented.

New perspectives on the theory of permeability and resistance in the study of drug transport and absorption.

Permeability coefficient (P) expressed as distance per unit time has been commonly interpreted to represent the velocity of drug movement across a heterogeneous medium such as skin and intestinal epithelium. The basis of such an interpretation is questioned on several grounds. For example, the basic assumption for calculating P (as defined conventionally) according to the Fick's law of diffusion requires the entire medium to be homogeneous and rate-limiting in transport. The theoretical basis of the widely used total resistance or resistance additivity concept is reviewed. Such a concept is shown to be applicable to the study of total transit time across the medium but may not be applicable to the study of steady-state flux or absorption across the medium under normal conditions. Based on the diffusional, compartmental, absorptive clearance or carrier-mediated-transport analysis it is shown that only the first transport resistance from the bulk medium across the surface (such as cellular membranes) of the permeation medium (such as a cell or a tissue) is usually the deciding factor in drug transport or absorption. Resistances on the other side of the surface barrier usually only affect the drug accumulation vs. time profile in the medium, but not the steady-state flux or absorption. The role of unstirred water layer adjacent to the internal capillary wall is postulated to play an important role in causing the blood-flow dependency in absorption, a phenomenon that cannot be rationalized by the conventional effective permeability concept. The conventional concept of sink conditions on the serosal side is questioned. The present analysis further supports the use of the absorptive or transport clearance concept in absorption or transport study. Effective permeability is regarded as a mathematical operator for transport across a barrier or a tissue, and may be unrelated to the Fick's law of diffusion under most conditions. The velocity unit for P is regarded simply as a "collapsed" unit based on the absorptive or transport clearance per unit gross surface area. It is hoped that this commentary will stimulate further research and discussions in this general area of drug transport and absorption. It appears that there is a need to experimentally confirm the total resistance theory in biological systems.