- Email: [email protected]
Comparison of the Intestinal Drug Permeation and Accumulation Between Normal Human Intestinal Tissues and Human Intestinal Tissues With Ulcerative Colitis
Journal Pre-proof Comparison of the intestinal drug permeation and accumulation between normal human intestinal tissues and human intestinal tissues with ulcerative colitis Daisuke Nakai, Masateru Miyake, Ayako Hashimoto PII:
S0022-3549(19)30823-8
DOI:
https://doi.org/10.1016/j.xphs.2019.12.015
Reference:
XPHS 1832
To appear in:
Journal of Pharmaceutical Sciences
Received Date: 29 October 2019 Revised Date:
2 December 2019
Accepted Date: 18 December 2019
Please cite this article as: Nakai D, Miyake M, Hashimoto A, Comparison of the intestinal drug permeation and accumulation between normal human intestinal tissues and human intestinal tissues with ulcerative colitis, Journal of Pharmaceutical Sciences (2020), doi: https://doi.org/10.1016/ j.xphs.2019.12.015. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
ID 19-972
Note
Comparison of the intestinal drug permeation and accumulation between normal human intestinal tissues and human intestinal tissues with ulcerative colitis
1)
1), 2)*
Daisuke Nakai , Masateru Miyake
, Ayako Hashimoto
2)
1)
Department of Pharmacy, Uppsala University, Husargatan 3, Uppsala, 75123, Sweden.
2)
Quality Assurance Section, Quality Assurance Department, Headquarters for Product Safety and
Quality Assurance, Otsuka Pharmaceutical Co. Ltd., Matsudani Itano-cho, Itano-gun Tokushima 779-0195, Japan.
*
To whom correspondence should be addressed Phone: +81-88-672 6070 Fax: +81-88-672 6086 E-mail: [email protected]
Revised version Date:December 3, 2019
1
ABSTRACT The purpose of this study was to examine drug absorption profile utilizing human intestinal tissues from ulcerative colitis (UC) patients and to compare with normal tissues from intestinal cancer patients. Human intestinal tissues from UC and cancer patients mounted in a mini-Ussing chamber were used to evaluate the permeation of drugs, including FD-4, a very low permeable marker, rebamipide, a low permeable marker, and metoprolol, a high permeable marker. The transport index (TI), an index of sum of permeated and tissue-accumulated molecules, of the model drugs was in accordance with their absorption rank order, and was almost kept constant irrespective of autopsy grade based on tissue fibrosis. showed the decreased X
corr
On the other hand, UC tissues of grade 2
, an index of permeated amount of molecules and increased T
corr
, an index
of tissue-accumulated molecules for every tested compound. Our finding of the transport characteristics in intestinal tissues of severe UC patients in mini-Ussing chamber system demonstrated that autopsy grade of UC patients didn’t drastically change membrane permeability of the tested compounds.
Furthermore, it was suggested that morphological changes of
intestinal tissues caused by fibrosis led to limited permeation and subsequently increased accumulation with little change of total absorption.
KEY WORDS Human absorption, Ulcerative colitis (UC), Human Tissues, Transport index (TI), Autopsy grade.
2
INTRODUCTION Ulcerative colitis (UC) and Crohn’s disease (CD) are the specific refractory disease at least in Japan. The number of patients regarding inflammatory bowel disease (IBD) such as UC and CD has 1
been increasing recently. The lesion sites of UC are specific in the large intestine, while the lesion sites of CD are not intestinal region specific to be observed in the upper, middle and lower intestinal 2
tract. Nejdfors et al. have reported that permeability of mannitol and FD-4 seemed to be affected by 3
the radiation therapy, suggesting that even slight injury to the intestinal mucosa would lead to change of the intestinal permeability. Therefore, it is important to understand the effect of the autopsy grades in UC patients on membrane permeability. So far whether intestinal permeability is increased in active disease region of UC patients is controversial when urinary excretion of sugars or 51Cr-EDTA as a 4-7
permeability index was examined after dosing of the marker substances in human.
Evaluation of
membrane permeability in Ussing chamber system could be a solution to the controversial conclusions since this system is able to provide more focused information on membrane permeability to examine the effect of disease state on membrane permeability. We have already established the novel human intestinal absorption system using mini-Ussing chamber by utilizing the human, dog, monkey and rat 8,9
intestinal tissues.
Also we have confirmed the validity and reproducibility of the transport index (TI)
utilizing the large intestinal tissues from UC or CD patients for the evaluation of intestinal 8
absorbability. In addition, we have also successfully conducted the simultaneous prediction of 10,11
intestinal absorption and metabolism using the mini-Ussing chamber system.
In the present study, we investigated whether autopsy grade in UC tissues affected the drug permeability and tissue accumulation through examination of permeability of three types of model drugs such as FD-4, a very low permeable marker via paracellular route, rebamipide, a low permeable 12
marker of a p-glycoprotein substrate,
and metoprolol, a high permeable marker via transcellular
route.
MATERIALS AND METHODS Materials 3
FD-4 was purchased from Sigma-Aldrich (Boston, MA, USA). H-metoprolol was purchased from PerkinElmer Sverige AB (Upplands Väsby, Sweden).
3
14
C-rebamipide was obtained from Otsuka
Pharmaceutical Co. Ltd. All other reagents used were of the highest purity.
