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1)
P-glycoprotein-mediated intestinal and biliary digoxin
transport in humans.
2)
Tacrolimus : effect of P-glycoprotein efflux on regional permeability of
tacrolimus in rats.
3)
An in
vitro perfusion model for the determination of absorption properties of
drugs in isolated rat small intestine.
4)
Intestinal metabolism of
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
in rats
5)
in vitro/ex vivo studies on the intestinal toxicity and
transport of covalently bound residues.
6)
Pretreatment with potent P-glycoprotein ligands may
increase intestinal secretion in rats.
7)
Optimization of the local inhibition of intestinal
adenosine deaminase (ADA) by erythro-9-(2-hydroxy-3-nonyl)adenine.
8)
Premicellar taurocholate enhances calcium uptake from
all regions of rat small intestine.
9)
Evaluation of a rat model for the study of local regulation of
intestinal blood flow: ex vivo asanguineous perfusion of the ileal vascular bed.
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isolated perfused rat gut used to study permeability
Region-dependent modulation
of intestinal permeability by drug efflux transporters: in vitro studies in
mdr1a(-/-) mouse intestine.
Stephens RH, Tanianis-Hughes J, Higgs NB, Humphrey M, Warhurst G.
Gut Barrier Group, University of Manchester and Salford Hospitals Trust,
Hope Hospital, Salford, United Kingdom.
Information on the extent to which xenobiotics interact with P-glycoprotein
(PGP) during transit through the intestine is crucial in determining the
influence of PGP on oral drug absorption. We have recently described a novel
use of isolated ileum from PGP-deficient mdr1a(-/-) mice to resolve PGP- and
non-PGP-dependent drug efflux and provide a definitive measure of intrinsic
drug permeability without recourse to inhibitors. The present study uses
this approach to investigate the impact of PGP on intestinal permeability of
paclitaxel and digoxin in different regions of the mouse intestine (jejunum,
ileum, and proximal and distal colon). Absorption of paclitaxel and digoxin
in tissues from wild-type mice was low and showed little regional variation.
In contrast, absorption of both drugs was markedly higher in mdr1a(-/-)
intestine, although the increase was highly region-dependent, with the ileum
and distal colon showing the greatest effect and much smaller changes in the
jejunum and proximal colon. These effects were accompanied by the abolition
of paclitaxel and digoxin secretion in mdr1a(-/-) mice, suggesting that
regional variations in intestinal permeability are masked by differential
PGP expression, confirmed by immunoblotting studies. Propranolol
permeability, which is not influenced by PGP, showed similar regional
variation in both wild-type and mdr1a(-/-) tissues, suggesting that
differences are at the level of transcellular permeability. These data
suggest that the ileum and the distal colon are regions of relatively high
transcellular permeability for xenobiotics that are compensated by enhanced
expression of PGP.
PMID: 12438532 [PubMed - indexed for MEDLINE]
1)
P-glycoprotein-mediated intestinal and biliary digoxin
transport in humans.
Drescher S, Glaeser H, Murdter T, Hitzl M, Eichelbaum M, Fromm MF.
Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology,
Auerbachstrasse 112, 70376 Stuttgart, Germany. siegfried.drescher@ikp-stuttgart.de
BACKGROUND AND AIMS: Intestinal transport by P-glycoprotein is a recently
recognized determinant of drug disposition. However, direct measurements of
transporter-mediated drug elimination into isolated segments of human small
intestine are lacking. METHODS: Using a recently developed intestinal
perfusion catheter, we perfused in healthy volunteers two 20-cm jejunal
segments with and without the P-glycoprotein inhibitor quinidine before and
during administration of the P-glycoprotein inducer rifampin (INN,
rifampicin). RESULTS: Within 3 hours after intravenous administration of
digoxin (1 mg), perfusate samples were collected. We found that 0.45% +/-
0.24% and 0.83% +/- 0.60% of the digoxin dose were eliminated into a jejunal
segment and into bile, respectively. Perfusion of the isolated segment with
quinidine reduced intestinal digoxin elimination (0.23% +/- 0.08%, P =.031).
