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European Journal of Pharmaceutical Sciences 17 (2002) 6371

www.elsevier.nl/locate/ejps

Mechanisms for absorption enhancement of inhaled insulin by sodium

taurocholatea , b b b ,1*Fredrik Johansson , Elisabeth Hjertberg , Stefan Eirefelt , Ann Tronde ,

b ,2Ursula Hultkvist Bengtsson

aPreformulation & Biopharmaceutics, AstraZeneca R&D Lund, SE-221 87Lund, Sweden

bDiscovery DMPK, AstraZeneca R&D Lund, SE-221 87Lund, Sweden

Received 22 April 2002; received in revised form 16 July 2002; accepted 17 July 2002

Abstract

The effects of sodium taurocholate (NaTC) on the absorption of inhaled insulin was investigated using both in vivo and in vitro

experiments. The absolute bioavailability of insulin when given as a nebulized solution (0.6 mM) to anesthetized intubated beagle dogs

was low (2.660.3%). However, when NaTC at different concentrations (232 mM) were included in the formulations the bioavailability

increased and at 32 mM it was about nine times higher (23.264.4%) than for pure insulin. In a similar concentration interval (2025

mM) NaTC decreased the transepithelial electrical resistance (TEER) across Caco-2 cell monolayers leading to an increased permeability

of insulin. At higher concentrations (above 30 mM) the viability of the Caco-2 cells decreased and the insulin permeability increased

dramatically. Furthermore, we show that NaTC in the concentration range 215 mM gradually decreases the aggregation state of insulin,

i.e., produces mono- or dimeric insulin from hexameric insulin. In conclusion, NaTC increases the bioavailability of nebulized insulin,

increases the permeability of insulin across Caco-2 cell monolayers, and decreases the aggregation state of insulin at similar

concentrations. We suggest that the main mechanisms behind the absorption enhancement of inhaled insulin by NaTC are the production

of insulin monomers and an opening of tight junctions between adjacent airway epithelial cells.

2002 Elsevier Science B.V. All rights reserved.

Keywords: Insulin; Inhalation; Absorption enhancer; Sodium taurocholate; Caco-2; Aggregation

1. Introduction than insulin administered via subcutaneous injection allow-

ing a better mealtime glucose control (Heinemann et al.,

The pulmonary route for the systemic delivery of 1997, 2000; Patton et al., 1999). A potential problem with

proteins and peptides has been suggested to be a valid topically administered insulin is the relatively low bio-

alternative to parenteral delivery (Wall, 1995; Patton and availability. There are several reports on the pulmonary

Plaz, 1992; Patton, 1996; Patton et al., 1999; Smith, 1997). absorption of insulin in both animal and human studies;

Potential advantages for systemic administration of pro- however, there are large differences in the reported bio-

teins and peptides via the lung include a large absorptive availabilities ranging from a few to almost 60% compared

area, a highly vascularized mucosa, relatively low levels of to subcutaneous injections (Wall, 1995; Patton, 1996).proteases and peptidases, and no hepatic first pass metabo- Heinemann et al. (1997) reported an absolute bioavail-

lism (Wall, 1995; Patton, 1996; Smith, 1997). Further- ability of 5.662.8% in man when insulin was administered

more, inhaled insulin is more rapidly absorbed and cleared as a dry powder formulation. When an absorption enhancer

(a bile salt) was included in the powder formulation at an

insulin to enhancer ratio of 3:1 (w/w) the bioavailability*Corresponding author. Tel.: 146-46-337-671; fax: 146-46-337-780. increased (10.265.0%, Heinemann et al., 2000) suggesting

E-mail address: [email protected] (F. Johan- the inclusion of absorption enhancers to be a valid mean tosson). increase the bioavailability of inhaled insulin in man.1Present address: Experimental Medicine, AstraZeneca R&D Lund, SE-

However, the use of absorption enhancers for the221 87 Lund, Sweden.2 pulmonary route may give rise to safety concerns regard-Present address: IBD Global Product Team, AstraZeneca R&D Lund,SE-221 87 Lund, Sweden. ing possible long-term effects. In order to be able to

0928-0987/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved.