Human Intestinal Tissues Human large intestinal tissues were taken from male or female patients, aged 32 to 69 years old, with UC and intestinal cancer (Table 1), with permission from the patients in accordance with the Declaration of Helsinki. All experiments were approved by the institutional review board of all institutions, and written informed consent was obtained from all patients.
All tissues chosen from the
isolated tissues were placed in ice-cold transport buffer immediately after removal from the patient’s body in the surgical operation room and were transferred to the laboratory within 1 hr. Transport buffer was composed of 128 mM NaCl, 5.1 mM KCl, 1.4 mM CaCl2, 1.3 mM MgSO4, 21 mM NaHCO3, 1.3 mM KH2PO4 and 10 mM NaH2PO4, adjusted to pH 7.4.
Autopsy diagnosis The measure of autoptical abnormality was quantified on an arbitrary scale of 0 - 2, in which no effect was scored as 0 and redding and slightly thickened mucosa was scored as 1, and a severe effect of ulcer and thickened mucosa by fibrosis was scored as 2. This evaluation was carried out by a medical doctor in a blinded fashion.
Transport Experiment in Mini-Ussing Chamber The mini-Ussing chamber was used for transport experiments and the isolated human large intestinal tissues, which were biopsies from patients and normal, were mounted following the method reported previously.
3,8,9
The tissues were transferred to the experimental room within 1 h. First of all,
isolated human large intestinal tissues, from which the muscle layer had been removed with fine tweezers, were mounted vertically in the mini-Ussing-type chambers, providing an exposed area of 2
0.07 cm for the transport study.
The apical and basal transport buffers were defined at 1.35 mL, and
the solution temperature was maintained at 37°C wit h a water-jacketed reservoir. The apical and basal buffers were containing 5 mM mannitol on apical one and 5 mM D-glucose and 1% (w/v) bovine serum albumin in basal one, respectively, and those buffers were gassed with 95% O2/5% CO2 before and during the transport experiment. FD-4 and the radioactive compounds were placed on the apical side
4
3
at a concentration of 10 mg/mL and 0.4 µCi/mL for H-metoprolol and 0.1 µCi/mL for
14
C-rebamipide,
respectively, and the transport study was performed for 2 hr. The samples were taken from basal side at 600 µL and replaced with an equivalent volume of fresh buffer at 0.5, 1, 1.5 and 2 hr, whereas the samples were also taken from apical side at 10 µL without replacing at 0.083, 0.25, 0.5, 1, 1.5 and 2 hr to estimate the change in drug concentration due to the possibility of the drug precipitation during the transport study. After taking the final sample, the tissue was homogenized with 1 mL basal transport buffer under ice-cold conditions to determine the accumulated amount of drugs.
Calculation of Transport Index We have already defined the transport index (TI), which is the percent of dose transported and tissue-accumulated corrected by the AUC value of the drug in the apical compartment (Eq. 1) in order to estimate the permeability based on the actual drug concentration in the apical compartment.
TI = X corr + T corr
corr
where X
and T
Eq. 1
corr
are the percent of the dose transported to the basal side compartment and that of
the dose accumulated in the tissue, respectively, which were corrected by the AUC of FD-4, rebamipide and metoprolol in the apical compartment of drugs (Eqs. 2 and 3).
X corr
T
corr
ti t i −1 2 ⋅ (C t0 ⋅ t ) ⋅ (C t3 ⋅ V ) 6 2 ⋅ (C t 0 apical 3 basal basal apical ⋅ (t 6 − t 3 )) ⋅ (C basal ⋅ Vbasal − C basal ⋅ (Vbasal − V sampled ) = 3 + ∑ t i −1 ti (C apical + C apical ) ⋅ (t i − t i −1 ) t i −1 ti i=4 (( C + C ) ⋅ ( t − t )) ∑ apical apical i i − 1 i =1 1 ⋅ t ⋅ 100 (%) Eq .2 0 C apical ⋅ Vapical
=
t0 2 ⋅ Capical ⋅ t6 ⋅ T 6
∑ (C i =1
ti −1 apical
+C
ti apical
) ⋅ (t i − t i−1 )
⋅
C
t0 apical
1 ⋅100 (%) ⋅Vapical
5
Eq.3
where
ti ti Capical and Cbasal mean the drug concentrations in the apical side and basal side at time ti,
Vbasal and Vsampled represent the volumes of apical, basal compartments, both
respectively. Vapical ,
1.35 mL, and sampled volume from basal compartment, 0.6 mL, respectively. T is the amount of the drug accumulated in the intestinal tissues in the end of transport study. Furthermore, t0 to t6 mean 0, 0.083, 0.25, 0.5, 1, 1.5 and 2 hrs, respectively.
Analytical Method The concentration of FD-4 in the sample solution was determined at Ex 495 nm and Em 535 nm using a microplate reader (DTX880; Beckman Coulter, Bromma, Sweden).
14
C-rebamipide and
3
H-metoprolol concentrations in the samples taken at the regular time intervals, samples were taken
from the apical and basal side and the tissue concentration of them using the human tissue were determined finishing the transport study.
14
3
C-rebamipide and H-metoprolol were analyzed using a
liquid scintillation counter (1900CA TriCarb; PerkinElmer Life Sciences, Upplands Väsby, Sweden).
Statistical Analysis Results are expressed as the mean ± S.E. of ≥3 experiments. Analysis of variance (ANOVA) was used to test the statistical significance of differences among groups. Statistical significance in the differences of the means was determined by Student’s t-test.