During rifampin, intestinal digoxin elimination was 0.80 +/- 0.59 (P =.383).
Enterocyte P-glycoprotein content correlated with the area under the plasma
concentration-time curve of digoxin (Spearman nonparametric correlation
coefficient [r(S)] = -0.73, P =.003) and digoxin nonrenal clearance (r(S) =
0.52, P =.056), as well as with intraluminal and plasma concentrations of
quinidine (r(S) = 0.55, P =.041 and r(S) = -0.67, P =.009, respectively).
CONCLUSION: Using segmental intestinal perfusion, we provide direct evidence
that intestinal P-glycoprotein mediates substantial drug elimination after
intravenous administration from the systemic circulation into the gut lumen
and prevents entry of luminally administered P-glycoprotein substrates into
the enterocytes. These data also highlight the relative importance of direct
intestinal drug secretion in comparison with drug elimination through bile.
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2) Tacrolimus is a class II low-solubility high-permeability
drug: the effect of P-glycoprotein efflux on regional permeability of
tacrolimus in rats.
Tamura S, Ohike A, Ibuki R, Amidon GL, Yamashita S.
Fujisawa Pharmaceutical Company, Ltd., 1-6, Kashima 2-Chome, Yodogawa-ku,
Osaka 532-8514, Japan. shigeki_tamura@po.fujisawa.co.jp
The objective of this study is to investigate the role of P-glycoprotein
(P-gp), a membrane efflux pump associated with multidrug resistance (MDR)
and a known substrate for tacrolimus, in determining the regional intestinal
permeability of tacrolimus in rats. Thus, isolated segments of rat jejunum,
ileum, or colon were perfused with tacrolimus solutions containing
polyethoxylated hydrogenated castor oil 60 surfactant, and with or without
verapamil, a P-gp substrate used to reverse the MDR phenotype. The results
indicated that the intrinsic permeability of tacrolimus in the jejunum,
calculated on the basis of the concentration of non-micellized free
tacrolimus, was quite high ( approximately 1.4 x 10(-4) cm/s). The apparent
permeability (P(app)) in the jejunum was unaffected by the presence of
verapamil; however, the P(app) in the ileum and the colon increased
significantly in the presence of verapamil and were similar to the values
observed in the jejunum. The results suggest that systemic absorption of
tacrolimus from the gastrointestinal tract could be significantly affected
by P-gp efflux mechanisms. It is also possible that differences in P-gp
function at various intestinal sites in a subject or at a given intestinal
site in various subjects could lead to large intra- and interindividual
variability in bioavailability of tacrolimus following oral administration.
Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association
.
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3)
An in
vitro perfusion model for the determination of absorption properties of
drugs in isolated rat small intestine.
Norta M, Schopke T.
Institut fur Pharmazie, Humboldt-Universitat zu Berlin.
A two-compartment model was developed to perform in vitro permeability
studies of drugs. Preparations of surviving small intestine are
continuously perfused under permanent oxygenation. The apparatus
especially allows the usage of variable volumes of donor and adaptable
length of intestinal segments ranging from 3 to 20 cm. Both the mucosal
and the serosal side of the apparatus are open for sample collection and
additional instrumentation. Due to the construction the use of
high-surface activity compounds is possible. Data may be derived as
absorption rates (micrograms.cm-1.min-1) and concentration increases vs.
time profiles.
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4)Intestinal metabolism of
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
in rats: Sex difference, inducibility and inhibition by phenethyliso-
thiocyanate.
Schulze J, Malone A, Richter E.
Walther-Straub-Institut fur Pharmakologie und Toxikologie, Ludwig
Maximillians-Universitat Munchen, Germany.
The intestinal metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK) was investigated in male and female Sprague-Dawley (SD)
rats and male F344 rats, using isolated perfused intestinal
segments. [1(-14)C]-NNK at 1 microM was metabolized by
alpha-hydroxylation, pyridine N-oxidation and carbonyl reduction.
