Effect of Polyols on Caco-2 Transport of Low-Permeability Drugs
Case Study

Effect of Polyols on Caco-2 Transport of Low-Permeability Drugs

Presented at the 13th PBP World meeting, 28 - 31 March 2022 | Rotterdam, The Netherlands



The Biopharmaceutics Classification System (BCS), based on aqueous solubility and on intestinal permeability, is an approach used to predict drug absorption during pharmaceutical development and to justify a waiver for the in vivo bioequivalence.1 For the formulation development, it is essential to know the possible impact of an excipient on the drug’s bioavailability. This is particularly important for BCS class III drugs that exhibit high solubility and low permeability. Some studies have shown that excipients are able to influence the drug absorption or its bioavailability through the modification of its solubility or through changes of the intestinal permeability or through the modulation of the gastrointestinal motility, via their osmotic loads in the small intestine.2,3,4,5 Another aspect is the potential of some excipients to modulate the intestinal permeability of the drug. A study carried out in humans, showed that commonly used excipients did not modulate the absorption of the BCS class 3 drugs cimetidine and acyclovir.6 Nevertheless, some excipients like TweenTM 80 (polysorbate 80) or docusate did have an impact on the Caco-2 permeability of drugs with low permeability such as furosemide, cimetidine and hydrochlorothiazide by inhibiting their active efflux, thus increasing the apical-to-basolateral directionpermeability.7 Due to contradictory results about the influence of ingredients on intestinal absorption, the impact of polyols in pharmaceutical formulations was studied. The aim of this work is to investigate the influence of four polyols: mannitol, maltitol, sorbitol and xylitol on the drug permeability. Seven active pharmaceutical ingredients (API) were tested, selected for their low permeability and their different physico-chemical properties: furosemide, amiloride, atenolol, ranitidine, nadolol, L-thyroxine and acyclovir. Permeability assays were performed with Caco-2 permeability model. 



All tested polyol samples were of pharmaceutical excipient quality. PEARLITOL® 200 SD mannitol, SweetPearl® P90 maltitol, NEOSORB® P 200 SD sorbitol and XYLISORB® 90 xylitol, supplied by Roquette Frères, Lestrem, France. The active substances came from Sigma Aldrich. The cytotoxicity of these seven drug substances, four excipients and combinations thereof was evaluated on Caco-2 cells, in order to make sure that the tested concentrations did not cause cellular damage. Any possible cytotoxicity was measured by ATP luminescence technology using Cell Titer-Glo Luminescent Cell Viability Assay (Promega/G7571). 
The relative quantification of each active substance and each polyol was performed using a 1290 Infinity Binary LC system (Agilent Technologies, Waldbronn, Germany) coupled to a Q-TRAP® 5500 mass spectrometer with an ESI Turbo V ion source (SCIEX, Foster City, CA, USA). Permeability assays were performed with Caco-2 cells, seeded in Multiscreen™ 96-well plates (Millipore), at days 21-25 post-seeding and performed in either apical to basolateral (A-B) direction or basolateral – apical (B-A) directions. All excipients and actives substances were tested at doses at which no cytotoxicity was observed; alone or in mix in buffer A for apical compartment (HBSS + 5 mM MES pH 6.5) and buffer B for basal compartment (HBSS + 10 mM Hepes pH 7.4) with a final concentration of DMSO of 1% (v/v). Incubation time for the permeability was as follows: A-B: 60 minutes 37 °C (5% CO2); B-A: 40 minutes 37 °C (5% CO2). Colchicine (PgP-transporter control) and ranitidine (very low permeability), labetalol (medium permeability) and propranolol (high permeability) were tested as four standard controls at concentration of 10 μM. Each experimental well was supplemented with one concentration of Lucifer yellow (LY), as an internal standard. Moreover, transepithelial electrical resistance (TEER) was measured in order to confirm the integrity and permeability of the monolayer, before and after the transfer. Apparent permeability coefficients (Papp) were calculated using analytical determination in A and B compartment for combinations of actives and polyols; this was in turn compared to the permeability of the active substance alone for apical to basolateral and basolateral to apical directions. The recovery of each compound was calculated between A and B compartment. The apparent permeability coefficient (Papp) of the test compound was calculated as described by Ma and co-workers.8



No cytotoxicity was detected for any of the four polyols and seven active substances, alone or in combination at doses up to 10 μM. LC-MS methods were optimized for the detection of four polyols and seven active substances in medium A and B for the permeability assay. For the permeability assay, no LY permeated through the Caco-2 cells monolayer. No modifications of electrical resistance TEER were detected, proving that the cell monolayer remained intact throughout the experiment. Neither the active substances nor the excipients or its combinations modulated the paracellular transport. Based on the Caco-2 permeability assay (see table 1), the active substances furosemide, amiloride, atenolol, ranitidine, nadolol, acyclovir and L-thyroxine did not cross from the apical to the basolateral region or from the basolateral to apical region. Combining these drugs substances with a polyol did not modulate the drug permeability in either direction under the chosen test conditions (see table 1). 