RESULTS corr
To confirm if TI, X
and T
corr
values in the large intestinal tissues of UC patients was changed
depending on autopsy grading, data obtained from human intestinal tissues were analyzed for normal and UC large intestinal segments in Figs. 1(A), 1(B) and 1(C). TI was almost kept constant irrespective of autopsy grading. In addition, T
corr
corr
value in Grade 2 was the largest among all grades and X
value
in grade 2 was the smallest among all grades (Figs. 1(A), 1(B) and 1(C) and Table 2).
DISCUSSION Our previous study is the first large compilation of human permeability data in the Ussing chamber 8
technique to develop a novel prediction system of human intestinal absorption. Therefore, our
6
mini-Ussing chamber technique makes it possible to project drug absorbability in humans more precisely, especially from the viewpoint of comparison of permeability in healthy tissues with that in disease tissues. Investigation of absorption profiles in intestinal tissues from IBD patients such as UC and CD would provide very useful information on selection of appropriate drugs for the treatment of IBD and useful information for pharmaceutical companies to develop effective drugs for IBD as well. In this study, autopsy grade by fibrosis was used to classify intestinal tissues of UC patients. It has been considered that the fibrosis must be the key factor to IBD. Indeed, submucosal fibrosis is well 13
correlated with the severity of intestinal inflammation.
Thus, the investigation of the change of
intestinal permeability from the viewpoint of severity of fibrosis is considered to provide one of the most important information to select the most appropriate drug for treatment of the disease depending on the severity. Additionally, as far as we know this is the first report to handle tissues without fibrosis from cancer patients as normal tissues. Our study demonstrated little change of total absorbability estimated by TI (%), while Xcorr and Tcorr values of the tested three model compounds in autopsy grade 2 significantly decreased and increased, respectively. Our observation indicated that permeability via both of paracellular route and transcellular route is kept constant even in UC patients. Taken together, morphological changes of intestinal tissues caused by fibrosis led to limited permeation and subsequently increased accumulation with little change of membrane permeability at the apical side. Little change of protein 14
expression of efflux transporters in UC tissues was observed.
This is consistent with our finding of 12
little change of rebamipide TI (%) even though rebamipide was shown to be a p-gp substrate.
As
little is known about permeability change and tissue accumulation on the disease grade, the new findings of this study would be worth a great deal to clinical research in order to make a new approach for producing medicines for IBD. Indeed, our findings in this study suggest that it is not required to propose different medication depending on severity of disease simply because membrane permeability was kept constant irrespective of autopsy grade. In the Ussing chamber system, tested molecules must pass through membrane including such as submucosal layer to appear in the basal side. Then, the reason why little change of total absorbed amount with decreased permeation and increased tissue accumulation in Grade 2 tissues was considered to be due to slower diffusion through membrane by fibrosis. Actually,
7
this consideration was partially supported by the fact that water diffusivity evaluated by Diffusion-weighted magnetic resonance imaging was slower in endoscopic inflamed segment 15
compared with normal segment.
In conclusion, the new findings on the drug absorption profile in intestinal tissues from normal and UC region demonstrated a change in drug tissue accumulation and permeation in accordance with disease progression.
Our finding of little change of TI index suggest that it is not required to propose
different medication depending on severity of disease simply because membrane permeability was kept constant irrespective of autopsy grade.
These findings would be quite useful to develop
therapeutic medicine for IBD, which is a well-known specific intractable diseases throughout the world.
Acknowledgements The authors are also very grateful for Dr. Anders Rönnblom MD and Dr. Fredrik Rorsman MD, who belong to Department of Medical Sciences, Uppsala University, in order to supply the human tissues from patients and also they judged the autopsy grade to all tissues we utilized in this study.
Conflicts of interest The authors declare no conflicts of interest.
8
REFERENCES 1) Piovani D, Danese S, Peyrin-Biroulet L, Nikolopoulos GK, Lytras T, Bonovas S. Environmental risk
factors
for
inflammatory
bowel
diseases:
an
umbrella
review
of
Meta-analyses,
Gastroenterology. 2019;157(3):647-659. 2) Haberman Y, Tickle TL, Dexheimer PJ, Kim MO, Tang D, Karns R, Baldassano RN, Noe JD, Rosh J, Markowitz J, Heyman MB, Griffiths AM, Crandall WV, Mack DR, Baker SS, Huttenhower C, Keljo DJ, Hyams JS, Kugathasan S, Walters TD, Aronow B, Xavier RJ, Gevers D, Denson LA. Pediatric Crohn disease patients exhibit specific ileal transcriptome and microbiome signature, J Clin Invest 2014;124(8):3617-3633. 3) Nejdfors P, Ekelund M, Weström BR, Willén R, Jeppsson B. Intestinal Permeability in Humans Is Increased After Radiation Therapy, Dis Colon Rectum 2000;43(11):1582-1587. 4) Miki K, Moore DJ, Butler RN, Southcott E, Couper RT, Davidson GP. The sugar permeability test reflects disease activity in children and adolescents with inflammatory bowel disease, J Pediatr 1998;133(6):750-754. 5) Arslan G, Atasever T, Cindoruk M, Yildirim IS. (51)CrEDTA colonic permeability and therapy response in patients with ulcerative colitis, Nucl Med Commun 2001;22(9):997-1001. 6) Peled Y, Watz C, Gilat T. Measurement of intestinal permeability using 51Cr-EDTA, Am J Gastroenterol 1985;80(10):770-773. 7) Büning C, Geissler N, Prager M, Sturm A, Baumgart DC, Büttner J, Bühner S, Haas V, Lochs H. Increased Small Intestinal Permeability in Ulcerative Colitis: Rather Genetic than Environmental and a Risk Factor for Extensive Disease? Inflamm Bowel Dis 2012;18(10):1932-1939. 8) Miyake M, Toguchi H, Nishibayashi T, Higaki K, Sugita A, Koganei K, Kamada N, Kitazume MT, Hisamatsu T, Sato T, Okamoto S, Kanai T, Hibi T. Establishment of novel prediction system of intestinal absorption in humans using human intestinal tissues, J Pharm Sci2013;102(8):2564-2571. 9) Miyake M, Koga T, Kondo S, Yoda N, Emoto C, Mukai T, Toguchi H. Prediction of drug intestinal absorption in human using the Ussing chamber system: A comparison of intestinal tissues from animals and humans, Eur J Pharm Sci 2016;96:373-380. 10) Miyake M, Kondo S, Koga T, Yoda N, Nakazato S, Emoto C, Mukai T, Toguchi H.