PII : S0928-0987(02)00133-1
mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]

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64 F. Johansson et al. / European Journal of Pharmaceutical Sciences 17 (2002) 6371

predict long-term effects of a particular absorption en- in Lund, Sweden (M241/93 and M21/ 97). The dogs were

hancer and to optimize the concentration of enhancer in the kept under standardized temperature (1920 8C), humidity

pharmaceutical formulation, it is important to understand (55615%), and light (12:12 h lightdark) conditions at the

the mechanism(s) for absorption enhancement. Veterinary Science Department at AstraZeneca R&D Lund.

Little is known about how absorption enhancers as, e.g., The dogs were starved for at least 16 h before exposure

NaTC, promote uptake of peptides and proteins from the and anesthetized with Plegicil vet. (0.1 ml intramuscular-

lung and there are several possible mechanisms that could ly, 10 mg/ml, Pherrovet, Malmo, Sweden) and Pentothal

be involved, e.g., alteration of the mucus layer, protection (25 mg/ml, 1 ml /kg intravenously, Abbott Laboratories,against enzymatic degradation, dissociation of high order IL, USA). The dogs were then immediately intubated

protein aggregates, and increased paracellular absorption (endotracheal tube, inner diameter 7.0 mm, Portex, Kent,

due to opening of tight junctions between epithelial cells UK) and exposed to the insulin or insulinNaTC aerosols

(for review, see, Lee et al., 1991; Gizurarson, 1990). for differing times. As an intravenous reference, insulin in

Due to the complex anatomical structure of the lung, 0.9% NaCl was infused for 5 min (0.2 U/kg, 0.5 U/ ml,

there are few methods to study these effects in a controlled 0.08 ml /kg per min) in the right foreleg vein.

manner using lung tissue (Wall, 1995). Therefore, we have The large animal inhalation system FIDO (Nerbrink et

used in vitro techniques, e.g., Caco-2 cells and circular al., 1997) was used to generate the aerosol and to measure

dichroism, to evaluate the mechanisms involved in the the inhaled dose delivered to the dogs. In short, FIDO

absorption enhancement of insulin by NaTC. In vitro data measures the aerosol concentration with a light scattering

on NaTC effects have been compared to bioavailability instrument (Casella 950 AMS, London, UK). Before

data obtained after administration of nebulized insulin/ exposure the Casella was calibrated by taking filter sam-NaTC solutions to beagle dogs. ples immediately after the Casella. The correlation factor

between measured concentration with the Casella and

actual concentration measured by filter sampling was used

in the dose measurement program. Tidal volume was2. Material and methods estimated from inhalation flow measured as differential

pressure (by a transducer) in a flow restriction placed just2.1. Chemicals before the endotracheal tube. Particle concentration and

tidal volume was used to estimate the inhaled doseHuman insulin of semisynthetic quality was obtained according to the following equation:

from Diosynth (Oss, The Netherlands). Sodium taurocho-

late was obtained from Biosynth (Staad, Switzerland). Inhaled dose5 (C3RMV3ET)/BW14

[ C]Mannitol was obtained from NEN, Life Science3Products (Boston, MA, USA). where C is the chamber concentration (mg/m ), RMV is

3the respiratory minute volume (m /min), ET is the expo-

sure time (min), and BW is the body weight (kg).2.2. Bioavailability studies in beagle dogs

The insulinsodium taurocholate solutions were aerosol-

ized by a PARI LC jet nebulizer (Pari-Werk, Germany).

The flow from the nebulizer was 3.2 l/min (1.0 Bar). The2.2.1. Test formulationstarget inhaled dose of insulin was 1 U/kg.Insulin was suspended in distilled water. The pH was

Venous blood samples were taken from the jugular veinadjusted using 1 M HCl until the insulin was dissolved at ainto heparinized BD Vacutainer tubes (BD VacutainerpH of about 3.2. Then the pH was raised to about 7.3 usingSystems, Plymouth, UK) before dosing and at 5, 10, 15,1 M NaOH. When included in the formulation, NaTC was25, 35, 50, 65, 95, 125 and 245 min after start (t50) ofadded to the insulin solution before the pH was finallyinhalation. The blood samples were immediately cen-adjusted to 7.4. The volume was adjusted with distilledtrifuged (4 8C, 12003g, Beckman GS-15R centrifuge,water to reach the required concentrations. The testedBeckman Instruments, Solna, Sweden), the plasma wassolutions were, pure insulin at 0.6 mM, 0.6 mM insulintransferred into new vials, frozen, and stored at 280 8Cwith 2.2 mM NaTC, 2.2 mM insulin with 8 mM NaTC, 4.4pending analysis. The samples were analysed, with respectmM insulin with 16 mM NaTC, and 8.8 mM insulin withto insulin, using an insulin RIA kit (Insulin RIA kit 100,32 mM NaTC. In all test solutions the insulinNaTC ratioPharmacia, Sweden). The bioavailability (F) of the inhaledwas kept at 3:1 (w/w).insulin formulations was approximated from the ratio of

the dose-adjusted AUC (area under curve) of the inhaled2.2.2. Nebulization experiments formulation to the dose-adjusted AUC for the i.v. dose