Jejunal segments from control female rats metabolized 26.2% of the
NNK during transepithelial transfer to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol
(NNAL, 12.2%), 4-(methylnitrosamino)-1-3-pyridyl-N-oxide)-1-butanone
(NNK-N-oxide, 7.7%), 4-oxo-4-(3-pyridyl)-butanol (KAlc, 2.7%), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanol
(NNAL-N-oxide, 1.8%), 4-oxo-4-(3-pyridyl)butyric acid (KA, 1.1%) and
4-hydroxy-4-(3-pyridyl)butyric acid (HA, 0.7%). Ileal segments
metabolized 20.8% of the NNK during absorption, with no difference
in metabolite distribution as compared to jejunal segments. In
control male SD and F344 rats, jejunal presystemic metabolism was
2.3-fold higher (56.4% and 60.8% respectively), mainly because of a
4-fold increase in NNAL formation (44.1% and 48.5%)> total NNK
metabolism was also induced in female rats by starvation (84.4%
metabolites), acetone (89.3%), phenobarbital PB, 75.3%) and Clophen
A50 (61%). PB and Clophen A50 induced N-oxidation to 38.9% (4 x) and
27.8% (3 x), and to a lesser extent NNAL formation and
alpha-hydroxylation (2 x), Starvation mainly increased N-oxidation
with a time-dependent increase from 1 day to 3 days of starvation (4
x and 8 x versus controls), whereas alpha-hydroxylation and NNAL
formation was elevated only after 1 day starvation. Acetone
pretreatment (3 days) stimulated all three pathways (NNAL 2 x,
N-oxidation 4 x, alpha-hydroxylation 4 x). In male F344 rats,
starvation and acetone induced N-oxidation (5 x and 7 x) and
alpha-hydroxylation (3 x and 5 x), and decreased NNAL formation by
40%, probably due to substrate competition or further metabolism of
NNAL. In acetone-induced female SD rats, NNK metabolism was
inhibited by in vivo pretreatment with phenethylisothiocyanate (PEITC)
or in vitro addition of 1% ethanol to the perfusate.
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Both inhibition
experiments reduced total metabolism by 20%; N-oxidation and
alpha-dhyroxylation were reduced to values found in control rats,
whereas NNAL formation increased from 31% to 51%.Inhibition of NNK
metabolism by PEITC im male F344 rats was less pronounced compared
to female SD rats; again a decrease in alpha-hydroxylation (6.7% to
3.3%) and N-oxidation (73.6% to 35.3) was accompanied by increased
NNAL formation (9.8% to 41.0%).(ABSTRACT TRUNCATED AT 400 WORDS)
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5) A contribution to safety assessment of veterinary
drug residues: in vitro/ex vivo studies on the intestinal toxicity and
transport of covalently bound residues.
Klee S, Baumung I, Kluge K, Ungemach FR, Horne E, O'Keeffe M, De
Angelis I, Vignoli AL, Zucco F, Stammati A.
Institute of Pharmacology, Pharmacy and Toxicology, University of
Leipzig, Germany. klee@rz.uni-leipzig.de
1. The gastrointestinal fate of protein-bound residues of the model
compound furazolidone (FZD) was investigated in vitro and ex vivo.
Protein-bound residues were generated in rat liver microsomes, isolated
by solvent extraction and digested with 0.5% hydrochloric acid and
Pronase E.
2. During digestion,
3-amino-2-oxazolidinone (AOZ), the side chain of furazolidone, was
partly released from bound residues.
3. The absorption of
free AOZ and digested protein-bound residues was tested in isolated
perfused rat gut segments (IPGS) and in the intestinal cell line Caco-2.
Free AOZ was transfered both in the IPGS model and in Caco-2 monolayer
cultures, while no indications for passage of bound residues were
obtained.
4. No acute toxicity of
AOZ or digested food residues respectively was observed in gut segments
and Caco-2 cells at concentrations that were substantially above maximum
residue levels to be expected in food of animal origin after
administration of therapeutic doses.
5. The results
demonstrate that digestive processes can alter the chemical nature of
drug residues and yield degradation products that may be bioavailable
for the consumer. Thus, the covalent binding of xenobiotics to
macromolecular tissue constituents cannot necessarily be regarded as an
irreversible endpoint of residue bioavailability and toxicity.