Evaluation of
intestinal metabolism and absorption using the Ussing chamber system equipped with intestinal
9
tissue from rats and dogs, Eur J Pharm Biopharm 2018;122:49-53. 11) Kondo S, Miyake M. Simultaneous prediction of intestinal absorption and metabolism using the mini-Ussing chamber system, J Pharm Sci 2019;108(1):763-769. 12) Miyake M, Nakai D. Effect of proinflammatory cytokine IL-6 on efflux transport of rebamipide in Caco-2 cells, Xenobiotica 2017;47(9):821-824. 13) Gordon IO, Agrawal N, Willis E, Goldblum JR, Lopez R, Allende D, Liu X, Patil DY, Yerian L, El-Khider F, Fiocchi C, Rieder F. Fibrosis in ulcerative colitis is directly linked to severity and chronicity of mucosal inflammation, Aliment Pharmacol Ther 2018;47(7):922-939. 14) Erdmann P, Bruckmueller H, Martin P, Busch D, Haenisch S, Müller J, Wiechowska-Kozlowska A, Partecke LI, Heidecke CD, Cascorbi I, Drozdzik M, Oswald S. Dysregulation of Mucosal Membrane Transporters and Drug-Metabolizing Enzymes in Ulcerative Colitis, J Pharm Sci 2019;108(2):1035-1046. 15) Yu LL, Yang HS, Zhang BT, Lv ZW, Wang FR, Zhang CY, Chen WB, Zhang HM. Diffusion-weighted magnetic resonance imaging without bowel preparation for detection of ulcerative colitis, World J Gastroenterol 2015;21(33):9785-9792.
10
FIGURE LEGENDS
Fig. 1
Transport index (TI) of FD-4, rebamipide and metoprolol across human large intestinal
tissues from UC patients.
(A) FD-4, (B) rebamipide, (C) metoprolol.
calculated in accordance with a previous paper [3], respectively. SE of ≥5 experiments. corr
X
;
,T
corr
*
†
corr
TI, X
and T
11
were
Each value of TI is the mean with
p <0.05 compared with Normal, p <0.05 compared with Grade 2.
.
corr
Keys:
,
Table 1 Patient characteristics and subject numbers Subject number
Autopsy grade
Autopsy grade
Autopsy grade
Total
0
1
2
Male
14
4
8
3
Female
8
4
3
1
8
8
---
---
14
---
11
4
Characteristics
Sex
Primary disease Cancer (Normal) Ulcerative colitis (UC)
Subjects ranged in age between 32 and 69 years old (mean age, 44.1). The tissues from one male UC patient were judged as grade 1 in non-inflamed tissue and grade 2 in inflamed tissue.
Table 2 Transport index (TI), X Model Drugs FD-4
Rebamipide
corr
and T
corr
TI % (number)
X
corr
corr
%
T
Autopsy Grade
%
0.355 ± 0.027 (31)
0.200 ± 0.029
0.155 ± 0.018
Normal
0.371 ± 0.018 (54)
0.226 ± 0.018
0.145 ± 0.006
0
0.458 ± 0.055 (19)
0.274 ± 0.064
0.184 ± 0.014
1
0.314 ± 0.038 (19)
0.080 ± 0.018
0.712 ± 0.047 (31)
0.460 ± 0.058
0.252 ± 0.029
Normal
0.787 ± 0.050 (54)
0.541 ± 0.049
0.246 ± 0.010
0
0.961 ± 0.101 (19)
0.636 ± 0.131
0.325 ± 0.038
1
0.639 ± 0.045 (12)
0.140 ± 0.021
*
0.499 ± 0.053
*
2
1.101 ± 0.071 (20)
0.595 ± 0.081
0.506 ± 0.060
Normal
1.135 ± 0.070 (31)
0.664 ± 0.082
0.471 ± 0.029
0
1.302 ± 0.133 (14)
0.852 ± 0.185
0.450 ± 0.057
1
*
Metoprolol
values for each model drug and each disease
*
0.905 ± 0.042 (11)
corr
corr
0.198 ± 0.031
*
*
0.234 ± 0.028
0.706 ± 0.048
*
*
2
2
Transport index (TI), X and T were calculated as shown in a previous paper [3], and are expressed as the mean ± SE. * p<0.05 compared with Normal. Number in the parentheses indicate total number of transport assays. Number of assays from one tissue was different depending on its tissue size.