Female Beagle dogs (Wema Hund, Osterbybruk, according to the equation.Sweden) were used in the bioavailability studies (n54).

The study was approved by the Animal Ethics Committee F5 (AUC / Dose ) /(AUC / Dose )inhaled inhaled i.v. i.v.

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F. Johansson et al. / European Journal of Pharmaceutical Sciences 17 (2002) 6371 65

142.3. Transport and cell toxicity experiments in Caco-2 [ C]mannitol concentrations were determined by liquid

cell monolayers scintillation (Packard 2200, CIAB, Stockholm, Sweden).14

Apparent permeabilities (P cm/ s) for [ C]mannitolapp,were calculated according to the following equation:2.3.1. Cell culture

Caco-2 cells were obtained from ATCC (American Type P 5dQ/ dt3 1/ACapp 0Culture Collection, Rockwell, MD, USA) and grown in

21

where dQ/dt is the steady-state flux (mol s ), C is theDulbeccos modified Eagle medium (DMEM, Life Tech- 0initial concentration in the donor chamber (mM), and A isnologies AB, Taby, Sweden) with 10% foetal calf serum,2

the area of the cell monolayer (cm ).1% non-essential amino acids, 100 U/ml penicillin and100 mg /ml streptomycin (Life Technologies, Taby,

Sweden). The cells were cultured in an atmosphere of 5% 2.3.4. Cell integrity: trypan blue (TB)CO 95% O at 37 8C on 12-well polycarbonate filters After completion of a Caco-2 experiment, the filters and2 2

2 (1.13 cm ; Transwell Clear, Costar, MA, USA) and the wells were washed twice with PBS. After the last

5 2seeded at a density of 3310 cells/ cm . The cells were wash, PBS was left in the basolateral chamber and 100 mlcultured on filters for 21 days to form differentiated TB (trypan blue solution, 0.4%, Sigma, Stockholm,confluent monolayers. The media on the apical and basola- Sweden) was applied apically. The plates were thenteral sides were changed on alternate days. Cells of incubated for 10 min at 37 8C. The TB solution waspassage number 6383 were used in the experiments. removed and the filters and wells were washed with PBS

as above. PBS was left basolaterally and the proportion of

coloured cells were visually determined using a light2.3.2. Preparation of insulin test solutionsmicroscope (Olympus CK2, LRI, Lund Sweden).Insulin was suspended in Hanks balanced salt solution

(HBSS) with 25 mM Hepes buffer. The pH was adjusted

using 1 M HCl until the insulin was dissolved at a pH of 2.3.5. Cell viability: neutral red (NR)

about 3.2. Then the pH was raised to about 7.3 by addition After completion of a Caco-2 experiment, the filters and

of 1 M NaOH. If included in the formulation, the enhancer wells were washed with PBS. PBS was left basolaterally

was added before the pH was finally adjusted to 7.4. The and 500 ml of a NR solution (Merck Certistain Neutral red,

volume was adjusted using HBSS with 25 mM Hepes, pH 125 ml of a 0.4% solution is mixed with 10 ml DMEM)

7.4. In all test solutions the insulinNaTC ratio was kept was added apically. The plates were incubated at 37 8C for

constant at 3:1 (w /w). 3 h, after which the filters and wells were washed with

PBS. Ethanolacetic acid (50%, 1%) was added (500 ml)

and the plates were left in darkness at room temperature2.3.3. Transport studiesfor 20 min. From every well, 250 ml were moved to aTransport studies were performed in HBSS (25 mM96-well plate and the absorbance was measured at 550 nmHepes, pH 7.4) at 37 8C (95% O , 5% CO ). Before the2 2on a Thermomax plate reader (Molecular Devices, CA,experiment 12-well clusters were preincubated with com-USA).plete growth medium for 15 min to saturate any free

