Profile of P‐glycoprotein Distribution in the Rat and Its Possible Influence on the Salbutamol Intestinal Absorption Irocess

Profile of P-Glycoprotein Distribution in the Rat and Its Possible Influence on the Salbutamol Intestinal Absorption Process ´ N RUIZ-CARRETERO,1 ADELA MARTI´N-VILLODRE,1 BELE´N VALENZUELA,1 AMPARO NA´CHER,1 PURIFICACIO ´ PEZ-CARBALLO,2 DOMINGO BARETTINO2 GRACIA LO 1

Department of Pharmacy and Pharmaceutics Technology, Faculty of Pharmacy, University of Valencia, Avd. Vicente Andre´s Estelle´s s/n, 46100 Burjassot, Valencia, Spain 2

Instituto de Biomedicina de Valencia (C.S.I.C.), Valencia, Spain

Received 25 September 2003; revised 13 January 2004; accepted 22 January 2004

ABSTRACT: The intrinsic absorption of salbutamol in different intestinal segments of the rat was measured and related with the corresponding intestinal P-glycoprotein (P-gp) expression levels. The apparent absorption rate constants (ka, h
1) observed in each fraction by means of the ‘‘in situ’’ rat gut absorption method after perfusion of a 0.29-mM isotonic solution of salbutamol were used as absorption indexes. In a separate series of studies, a semiquantitative analysis of the mRNA expression of P-gp by means of polymerase chain reaction and Western blot with an antibody raised against the P-gp were also performed. The ‘‘in situ’’ ka values determined in the different segments (h
1) showed that the absorption is not homogeneous along the intestinal tract, that is, 0.499  0.054 for colon, 0.474  0.052 for the proximal segment, 0.345  0.014 for the mean, and 0.330  0.023 for the distal fraction. Addition of verapamil to the perfusion fluid did provide a better absorption of salbutamol in the distal segment. The analysis of the mRNA expression and levels of P-gp showed that the enzyme content in each section of the intestine was inversely related to salbutamol absorption. ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1641–1648, 2004

Keywords: salbutamol; verapamil; intestinal absorption; intestinal secretion; Pglycoprotein (P-gp) expression; bioavailability; mRNA; reverse transcription-polymerase chain reaction (RT-PCR); Western blot

INTRODUCTION Salbutamol is a b-2-adrenergic agonist widely used in the treatment of asthmatic disorders and chronic obstructive lung diseases. The absolute bioavailability of salbutamol when administered in conventional oral dosage forms has shown to be incomplete and rather irregular, this being attributed to presystemic metabolism, mainly due

Correspondence to: Adela Martı´n-Villodre (Telephone: 34-6354-4912; Fax: 34-6-354-4911; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 93, 1641–1648 (2004) ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association

to sulfation in the duodenal mucosa,1,2 and to a poor intestinal permeation capacity.3 In a previous article,4 we demonstrated that the absorption process of aqueous salbutamol solutions along the whole length of the rat small intestine is modulated by an active secretion process of the drug from the enterocytes to the luminal fluid, in which P-glycoprotein (P-gp) could be involved. The efflux can contribute to explanation of the poor and variable peroral bioavailability of salbutamol. P-gp belongs to the adenosine 50 -triphosphate– binding cassette superfamily of membrane transport proteins.5 The intestinal epithelium is one of the normal tissues expressing the multidrug

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resistance (MDR) gene that gives the multidrug resistant phenotype to a variety of tumors. The MDR gene encodes P-gp, a transmembrane efflux pump.6–8 The present study examines the relationship of P-gp expression in segments of the rat intestine and the absorption of salbutamol. Additionally, the role of P-gp in modulating the absorption of salbutamol is examined using in situ perfusion and P-gp inhibitors.