Fig. 1; Nakai et al
0.2
0
1.2
0.8
0.4
0
B
C
*
2.0 Transport Index (%)
0.4
A Transport Index (%)
Transport Index (%)
0.6
1.5
1.0
0.5
0
*
S0022-3549(19)30823-8
DOI:
https://doi.org/10.1016/j.xphs.2019.12.015
Reference:
XPHS 1832
To appear in:
Journal of Pharmaceutical Sciences
Received Date: 29 October 2019 Revised Date:
2 December 2019
Accepted Date: 18 December 2019
Please cite this article as: Nakai D, Miyake M, Hashimoto A, Comparison of the intestinal drug permeation and accumulation between normal human intestinal tissues and human intestinal tissues with ulcerative colitis, Journal of Pharmaceutical Sciences (2020), doi: https://doi.org/10.1016/ j.xphs.2019.12.015. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
ID 19-972
Note
Comparison of the intestinal drug permeation and accumulation between normal human intestinal tissues and human intestinal tissues with ulcerative colitis
1)
1), 2)*
Daisuke Nakai , Masateru Miyake
, Ayako Hashimoto
2)
1)
Department of Pharmacy, Uppsala University, Husargatan 3, Uppsala, 75123, Sweden.
2)
Quality Assurance Section, Quality Assurance Department, Headquarters for Product Safety and
Quality Assurance, Otsuka Pharmaceutical Co. Ltd., Matsudani Itano-cho, Itano-gun Tokushima 779-0195, Japan.
*
To whom correspondence should be addressed Phone: +81-88-672 6070 Fax: +81-88-672 6086 E-mail: [email protected]
Revised version Date:December 3, 2019
1
ABSTRACT The purpose of this study was to examine drug absorption profile utilizing human intestinal tissues from ulcerative colitis (UC) patients and to compare with normal tissues from intestinal cancer patients. Human intestinal tissues from UC and cancer patients mounted in a mini-Ussing chamber were used to evaluate the permeation of drugs, including FD-4, a very low permeable marker, rebamipide, a low permeable marker, and metoprolol, a high permeable marker. The transport index (TI), an index of sum of permeated and tissue-accumulated molecules, of the model drugs was in accordance with their absorption rank order, and was almost kept constant irrespective of autopsy grade based on tissue fibrosis. showed the decreased X
corr
On the other hand, UC tissues of grade 2
, an index of permeated amount of molecules and increased T
corr
, an index
of tissue-accumulated molecules for every tested compound. Our finding of the transport characteristics in intestinal tissues of severe UC patients in mini-Ussing chamber system demonstrated that autopsy grade of UC patients didn’t drastically change membrane permeability of the tested compounds.
Furthermore, it was suggested that morphological changes of
intestinal tissues caused by fibrosis led to limited permeation and subsequently increased accumulation with little change of total absorption.
KEY WORDS Human absorption, Ulcerative colitis (UC), Human Tissues, Transport index (TI), Autopsy grade.
2
INTRODUCTION Ulcerative colitis (UC) and Crohn’s disease (CD) are the specific refractory disease at least in Japan. The number of patients regarding inflammatory bowel disease (IBD) such as UC and CD has 1
been increasing recently. The lesion sites of UC are specific in the large intestine, while the lesion sites of CD are not intestinal region specific to be observed in the upper, middle and lower intestinal 2
tract. Nejdfors et al. have reported that permeability of mannitol and FD-4 seemed to be affected by 3
the radiation therapy, suggesting that even slight injury to the intestinal mucosa would lead to change of the intestinal permeability. Therefore, it is important to understand the effect of the autopsy grades in UC patients on membrane permeability. So far whether intestinal permeability is increased in active disease region of UC patients is controversial when urinary excretion of sugars or 51Cr-EDTA as a 4-7
permeability index was examined after dosing of the marker substances in human.
Evaluation of
membrane permeability in Ussing chamber system could be a solution to the controversial conclusions since this system is able to provide more focused information on membrane permeability to examine the effect of disease state on membrane permeability. We have already established the novel human intestinal absorption system using mini-Ussing chamber by utilizing the human, dog, monkey and rat 8,9
intestinal tissues.
Also we have confirmed the validity and reproducibility of the transport index (TI)
utilizing the large intestinal tissues from UC or CD patients for the evaluation of intestinal 8
absorbability. In addition, we have also successfully conducted the simultaneous prediction of 10,11
intestinal absorption and metabolism using the mini-Ussing chamber system.
In the present study, we investigated whether autopsy grade in UC tissues affected the drug permeability and tissue accumulation through examination of permeability of three types of model drugs such as FD-4, a very low permeable marker via paracellular route, rebamipide, a low permeable 12
marker of a p-glycoprotein substrate,
and metoprolol, a high permeable marker via transcellular
route.
MATERIALS AND METHODS Materials 3
FD-4 was purchased from Sigma-Aldrich (Boston, MA, USA). H-metoprolol was purchased from PerkinElmer Sverige AB (Upplands Väsby, Sweden).
3
14
C-rebamipide was obtained from Otsuka
Pharmaceutical Co. Ltd. All other reagents used were of the highest purity.
Human Intestinal Tissues Human large intestinal tissues were taken from male or female patients, aged 32 to 69 years old, with UC and intestinal cancer (Table 1), with permission from the patients in accordance with the Declaration of Helsinki. All experiments were approved by the institutional review board of all institutions, and written informed consent was obtained from all patients.