binding sites that can cause adhesion of proteins. The2.4. Cell morphology: electron microscopygrowth medium was then changed for 1.5 ml HBSS (25

mM Hepes, pH 7.4), the cell filters were placed in theEpithelial samples were obtained by carefully cuttingclusters and HBSS (25 mM Hepes, pH 7.4) added to the

out circular sheets from the Caco-2 cell monolayers andapical side. After 15 min of incubation the transepithelialfilters. The samples were placed in fixative (PBS buffer,electrical resistance (TEER) was measured using an Endo-pH 7.4, containing 3% (v/v) formaldehyde and 1% (v/v)hm 12/EVOM chamber (WPI, UK) and the apical solu-glutaraldehyde) overnight, rinsed in PBS buffer and dehy-tions were replaced with the test solutions. After 1 h of

drated through a series of ethanol solutions (50, 75, 95,incubation the basolateral solutions were withdrawn for100% (v/v)ethanol, 2310 min in each solution). Theanalysis of amount of transported insulin. In controlspecimens were dried in a critical point drier using liquidexperiments the apparent permeability (P ) forapp

14 CO as the transitional fluid. Samples were coated with a[ C]mannitol were determined by taking samples from the 220-nm thick layer of gold using a gold sputter coater andbasolateral solutions at 5, 10, 20, 30 and 60 min. After theexamined in a Philips 515 SEM and a Hitachi fieldlast sampling time, the basolateral and apical solutionsemission scanning electron microscope.were replaced with fresh HBSS, and after 15 min of

incubation the TEER was measured. After completion of

the experiment Caco-2 cell integrity and viability were 2.5. Aggregation state of insulin: circular dichroism

determined using trypan blue and neutral red, respectively. (CD)

The samples were analysed with respect to insulin using an

Insulin RIA kit (Insulin RIA kit 100, Pharmacia, Sweden). A CD spectropolarimeter (Jasco J-715, Skafte, Gothen-

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burg, Sweden) was used to measure the effect of NaTC on 3.2. Effect of NaTC alone on Caco-2 cell monolayer

the aggregation state of insulin. Solutions containing 2 mM integrity and viability

insulin and different concentrations of NaTC (2.530 mM)

were scanned from 300 to 250 nm at room temperature and The effect of NaTC on epithelial integrity was investi-

with a scanning speed of 50 nm/ min using a cuvette with a gated using Caco-2 cell monolayers. Fig. 2A shows the

pathlength of 0.1 cm. effect on transepithelial electrical resistance (TEER) and

cell viability after a 1-h incubation with increasing con-

centrations of NaTC. At concentrations above 12 mM,3. Results NaTC reduced the TEER and at about 20 mM a minimum

plateau was reached. In this concentration range, the cell

3.1. NaTC increases the bioavailability of nebulized integrity and viability, measured as the accumulation of

insulin TB into the cells and the accumulation of NR (NR

accumulates in viable cells; Borenfreund and Puerner,

Fig. 1 shows the effect of increasing concentrations of 1985), were rather unaffected. However, as the NaTC

NaTC on the absolute bioavailability of insulin given as concentration was raised to above about 24 mM, there was

nebulized solutions to anesthetized, intubated beagle dogs. a rapid increase in cell permeability and a corresponding

In all nebulized solutions the ratio of insulinNaTC was decrease in cell viability (Fig. 2A,B).

kept constant at 3:1 (w/ w). Hence, both the NaTC and Fig. 2B shows the effect of NaTC on the apparent

insulin concentrations were varied but the administered

dose of insulin was kept constant by varying the nebuliza-tion time. The bioavailability of pure insulin was

2.660.3%. Addition of NaTC to the nebulized insulin

solutions increased the bioavailability, and at a NaTC

concentration of 32 mM a bioavailability of 23.264.4%

was obtained. The bioavailability of insulin, administered

to the lungs as aerosolized powder is also shown in Fig. 1.

In this case, high local concentrations of insulin can be

reached; however, the bioavailability was still low

(3.8161.12%) indicating that the increasing concentration

of insulin in the nebulized solutions did not affect the

bioavailability of nebulized insulin.