6) Pretreatment with potent P-glycoprotein ligands may
increase intestinal secretion in rats.
Hanafy A, Langguth P, Spahn-Langguth H.
Department of Pharmaceutical Chemistry, Martin-Luther-University
Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, D-06120 Halle/Saale,
Germany.
The expression of P-glycoprotein is induced in cell cultures upon
exposure to various inducers. Therefore, the aim of the present study
was to evaluate the in-vivo relevance of this observation, i.e. the
influence of chronic pretreatments with selected drugs
-- all of which are
ligands to P-glycoprotein (P-gp) as demonstrated in radioligand binding
studies and all of which have some or a considerable effect on P-gp
expression in Caco-2 cells.
-- on the effective
intestinal permeabilities of the model compound talinolol in rats
employing in-situ single-pass intestinal perfusion of three different
gut segments. Talinolol was selected, because it shows high
selectivity for one of the exsorptive transporters (P-gp) and its
intestinal permeability is very sensitive to changes in exsorption when
the perfusate concentration is low. Prior to the induction study the
perfusion model was optimized regarding the type and concentration of a
competitive inhibitor which may be used to block the exsorption-related
permeability reduction (through intestinal exsorption) during an ongoing
perfusion and would permit an intra-individual comparison of the
effective permeability without and with blockade of exsorption. While
repetitive verapamil and talinolol dosing had no statistically
significant exsorption-inducing effect, vinblastine and rifampicin
pretreatments resulted in decreased intestinal talinolol permeabilities
in the three tested gut segments, duodenum, jejunum, and colon [e.g.,
S-talinolol in jejunum: control, 2.50 x 10(-4) cm/s; vinblastine
induction, 1.48 x 10(-4) cm/s (P<0.05); rifampicin induction, 1.51 x
10(-4) cm/s (P<0.05)]. Addition of an efficient secretion inhibitor
(vinblastine) to the perfusate permitted the determination of the impact
of inhibitable secretory processes on the total effective permeabilities
and an estimation of passive permeability in the respective individual.
The inhibitable permeability fractions were higher for vinblastine than
for any other pretreatment and the difference from control pretreatment
was statistically significant for all intestinal segments (duodenum,
61.8%; jejunum, 63.1%; colon, 43,7%; S-talinolol). Statistically
significant differences were also detected for rifampicin in the
perfused duodenum and jejunum (33.1 and 27.5% increase in inhibitable
fraction, respectively, for S-talinolol). These differences are
explained by a significant induction of outside-directed transport in
the intestinal enterocytes by vinblastine and rifampicin.
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7) Optimization of the local inhibition of intestinal
adenosine deaminase (ADA) by erythro-9-(2-hydroxy-3-nonyl)adenine:
enhanced oral delivery of an ADA-activated prodrug for anti-HIV therapy.
Singhal D, Anderson BD.
Department of Pharmaceutics and Pharmaceutical Chemistry, University of
Utah, Salt Lake City, Utah 84112, USA.
Previous in situ perfusion studies in rat ileal segments have
demonstrated that high concentrations (>40 microg/mL) of
erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), a semitight binding
inhibitor of adenosine deaminase (ADA), are effective in completely
inhibiting the intestinal metabolism of 6-chloro-2',3'-dideoxypurine
(6-Cl-ddP), an ADA activated prodrug of the anti-HIV agent 2',
3'-dideoxyinosine (ddI) designed for improved targeting to the central
nervous system. However, the intestinal absorption of EHNA results in
complete inhibition of the ADA activity in the mesenteric blood draining
the isolated intestinal segment being perfused and may lead to complete
inhibition of ADA present in the systemic circulation and other sites,
an unacceptable outcome since bioconversion in the target tissue is
required for prodrug efficacy. This study examines the feasibility of
locally inhibiting ADA present in the intestinal wall using EHNA to
increase the intestinal absorption of 6-Cl-ddP. Transport experiments
conducted in isolated ileal segments from mesenteric cannulated rats
using perfusate containing prodrug and various concentrations of EHNA
demonstrated that a 0.1 microg/mL logarithmic mean lumenal concentration
of EHNA was effective in increasing the intestinal bioavailability of
Cl-ddP to > 90%. Intestinal uptake parameters for EHNA and
pharmacokinetic parameters generated in vivo in chronically catheterized
rats given intravenous infusions ranging from 12.5 to 310 microg/kg/min
were used to demonstrate that <10% of systemic ADA would be inhibited
at steady state using the optimal perfusate concentration of EHNA. Thus,
in continuous perfusions it is possible to increase the intestinal
bioavailability of 6-Cl-ddP to >90% with minimal (<10%) inhibition
of systemic ADA. Local inhibition of enzymes may be an effective
strategy to increase the oral bioavailability of tissue enzyme-activated
prodrugs or other drugs which may also be substrates
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8) Premicellar taurocholate enhances calcium uptake from
all regions of rat small intestine.