All tissues chosen from the
isolated tissues were placed in ice-cold transport buffer immediately after removal from the patient’s body in the surgical operation room and were transferred to the laboratory within 1 hr. Transport buffer was composed of 128 mM NaCl, 5.1 mM KCl, 1.4 mM CaCl2, 1.3 mM MgSO4, 21 mM NaHCO3, 1.3 mM KH2PO4 and 10 mM NaH2PO4, adjusted to pH 7.4.
Autopsy diagnosis The measure of autoptical abnormality was quantified on an arbitrary scale of 0 - 2, in which no effect was scored as 0 and redding and slightly thickened mucosa was scored as 1, and a severe effect of ulcer and thickened mucosa by fibrosis was scored as 2. This evaluation was carried out by a medical doctor in a blinded fashion.
Transport Experiment in Mini-Ussing Chamber The mini-Ussing chamber was used for transport experiments and the isolated human large intestinal tissues, which were biopsies from patients and normal, were mounted following the method reported previously.
3,8,9
The tissues were transferred to the experimental room within 1 h. First of all,
isolated human large intestinal tissues, from which the muscle layer had been removed with fine tweezers, were mounted vertically in the mini-Ussing-type chambers, providing an exposed area of 2
0.07 cm for the transport study.
The apical and basal transport buffers were defined at 1.35 mL, and
the solution temperature was maintained at 37°C wit h a water-jacketed reservoir. The apical and basal buffers were containing 5 mM mannitol on apical one and 5 mM D-glucose and 1% (w/v) bovine serum albumin in basal one, respectively, and those buffers were gassed with 95% O2/5% CO2 before and during the transport experiment. FD-4 and the radioactive compounds were placed on the apical side
4
3
at a concentration of 10 mg/mL and 0.4 µCi/mL for H-metoprolol and 0.1 µCi/mL for
14
C-rebamipide,
respectively, and the transport study was performed for 2 hr. The samples were taken from basal side at 600 µL and replaced with an equivalent volume of fresh buffer at 0.5, 1, 1.5 and 2 hr, whereas the samples were also taken from apical side at 10 µL without replacing at 0.083, 0.25, 0.5, 1, 1.5 and 2 hr to estimate the change in drug concentration due to the possibility of the drug precipitation during the transport study. After taking the final sample, the tissue was homogenized with 1 mL basal transport buffer under ice-cold conditions to determine the accumulated amount of drugs.
Calculation of Transport Index We have already defined the transport index (TI), which is the percent of dose transported and tissue-accumulated corrected by the AUC value of the drug in the apical compartment (Eq. 1) in order to estimate the permeability based on the actual drug concentration in the apical compartment.
TI = X corr + T corr
corr
where X
and T
Eq. 1
corr
are the percent of the dose transported to the basal side compartment and that of
the dose accumulated in the tissue, respectively, which were corrected by the AUC of FD-4, rebamipide and metoprolol in the apical compartment of drugs (Eqs. 2 and 3).
X corr
T
corr
ti t i −1 2 ⋅ (C t0 ⋅ t ) ⋅ (C t3 ⋅ V ) 6 2 ⋅ (C t 0 apical 3 basal basal apical ⋅ (t 6 − t 3 )) ⋅ (C basal ⋅ Vbasal − C basal ⋅ (Vbasal − V sampled ) = 3 + ∑ t i −1 ti (C apical + C apical ) ⋅ (t i − t i −1 ) t i −1 ti i=4 (( C + C ) ⋅ ( t − t )) ∑ apical apical i i − 1 i =1 1 ⋅ t ⋅ 100 (%) Eq .2 0 C apical ⋅ Vapical
=
t0 2 ⋅ Capical ⋅ t6 ⋅ T 6
∑ (C i =1
ti −1 apical
+C
ti apical
) ⋅ (t i − t i−1 )
⋅
C
t0 apical
1 ⋅100 (%) ⋅Vapical
5
Eq.3
where
ti ti Capical and Cbasal mean the drug concentrations in the apical side and basal side at time ti,
Vbasal and Vsampled represent the volumes of apical, basal compartments, both
respectively. Vapical ,
1.35 mL, and sampled volume from basal compartment, 0.6 mL, respectively. T is the amount of the drug accumulated in the intestinal tissues in the end of transport study. Furthermore, t0 to t6 mean 0, 0.083, 0.25, 0.5, 1, 1.5 and 2 hrs, respectively.
Analytical Method The concentration of FD-4 in the sample solution was determined at Ex 495 nm and Em 535 nm using a microplate reader (DTX880; Beckman Coulter, Bromma, Sweden).
14
C-rebamipide and
3
H-metoprolol concentrations in the samples taken at the regular time intervals, samples were taken
from the apical and basal side and the tissue concentration of them using the human tissue were determined finishing the transport study.
14
3
C-rebamipide and H-metoprolol were analyzed using a
liquid scintillation counter (1900CA TriCarb; PerkinElmer Life Sciences, Upplands Väsby, Sweden).
Statistical Analysis Results are expressed as the mean ± S.E. of ≥3 experiments. Analysis of variance (ANOVA) was used to test the statistical significance of differences among groups. Statistical significance in the differences of the means was determined by Student’s t-test.
RESULTS corr
To confirm if TI, X
and T
corr
values in the large intestinal tissues of UC patients was changed
depending on autopsy grading, data obtained from human intestinal tissues were analyzed for normal and UC large intestinal segments in Figs. 1(A), 1(B) and 1(C). TI was almost kept constant irrespective of autopsy grading. In addition, T
corr
corr
value in Grade 2 was the largest among all grades and X
value
in grade 2 was the smallest among all grades (Figs. 1(A), 1(B) and 1(C) and Table 2).