Table 1. Mean Papp (apparent permeability coefficient) of seven active substances in Apical to Basolateral and Basolateral to Apical Caco-2 permeability assay, either alone or in the presence of an excipient (ND: not determined).


(x10-6 cm.s-1)
 Apical to Basolateral Compartment  Basolateral to Apical Compartment
 No excipient  Mannitol  Maltitol  Sorbitol Xylitol   No excipient  Mannitol  Maltitol Sorbitol  Xylitol 
 Furosemide  0.08  0.07  0.08  0.07 0.07   5.88 5.16   5.83 5.86   5.41
 Amiloride  0.15 0.23   0.22 0.21   0.24 0.46   0.28 0.34   0.29  0.24
 Atenolol  0.16  0.17 0.2   0.2 0.18   0.14 0.19   0.22 0.21  0.23
 Ranitidine  0.15 0.16 0.19 0.18 0.14 0.4 0.44 0.39 0.4 0.45
 Nadolol  0.06 0.08 0.08 0.08 0.11 0.1 0.18 0.17 0.19 0.32
 Acyclovir 0.15 0.17 0.08 0.22 0.14 0.36 0.31 0.14 0.09 0.17
 L-Thyroxine 0.74 0.75 ND 0.71 ND 0.4 0.29 ND 0.28 ND



The permeability of four BCS Class III drugs (including acyclovir, atenolol and nadolol) in presence of five different excipients (lactose, povidone, hydroxypropyl methylcellulose, sodium lauryl sulfate and PEG 400) was always studied, using two different models: Caco-2 cell monolayers as well as in situ rat intestinal perfusion. No permeability increase of any of the active substances was observed in the presence of any of the tested excipients in either of the models.9
This study focused on other commonly used excipients, mannitol, maltitol, sorbitol and xylitol. All these polyols did not change the permeability of furosemide, amiloride, atenolol, ranitidine, nadolol, acyclovir and L-thyroxine. Moreover, the presence of polyols did not alter the efflux of the active principle (basolateral to apical) (table 1). 
These results show that, under the tested experimental conditions and concentrations, the intestinal permeability of these four BCS Class III compounds is not modulated by the presence of polyols. Sorbitol, mannitol and xylitol are suitable for formulation with low permeability API.



1. Amidon G.L., Lennernäs H., Shah V.P. and Crison J.R. A theoretical basis for a biopharmaceutics drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm. Res. (1995) 12, 13–20.


2. Adkin D.A., Davis S.S., Sparrow R.A., Huckle P.D., Philips A.J. and Wilding I.R. The effects of pharmaceutical excipients on small intestinal transit. Br. J. Clin. Pharmacol. (1995) 39,381–387.


3. Ashiru D.A., Patel R. and Basit A.W. Polyethylene glycol 400 enhances the bioavailability of a BCS Class III drug (ranitidine) in male subjects but not females. Pharm. Res. (2008) 25,2327–2333.


4. Adkin A.D., Davis S. S., Sparrow R. A., Huckle P. D., Phillips A. J. and Wilding I. R. The Effect of Mannitol on the Oral Bioavailability of Cimetidine. J. Pharm. Sc. (1995) 39, 381-387.


5. Chen M.L., Sadrieh N. and Yu L. Impact of Osmotically Active Excipients on Bioavailability and Bioequivalence of BCS Class III Drugs. The AAPS Journal. (2013) 15, 1043-1050.


6. Vaithianathan S., Haidar S.H., Zhang X., Jiang W., Avon C., Dowling T.C, Shao C., Kane M., Hoag S.W., Flasar M.H., Ting T.Y., Polli J.E. Effect of Common Excipients on the Oral Drug Absorption of Biopharmaceutics Classification System Class 3 Drugs Cimetidine and Acyclovir. J. Pharm. Sc. (2016) 105,996-1005.


7. Rege B. D., Yu L.X., Hussain A. S. and Polli J.E. Effect of Common Excipients on Caco-2 Transport of Low-Permeability Drugs. J. Pharm. Sc. (2001) 90, 1770-1786.


8. Ma B., Wang J., Sun J., Li M., Xu H., Sun G. and Sun X Permeability of rhynchophylline across human intestinal cell in vitro. Int. J. Clin. Exp. Pathol. (2014), 7(5), 1957-1966.


9. Parr A, Hidalgo I.J., Bode C., Brown W., Yazdanian M., Gonzalez M.A., Sagawa K., Miller K., Jiang W. and Stippler E.S. The Effect of Excipients on the Permeability of BCS Class III Compounds and Implications for Biowaivers. Pharm. Res. (2016) 33, 167-176.


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®Registered trademark(s) of Roquette Frères. 

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