Fig. 2. Effect of NaTC on Caco-2 cell monolayer viability and per-

Fig. 1. Effect of NaTC on the absolute bioavailability of nebulized meability. Caco-2 cell monolayers were incubated for 1 h with an

insulin. Solutions containing insulin and different concentrations of NaTC increasing concentration of NaTC in the absence (A) or presence (B) of14

were given as nebulized solutions (open circles) or powder (closed circle) [ C]mannitol. TEER as well as trypan blue, and neutral red accumulation14

to anesthetized, intubated beagle dogs (n54). were determined (A). The P for [ C]mannitol was determined (B).app

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14permeability (P ) for [ C]mannitol. At concentrationsappabove 12 mM, NaTC gradually increased the P ofapp

14 14[ C]mannitol. The increase in [ C]mannitol transport rate

corresponded to the gradual reduction in TEER (Fig. 2A).

Higher concentrations of NaTC (above ca. 22 mM)14

dramatically increased the permeability of [ C]mannitol

(data not shown).

3.3. NaTC enhances insulin transport across Caco-2 cell

monolayers

Caco-2 cells were incubated with increasing concen-

tration of NaTC in the presence of insulin. The ratio of

insulinNaTC was kept at 3:1 (w/w) as in the in vivo

studies. The Caco-2 cell monolayers were not affected by

the highest insulin concentration (8.8 mM) in the absence

of NaTC (data not shown). Results showed a reduction in

TEER at NaTC concentrations between 25 and 30 mM

which coincided with a marked increase in both insulinand mannitol transport (Fig. 3A,B). As the concentration

of NaTC exceeded 32 mM, the viability of cell layers was

close to zero (Fig. 3A) and a dramatic increase in both

insulin and mannitol permeability was observed (data not

shown). In Fig. 3A, an apparent increase in insulin

transport is evident at 16 mM NaTC. However, this

unexpected increase could not be repeated and is consid-

ered as an experimental outlier.Fig. 3. Effect of NaTC on membrane permeability, cell viability and

transport of insulin across Caco-2 cell monolayers. Caco-2 cell mono-

layers were incubated for 1 h with insulin and increasing concentration of3.4. Effect of NaTC on Caco-2 cell monolayer14

NaTC in the absence (A) and presence (B) of [ C]mannitol. An insulinmorphologyNaTC ratio of 3:1 was used. The amount of insulin at the basolateral side

as well as TEER, trypan blue, and neutral red accumulations were14Caco-2 cell monolayers were incubated with 24 mM determined (A). The P for [ C]mannitol was determined (B).app

NaTC in the presence (Fig. 4A) and absence (Fig. 4B) of

6.6 mM insulin. After the incubation the Caco-2 cells were

fixated and examined by scanning electron microscopy.

Severe epithelial exfoliation was observed in the cell layerschange further, indicating that most of the insulin wasincubated with pure NaTC (24 mM, Fig. 4B) In contrast,present as monomers or possibly as dimers.no apparent disturbance of the epithelial lining was ob-

served in the samples after incubation with the insulin

NaTC (6.6:24 mM) formulation (Fig. 4A).

4. Discussion

3.5. NaTC deaggregates high order aggregates of

insulin There are several reports on the pulmonary absorption ofinsulin in both animals and humans. However, there are

The dissociation of insulin hexamers can be monitored large differences in the reported bioavailabilities ranging

by circular dichroism. Attenuation of a band at 276 nm can from a few to almost 60% compared to subcutaneous

be correlated with deaggregation, while a strengthening of injections (Wall, 1995; Patton, 1996). In this study,

the same band can be correlated with an association of nebulized insulin with increasing concentrations of NaTC

insulin monomers (Li et al., 1992; Shao et al., 1993; Ogiso was given to anesthetized, intubated beagle dogs. The ratio

et al., 1996). When 2.0 mM insulin was incubated with between insulin and NaTC was kept constant. Hence, for

increasing concentrations of NaTC, there was a clear all the dogs to receive the same target dose, the time of the

attenuation of the CD spectra at 276 nm (Fig. 5). At NaTC nebulization was varied. The bioavailability of pure insulin

concentrations above 15 mM the CD-spectra did not was low (about 2.6%). This is somewhat lower than the

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bioavailability of insulin increased and at 32 mM NaTC it

was about 23% (Fig. 1), suggesting NaTC to be an

efficient absorption enhancer for nebulized insulin.

In this study we used Caco-2 cell monolayers as a

general epithelial model to study the effects of NaTC on

both epithelial integrity/viability and transport of insulin.