MATERIALS AND METHODS Absorption Studies Absorption tests were performed on male Wistar rats belonging to the colony of our Pharmacy Faculty, weighing 200–300 g, fasted for 20 h but with free access to water. Anesthesia was induced 1 h before surgery by an intraperitoneal injection of 40 mg/kg pentobarbital. All pharmacokinetic studies adhered to ‘‘Principles of Laboratory Animal Care,’’ and the study with animals was approved by the Research Commission of the Department Council. The in situ rat gut preparation,9 modified as previously reported,10–12 was used. Because the whole length of the rat small intestine is about 1 m, it can be divided in three fractions of equal lengths, that is, proximal, mean, and distal (L & 33 cm). The whole colon (L & 15 cm) was also used. Perfusion was performed in one of these segments, after measuring and cannulation. The selected fraction was first rinsed with 25 mL of physiological saline to eliminate fecal residues and debris. In a previous article,4 we demonstrated that the incorporation process of the salbutamol along the whole length of the small intestine is saturable. Moreover, salbutamol is acting as a substrate of an intestinal secretory transport, which probably includes the P-gp enzyme to some extend, because verapamil has been shown to inhibit the secretion process by dose-dependent competition.4 Then, in the first series of experiments (n ¼ 8), 5 mL of an isotonic, buffered salbutamol (Laboratory Aldo Unio´n, Barcelona, Spain) solution 0.29 mM (near the allometric oral dose), previously thermostated to 378C, was perfused in the proximal, mean, distal, and colon segments. For the second series, 5 mL of a 0.29-mM salbutamol solution in the presence of 10 mM verapamil was isotonized and perfused in proximal and distal intestinal segments. The influence of verapamil on the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 6, JUNE 2004

salbutamol absorption process was characterized by statistical comparison [analysis of variance (ANOVA)] between the ka values found in the presence and in the absence of the P-gp inhibitor in these segments. The pH was adjusted to the mean value obtained for each intestinal segment in preliminary five-rat experiments. Results were as listed: 6.50  0.08 (proximal); 6.72  0.13 (mean); 6.92  0.13 (distal); and 6.72  0.08 (colon). In the proximal segment tests, the biliary conduct was ligated to prevent reabsorption phenomena. Every 5 min for a total time of 30 min, 200-mL samples of the perfusate were taken and the salbutamol concentration in each sample was measured. Water reabsorption was evaluated for each animal by using a procedure already reported.10,13 From the values of the mean remaining volume at 0 min (n ¼ 5), and the individual volume at 30 min, the actual salbutamol concentration in samples can be properly corrected. Analytical Procedures Intestinal samples were assayed for salbutamol content by high-performance liquid chromatography fluorometry, which provides an excellent separation and quantification technique. The validated method used was described in detail in a previous article.4 Accuracy was <3.33% and the precision was <3.45%. The limit of quantification was 0.1 mM. These features were believed to be completely acceptable from a biopharmaceutical viewpoint.14

Absorption Rate Constant Measurements Intestinal absorption of salbutamol was quantified using the apparent first-order rate constant, ka, according to the classical expression: A ¼ A0  e
ka  t

ð1Þ

where A represents the concentration of salbutamol remaining in the luminal fluid at the sampling time, t, corrected for water reabsorption, and A0 is the calculated intercept at zero time, which is usually lower than the initial concentration perfused due to the fast initial membrane adsorption of the solute and/or to sample dilution in the residual rinsing fluid.11,13 To overcome these effects, only samples collected between 5 and 30 min were used for calculations (i.e., the initial nonperfused sample was excluded).

P-GLYCOPROTEIN DISTRIBUTION IN THE RAT

Both parameters A0 and ka were calculated for each animal by nonlinear ordinary least-squares regression procedure using SPSS 9.0 software. Parameter values were also calculated from the average remaining concentrations obtained for the eight animals of each group at each time, t. The correlation coefficients, r, between theoretically predicted and experimentally obtained values and the residual distribution were used to assess the goodness of the fits. However, the ka values obtained for each data set were statistically compared using a one-way ANOVA test. Results were assumed to be significant for a 95% probability value ( p < 0.05). To estimate the statistical differences among absorption rate constants, the Scheffe´ test was used. Analysis of MDR Gene Expression To estimate the amounts of the MDR gene mRNA encoding P-gp, we performed a semiquantitative reverse transcription–polymerase chain reaction (RT-PCR) analysis. The experiment is based on the fact that end-time PCR reactions are not suitable for quantitation, because amplification of the PCR product reaches a plateau after a certain number of cycles and therefore the amount of product present after the end of the reaction is not necessarily proportional to the initial number of target molecules present in the sample. The most quantitative parameter in a PCR reaction is the cycle number in which the amplified product is detectable, that is proportional to the initial number of target molecules. This is the parameter used in all the quantitative real-time PCR systems presently marketed. Using this parameter, the absolute number of copies of a sequence present in a sample can be calculated, provided that a set of standard samples of known copy number is available. If this is not possible, the relative amounts of a sequence in a set of samples can be obtained by comparison between the cycle numbers where the amplification product could be detected in each sample, assuming that a difference of n cycles represents in copy number a difference of 2n.