Sanyal AJ, Hirsch JI, Moore EW.
Department of Medicine, Medical College of Virginia, Virginia
Commonwealth University, Richmond.
BACKGROUND/AIMS: The specific components of bile, which is necessary for
normal calcium absorption, are unknown. We have previously shown that
Ca2+ is bound with high affinity by premicellar taurocholate. The
current studies examined the effects of taurocholate on intestinal
calcium transport.
METHODS: Intestinal Ca2+
uptakes were measured from proximal, mid, and distal small intestinal
segments perfused with solutions containing 45CaCl2 (0.1-1 mmol/L),
taurocholate (0-10 mmol/L), trihydroxymethylaminomethane buffer (pH 7),
phenolsulfonpthalein (nonabsorbable marker), and NaCl (total ionic
strength, 0.16 mol/L) for four randomized perfusion periods. In other
studies, the proximal small intestine was divided into two equal
segments and perfused with either 45CaCl2 or 45CaCl2 plus taurocholate
(2.5-5 mmol/L). Calcium absorption was measured from the difference in
uptake and calcium concentration retained in mucosa. Finally, effects of
taurocholate on Ca2+ uptake across isolated brush border membrane
vesicles were measured.
RESULTS: Premicellar
taurocholate produced an approximately 1.7-2-fold enhancement (P <
0.01) in Ca2+ uptake in all regions, with lesser contributions from
micellar taurocholate. These effects resulted in a net increase in
calcium absorption. Premicellar taurocholate also significantly
increased calcium uptake across brush border vesicles.
CONCLUSIONS: Premicellar
taurocholate significantly enhances calcium uptake into, and absorption
across, enterocytes. The mechanisms remain to be experimentally verified.
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9) Evaluation
of a rat model for the study of local regulation of intestinal blood
flow: ex vivo asanguineous perfusion of the ileal vascular bed.
Nyren O, Blank MA, Jaffe BM.
Department of Surgery, State University of New York Health Science
Center, Brooklyn 11203.
A new model of ex vivo vascularly perfused, isolated rat ileum was
developed and evaluated. Segments of distal ileum (approximately 5 cm)
from male Wistar rats were isolated on their vascular pedicles.
Perfusion through an aortic cannula with oxygenated (95% O2, 5% CO2)
Krebs solution containing 5% bovine albumin, 5.6 mM glucose, and 25 mM
mannitol at 37 degrees C was initiated immediately after interruption of
blood flow. The bowel preparations, including the abdominal aorta, were
then transferred to a perfusion chamber. Perfusion pressure was
maintained by gravity at 40 mm Hg. Flow was measured with an
electromagnetic flow probe. The portal vein, together with the
lymphatics, drained freely into collection tubes. The bowel lumen was
perfused at 0.85 ml/min with isotonic modified Krebs solution containing
[14C]polyethylene glycol, and the luminal perfusion pressure was
monitored. Luminal effluents were collected through a large-bore outlet
tubing. As determined by histology, O2 consumption, vascular reactivity,
and mucosal permeability, the preparations were viable for at least 60
min of perfusion. With this model, a vasoconstrictor effect of the alpha
2-adrenoceptor agonist clonidine was documented for the first time in
isolated rat bowel.
PMID: 1434595 [PubMed - indexed for MEDLINE]
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