Caco-2 cell monolayers is a well characterized model for

intestinal drug absorption (Artursson, 1991). AlthoughCaco-2 cells originate from the intestine rather than the

airways, it is likely to show similar transport characteris-

tics as airway cell cultures, e.g., Calu-3 and 16HBE14o-,

when it comes to passive transcellular and paracellular

diffusion.

NaTC, showed a non-linear concentration dependent14

absorption enhancement of [ C]mannitol, but also a

concentration dependent toxicity when administered to

Caco-2 cell monolayers. These effects were seen at NaTC

concentrations above the critical micelle concentration

(CMC is about 2 mM in HBSS at 37 8C, Stefan Ulvenlund,

unpublished results). In the presence of NaTC concen-trations of 1522 mM there was a moderate increase in the

14transport of [ C]mannitol. In this concentration range, a

marked decrease in TEER was also observed. At con-14

centrations above 22 mM the transport of [ C]mannitol

increased dramatically (data not shown) and this increase

correlated rather well with a decreased cell viability

measured as the accumulation of trypan blue and neutralFig. 4. Scanning electron micrographs of Caco-2 cell monolayers grown

red. These findings are in accordance with earlier Caco-2on filters. The Caco-2 cell monolayers were incubated for 1 h with 24

studies. Anderberg et al. (1992) found 21 mM of NaTC tomM NaTC and 6.6 mM insulin (A) or with 24 mM NaTC (B) priormoderately increase the apparent permeability offixation.

14 14[ C]mannitol and [ C]PEG 4000, whereas higher con-

centrations (50 mM) led to a dramatic increase in P forvalues reported for man when insulin was administered as appboth substances. Werner et al. (1996) showed that NaTCa powder aerosol (5.662.8%; Heinemann et al., 1997).

reduced TEER in the concentration range 1520 mM andHowever, as NaTC was included in the formulations thein this range they also found an increase in the permeabili-

ty of a peptidomimetic thrombin inhibitor, CRC 220, and

sulforhodamine. At higher NaTC concentrations they saw

a further increased permeability but also decreased cell

viability. Meaney and ODriscoll (2000) showed a de-3

crease in TEER and an increased P for [ H]mannitol,app3 14

[ H]PEG 900 and [ C]PEG 4000 in the presence of 20

mM NaTC. In another study, NaTC at 10 and 15 mM was

also shown to enhance the permeability of a cyclopeptidic-

ab -agonist and FITC-dextran 4400 as well as decreasing3

the TEER (Kamm et al., 2000). Adjacent to the tightjunction in the cytoplasm is an actin myosin ring, which

circumscribes the cell (the perijunctional ring). This ring is

associated with the plasma membrane and can contract.

Such contractions have been correlated with the loosening

of the tight junction. This ring can be visualized by

staining filamentous actin with fluorescent-labelled phal-

loidin (Hochman and Artursson, 1994). Staining of Caco-2

cells with this labelled compound in the presence of

different concentrations of NaTC has shown that, at lowFig. 5. Near-UV circular dichroism spectra of pure insulin and insulin in

concentrations of NaTC (,20 mM), the staining becamethe presence of increasing concentrations of NaTC. The insulin solutions(2 mM insulin) were scanned from 300 to 250 nm. fuzzy and not all cells were surrounded by a fluorescent

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belt but the cells looked viable. At higher concentrations of about 2.6 nm (Chien, 1996). The pore size through the

NaTC (3040 mM) the defined actin structures disap- tight junctions in the lung has been suggested to be in the

peared and the cells were damaged (Werner et al., 1996). order of 0.52 nm (Patton, 1996). This would make it

After exposure to 20 mM NaTC the effect on the tight unlikely for an insulin hexamer to be absorbed across the

junction was reversible. Taken together, the results suggest epithelium and a dissociation of the aggregates would be

that the NaTC induced decrease in TEER can be coupled likely to enhance its absorption. Liu et al. (1993) found

to an opening of tight junctions between adjacent cells. both hexameric and dimeric insulin to be rapidly absorbed

Hence, the NaTC concentration interval where the TEER is when instilled to the rat lung. However, the absorption oflow but the epithelial cell integrity and viability maintained hexameric insulin was slightly delayed compared to the

(1222 mM) could represent a range where an increased case with dimeric insulin. These results suggest the

paracellular transport of hydrophilic molecules could occur aggregation state of insulin to affect the absorption. The

without any damage to the epithelial cells. dissociation of insulin hexamers can be monitored by CD.