DISCUSSION Our previous study is the first large compilation of human permeability data in the Ussing chamber 8
technique to develop a novel prediction system of human intestinal absorption. Therefore, our
6
mini-Ussing chamber technique makes it possible to project drug absorbability in humans more precisely, especially from the viewpoint of comparison of permeability in healthy tissues with that in disease tissues. Investigation of absorption profiles in intestinal tissues from IBD patients such as UC and CD would provide very useful information on selection of appropriate drugs for the treatment of IBD and useful information for pharmaceutical companies to develop effective drugs for IBD as well. In this study, autopsy grade by fibrosis was used to classify intestinal tissues of UC patients. It has been considered that the fibrosis must be the key factor to IBD. Indeed, submucosal fibrosis is well 13
correlated with the severity of intestinal inflammation.
Thus, the investigation of the change of
intestinal permeability from the viewpoint of severity of fibrosis is considered to provide one of the most important information to select the most appropriate drug for treatment of the disease depending on the severity. Additionally, as far as we know this is the first report to handle tissues without fibrosis from cancer patients as normal tissues. Our study demonstrated little change of total absorbability estimated by TI (%), while Xcorr and Tcorr values of the tested three model compounds in autopsy grade 2 significantly decreased and increased, respectively. Our observation indicated that permeability via both of paracellular route and transcellular route is kept constant even in UC patients. Taken together, morphological changes of intestinal tissues caused by fibrosis led to limited permeation and subsequently increased accumulation with little change of membrane permeability at the apical side. Little change of protein 14
expression of efflux transporters in UC tissues was observed.
This is consistent with our finding of 12
little change of rebamipide TI (%) even though rebamipide was shown to be a p-gp substrate.
As
little is known about permeability change and tissue accumulation on the disease grade, the new findings of this study would be worth a great deal to clinical research in order to make a new approach for producing medicines for IBD. Indeed, our findings in this study suggest that it is not required to propose different medication depending on severity of disease simply because membrane permeability was kept constant irrespective of autopsy grade. In the Ussing chamber system, tested molecules must pass through membrane including such as submucosal layer to appear in the basal side. Then, the reason why little change of total absorbed amount with decreased permeation and increased tissue accumulation in Grade 2 tissues was considered to be due to slower diffusion through membrane by fibrosis. Actually,
7
this consideration was partially supported by the fact that water diffusivity evaluated by Diffusion-weighted magnetic resonance imaging was slower in endoscopic inflamed segment 15
compared with normal segment.
In conclusion, the new findings on the drug absorption profile in intestinal tissues from normal and UC region demonstrated a change in drug tissue accumulation and permeation in accordance with disease progression.
Our finding of little change of TI index suggest that it is not required to propose
different medication depending on severity of disease simply because membrane permeability was kept constant irrespective of autopsy grade.
These findings would be quite useful to develop
therapeutic medicine for IBD, which is a well-known specific intractable diseases throughout the world.
Acknowledgements The authors are also very grateful for Dr. Anders Rönnblom MD and Dr. Fredrik Rorsman MD, who belong to Department of Medical Sciences, Uppsala University, in order to supply the human tissues from patients and also they judged the autopsy grade to all tissues we utilized in this study.
Conflicts of interest The authors declare no conflicts of interest.
8
REFERENCES 1) Piovani D, Danese S, Peyrin-Biroulet L, Nikolopoulos GK, Lytras T, Bonovas S. Environmental risk
factors
for
inflammatory
bowel
diseases:
an
umbrella
review
of
Meta-analyses,
Gastroenterology. 2019;157(3):647-659. 2) Haberman Y, Tickle TL, Dexheimer PJ, Kim MO, Tang D, Karns R, Baldassano RN, Noe JD, Rosh J, Markowitz J, Heyman MB, Griffiths AM, Crandall WV, Mack DR, Baker SS, Huttenhower C, Keljo DJ, Hyams JS, Kugathasan S, Walters TD, Aronow B, Xavier RJ, Gevers D, Denson LA. Pediatric Crohn disease patients exhibit specific ileal transcriptome and microbiome signature, J Clin Invest 2014;124(8):3617-3633. 3) Nejdfors P, Ekelund M, Weström BR, Willén R, Jeppsson B. Intestinal Permeability in Humans Is Increased After Radiation Therapy, Dis Colon Rectum 2000;43(11):1582-1587. 4) Miki K, Moore DJ, Butler RN, Southcott E, Couper RT, Davidson GP. The sugar permeability test reflects disease activity in children and adolescents with inflammatory bowel disease, J Pediatr 1998;133(6):750-754. 5) Arslan G, Atasever T, Cindoruk M, Yildirim IS. (51)CrEDTA colonic permeability and therapy response in patients with ulcerative colitis, Nucl Med Commun 2001;22(9):997-1001. 6) Peled Y, Watz C, Gilat T. Measurement of intestinal permeability using 51Cr-EDTA, Am J Gastroenterol 1985;80(10):770-773. 7) Büning C, Geissler N, Prager M, Sturm A, Baumgart DC, Büttner J, Bühner S, Haas V, Lochs H. Increased Small Intestinal Permeability in Ulcerative Colitis: Rather Genetic than Environmental and a Risk Factor for Extensive Disease? Inflamm Bowel Dis 2012;18(10):1932-1939. 8) Miyake M, Toguchi H, Nishibayashi T, Higaki K, Sugita A, Koganei K, Kamada N, Kitazume MT, Hisamatsu T, Sato T, Okamoto S, Kanai T, Hibi T. Establishment of novel prediction system of intestinal absorption in humans using human intestinal tissues, J Pharm Sci2013;102(8):2564-2571. 9) Miyake M, Koga T, Kondo S, Yoda N, Emoto C, Mukai T, Toguchi H. Prediction of drug intestinal absorption in human using the Ussing chamber system: A comparison of intestinal tissues from animals and humans, Eur J Pharm Sci 2016;96:373-380. 10) Miyake M, Kondo S, Koga T, Yoda N, Nakazato S, Emoto C, Mukai T, Toguchi H.