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RNA samples was checked by electrophoresis in agarose gel, and RNA concentration was estimated spectrophotometrically. For cDNA synthesis, 2 mg of total RNA and 0.5 mg of dT16 primer were preheated at 708C in DEPC-treated water and cooled on ice. Reaction solutions (20 mL) contained 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM Mg Cl2, 10 mM DTT, 40 units of RNAsin (Promega, Barcelona, Spain), 500 mM each dNTP and 300 units of Superscript Reverse Transcriptase (Life Technologies, Barcelona, Spain). The reactions were incubated at 428C for 1 h. Semiquantitative RT-PCR Analysis Oligonucleotide primers were designed to detect the mRNA encoding P-gp, transcribed from the MDR gene. Primer MDR-U (50 -CAT CGC CTA CGG AGA CAA CA-30 ) corresponds to nucleotides 3489-3508 and primer MDR-L (50 -CTG CGT TCT GGA TGG TGG AC-30 ) corresponds to nucleotides 3801-3820 of the rat MDR-1 cDNA sequence.16 The primers gave rise to a 331 nucleotide PCR product. Primers CYC-U (50 -CGT CTG CTT CGA GCT GTT TG-30 ) and CYC-L (50 -GTA AAA TGC CCG CAA GTC AA-30 ) correspond to nucleotides 99-118 and 543-562, respectively, of the rat cyclophilin A cDNA,17 and generated a 463 nucleotide product. PCR reactions (25 mL) were set in 75 mM TrisHCl (pH 9.0), 2 mM MgCl2, 50 mM KCl, 20 mM (NH4)2SO4, 0.001% bovine serum albumin, 200 mM each dNTP, 5 pmol each primer, 1 unit of Taq DNA polymerase (Biotools, Madrid, Spain), using as template 1 mL of the above Reverse Transcription reactions. Parallel amplification reactions were set for MDR and cyclophilin A that was used as internal control. After an initial denaturing step at 948C, 2 min, 32 cycles of 948C, 10 s, 588C, 15 s, and 728C, 20 s, were performed. Samples from the PCR reactions (3.5 mL) were taken after 22, 24, 26, 28, 30, and 32 cycles, and analyzed by electrophoresis in 1.5% agarose gels stained with ethidium bromide. Western Blot

RNA Preparation and cDNA Synthesis Animals were anesthetized and the different organs and tissues dissected. Total RNA from these tissues was obtained by the method of guanidinium isothiocyanate-phenol according to Chomczynski and Sacchi.15 The integrity of the

Tissues and organs were dissected as above, washed in saline, and frozen under liquid nitrogen. Tissues were ground in a mortar under liquid nitrogen, and lysated in RIPA buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM ethyleneglycotetraacetic JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 6, JUNE 2004

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acid, 10 mM NaF, 1% TritonX100, 1% deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM sodium orthovanadate, 10 mg/mL leupeptin, 10 mg/mL aprotinin, 100 mM phenyl methyl sulfonyl fluoride): Determination of the protein content was made with a detergent-compatible BioRad DC protein assay. Equal amount of extracts (30 mg of protein) were denaturized in sample buffer and loaded to 7% acrylamide sodium dodecyl sulfate– polyacrylamide gel electrophoresis gels according to Laemmli.18 Electrophoresed proteins were electro-transferred to nitrocellulose membranes (HybondECL; Amersham Pharmacia Biotech). Membrane was blocked with phosphate-buffered saline (PBS), 0.1% Tween 20, and 5% nonfat dry milk for at least 1 h and then incubated with rabbit antibodies raised against human MDR (H241, Santa Cruz Biotechnology; dilution 1:1000) in the above solution at 48C for 16 h. After washing with PBS–0.1% Tween 20, the filter was incubated with horseradish peroxidase– conjugated donkey antibodies raised against rabbit immunoglobulin G (Amersham Pharmacia Biotech) in PBS, 0.1% Tween 20, and 5% nonfat dry milk (dilution 1:1000) for 1 h at room temperature. Luminescent signal development with electrochemiluminescence (ECL) (Amersham Pharmacia Biotech) was performed as recommended by the manufacturer, and filters were exposed to X-ray films in the dark.