When insulin was included in the incubation medium, a Attenuation of a band at 276 nm is correlated with14

moderate increase in both insulin and [ C]mannitol deaggregation, while a strengthening of the same band is

permeability across Caco-2 monolayers was obtained at correlated with an association of insulin monomers (Li et

moderate concentrations of NaTC (2530 mM). This al., 1992; Shao et al., 1993; Ogiso et al., 1996). In this

increase in permeability coincided with a decrease in work we used circular dichroism to show that NaTC does

TEER. At higher concentrations of NaTC, a dramatic promote the dissociation of insulin. At NaTC concen-14

increase in insulin and [ C]mannitol permeability, and a trations between 2.5 and 15 mM, there was a gradual

corresponding decrease in cell viability were seen. From attenuation of the band at 276 nm in the insulin (2.2 mM)this we suggest that NaTC increases the permeability of CD spectra. At NaTC concentrations between 15 and 30

insulin across Caco-2 cell monolayers by opening of tight mM there was no further changes in the CD spectra

junctions. NaTC increased the bioavailability of nebulized indicating that most insulin molecules were in the mono-

insulin at similar concentrations and hence, we suggest that mer or possibly dimer form already at a NaTC con-

NaTC promote absorption of insulin from the lung partly centration of 15 mM. This is in accordance with earlier

by opening of tight junctions between airway epithelial findings that several bile salts, and among them NaTC,

cells. exhibits considerable insulin dissociating power (Li et al.,

The presence of insulin also affected the dose-dependent 1992; Yamamoto et al., 1992; Shao et al., 1993).

toxicity of NaTC on the Caco-2 cell monolayers. When As the NaTC concentrations needed to dissociate insulin

comparing different experiments run in the absence and aggregates were in the same range as the concentrations

presence of insulin, it was obvious that the concentration that enhanced the absorption of nebulized insulin, the

interval at which TEER as well as cell viability were dissociation of high order insulin aggregates are likely to

affected, shifted towards higher concentrations in the be a part of the mechanism by which NaTC possess its

presence of insulin. When insulin was present, TEER effect on the bioavailability of insulin administered by the

approached zero at a NaTC concentration of about 25 mM, pulmonary route.

whereas the cells appeared intact and viable to about 30 Insulin has been shown to be degraded in lung and other

mM. In the absence of insulin, corresponding values were mucosal homogenates (Yamamoto et al., 1990, 1994a;

significantly lower, 20 and 24 mM, respectively. Further- Fukuda et al., 1995; Hsu and Bai, 1998; Shen et al., 1999).

more, higher concentrations of NaTC were needed to Various protease inhibitors, e.g., the bile salt sodium14

increase the P for [ C]mannitol if insulin was present glycocholate (NaGC), inhibit the insulin degradation inappin the incubation media. This was confirmed using scan- different homogenates (Yamamoto et al., 1990, 1994a;

ning electron microscopy. Severe epithelial exfoliation was Fukuda et al., 1995; Shen et al., 1999). However, as

observed in cell monolayers incubated with pure NaTC (24 pointed out by Wall (1995), detection of protease activity

mM), whereas no apparent disturbance of the epithelial in mucosal homogenates does not reveal whether the

monolayer was observed in the samples containing insulin enzymes responsible for the activity will have access to the(24 mM NaTC, 6.6 mM insulin). One explanation to these inhaled insulin and influence its absorption. Assuming that

observations is that insulin interacts with, and decreases insulin, being a large hydrophilic substance, is absorbed

the free concentration of NaTC, i.e., NaTC exerts its effect through the tight junctions between epithelial cells, the

at the same free concentration both in absence and relevant enzymes in normal lungs exposed to insulin will

presence of insulin. be those present in the airway surface liquid layer and at

Insulin exists as monomers only at very low concen- epithelial and endothelial cell surfaces. Protease inhibitors

trations (,0.1 mM), at higher concentrations insulin that inhibit insulin degradation in lung homogenates do,

dimerizes, and in the pH range 4 8 in the presence of however, also increase the bioavailability of inhaled in-21

Zn , three dimers assemble further to form hexamers at sulin (Okumura et al., 1992; Yamamoto et al., 1994b,

concentrations above 10 mM (Chien, 1996). The diameter 1996; Hsu and Bai, 1998; Shen et al., 1999). As this was

of an insulin hexamer is about 5.6 nm and of a monomer seen for a bile salt, NaGC, it is likely that inhibition of

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