Evaluation of
intestinal metabolism and absorption using the Ussing chamber system equipped with intestinal
9
tissue from rats and dogs, Eur J Pharm Biopharm 2018;122:49-53. 11) Kondo S, Miyake M. Simultaneous prediction of intestinal absorption and metabolism using the mini-Ussing chamber system, J Pharm Sci 2019;108(1):763-769. 12) Miyake M, Nakai D. Effect of proinflammatory cytokine IL-6 on efflux transport of rebamipide in Caco-2 cells, Xenobiotica 2017;47(9):821-824. 13) Gordon IO, Agrawal N, Willis E, Goldblum JR, Lopez R, Allende D, Liu X, Patil DY, Yerian L, El-Khider F, Fiocchi C, Rieder F. Fibrosis in ulcerative colitis is directly linked to severity and chronicity of mucosal inflammation, Aliment Pharmacol Ther 2018;47(7):922-939. 14) Erdmann P, Bruckmueller H, Martin P, Busch D, Haenisch S, Müller J, Wiechowska-Kozlowska A, Partecke LI, Heidecke CD, Cascorbi I, Drozdzik M, Oswald S. Dysregulation of Mucosal Membrane Transporters and Drug-Metabolizing Enzymes in Ulcerative Colitis, J Pharm Sci 2019;108(2):1035-1046. 15) Yu LL, Yang HS, Zhang BT, Lv ZW, Wang FR, Zhang CY, Chen WB, Zhang HM. Diffusion-weighted magnetic resonance imaging without bowel preparation for detection of ulcerative colitis, World J Gastroenterol 2015;21(33):9785-9792.
10
FIGURE LEGENDS
Fig. 1
Transport index (TI) of FD-4, rebamipide and metoprolol across human large intestinal
tissues from UC patients.
(A) FD-4, (B) rebamipide, (C) metoprolol.
calculated in accordance with a previous paper [3], respectively. SE of ≥5 experiments. corr
X
;
,T
corr
*
†
corr
TI, X
and T
11
were
Each value of TI is the mean with
p <0.05 compared with Normal, p <0.05 compared with Grade 2.
.
corr
Keys:
,
Table 1 Patient characteristics and subject numbers Subject number
Autopsy grade
Autopsy grade
Autopsy grade
Total
0
1
2
Male
14
4
8
3
Female
8
4
3
1
8
8
---
---
14
---
11
4
Characteristics
Sex
Primary disease Cancer (Normal) Ulcerative colitis (UC)
Subjects ranged in age between 32 and 69 years old (mean age, 44.1). The tissues from one male UC patient were judged as grade 1 in non-inflamed tissue and grade 2 in inflamed tissue.
Table 2 Transport index (TI), X Model Drugs FD-4
Rebamipide
corr
and T
corr
TI % (number)
X
corr
corr
%
T
Autopsy Grade
%
0.355 ± 0.027 (31)
0.200 ± 0.029
0.155 ± 0.018
Normal
0.371 ± 0.018 (54)
0.226 ± 0.018
0.145 ± 0.006
0
0.458 ± 0.055 (19)
0.274 ± 0.064
0.184 ± 0.014
1
0.314 ± 0.038 (19)
0.080 ± 0.018
0.712 ± 0.047 (31)
0.460 ± 0.058
0.252 ± 0.029
Normal
0.787 ± 0.050 (54)
0.541 ± 0.049
0.246 ± 0.010
0
0.961 ± 0.101 (19)
0.636 ± 0.131
0.325 ± 0.038
1
0.639 ± 0.045 (12)
0.140 ± 0.021
*
0.499 ± 0.053
*
2
1.101 ± 0.071 (20)
0.595 ± 0.081
0.506 ± 0.060
Normal
1.135 ± 0.070 (31)
0.664 ± 0.082
0.471 ± 0.029
0
1.302 ± 0.133 (14)
0.852 ± 0.185
0.450 ± 0.057
1
*
Metoprolol
values for each model drug and each disease
*
0.905 ± 0.042 (11)
corr
corr
0.198 ± 0.031
*
*
0.234 ± 0.028
0.706 ± 0.048
*
*
2
2
Transport index (TI), X and T were calculated as shown in a previous paper [3], and are expressed as the mean ± SE. * p<0.05 compared with Normal. Number in the parentheses indicate total number of transport assays. Number of assays from one tissue was different depending on its tissue size.
Fig. 1; Nakai et al
0.2
0
1.2
0.8
0.4
0
B
C
*
2.0 Transport Index (%)
0.4
A Transport Index (%)
Transport Index (%)
0.6
1.5
1.0
0.5
0
*