RESULTS In Situ Absorption Tests The apparent absorption rate constants (ka, h
1, already corrected for reabsorption) determined for salbutamol after perfusion of 0.29-mM drug solutions in different segments of the small intestine and colon of the rat are shown in Table 1. The statistical test performed revealed statistically significant differences between the ka values obtained in the different intestinal fractions tested. In brief, the absorption rates of salbutamol in the colon and/or in the proximal fraction of the rat small intestine are not statistically different ( p ¼ 0.644) and significantly greater than that observed in the mean and/or distal segments, for which no significant differences were found ( p ¼ 0.888). The working equations calculated from the mean remaining salbutamol concentrations at each time unity ðM; for n ¼ 8Þ were as follows: JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 6, JUNE 2004

Table 1. Average Absorption Rate Constants (h
1  SD) in Each Intestinal Segment Tested for Salbutamol 0.29 mMa Statistical Analysis Intestinal Segments

ka (h
1)  SD

Colonb Proximal

0.499  0.054 0.474  0.052

Mean Distalb

0.344  0.014 0.332  0.023

F

p

37.016

0.000

a Statistical analysis of differences (one-way ANOVA test) are also shown. b The Scheffe´ test showed statistical differences among ka values in colon and mean/distal values; also in proximal and mean/distal values.

   

Colon: A ¼ 92.65  e
0.499  t Proximal: A ¼ 89.03  e
0.474  t Mean: A ¼ 84.50  e
0.345  t Distal: A ¼ 82.80  e
0.330  t

As can be seen, the resulting absorption rate constants are practically the same as those obtained as the average of the individual ka values shown in Table 1. When the absorption experiments were performed in proximal and distal segments in the presence of verapamil, the ka values were 0.471  0.051 and 0.734  0.093 h
1, respectively. As can be seen in the Table 2, the one-way ANOVA test shows significant differences between ka values found in the presence and in the absence of the P-gp inhibitor (verapamil) in the distal segment. Accordingly, in the proximal segment, the same test showed no significant differences between the ka values of salbutamol 0.29 mM tested solution, obtained in the absence and in the presence of verapamil ( p ¼ 0.996). Estimation of P-gp mRNA and Protein Levels According to the semiquantitative RT-PCR analysis shown in the Figure 1, the MDR mRNA levels are highest in the distal portion of the rat small intestine. Referred to the levels found in the proximal part of the small intestine, the MDR mRNA content is equivalent in colon, about 2-fold higher in the mean section of the small intestine, around 4-fold higher in the buccal mucosa, around 16-fold higher in the lung, and about 32-fold higher in the distal portion of the small intestine. In stomach, P-gp transcript was not detected. As control for the amount of RNA and the correct

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Table 2. Average Absorption Rate Constants (h
1  SD) Found in Proximal and Distal Segments for Salbutamol 0.29 mM, in the Absence and in the Presence of Verapamil 10 mMa Statistical Analysis Intestinal Segments

Inhibitor Concentration (mM)

ka (h
1)  SD

Proximal

0

0.474  0.052

10 0

0.471  0.050 0.332  0.023

10

0.734  0.093

Distal

F

p

0.002

0.996

143.067

0.000

a

Statistical analysis of differences were performed using the one-way ANOVA test.

performance of the RT reaction, we performed a parallel analysis with primers specific for the transcript of the housekeeping gene cyclophilin A. As shown in Figure 1, cyclophilin A mRNA levels were equivalent in all tissues tested.

To confirm these results, analysis of P-gp levels in different tissues by Western blot was performed. As shown in Figure 2, it is confirmed that the greatest level of P-gp is obtained in the distal segment of the rat small intestine.

Figure 1. Estimation of MDR mRNA levels in different tissues by semiquantitative PCR. cDNA samples from proximal, mean, and distal small intestine, colon, stomach, oral mucosa, and lung were analyzed. Samples from the corresponding MDR and cyclophilin (CYC) PCR reactions were taken after the number of cycles indicated in the figure, and electrophoresed in 1.5% agarose gels stained with ethidium bromide. PCR products of 331 (MDR) and 463 (CYC) nucleotides were obtained. Molecular weight markers (M) were 100 bp DNA ladder (Life Technologies). For better sensitivity, the figure shows inverted pictures of the ethidium bromide stained gels. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 6, JUNE 2004

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Figure 2. Analysis of P-gp levels in different tissues by Western blotting. Protein extracts from oral mucosa, lung, stomach, and colon, and proximal, mean, and distal small intestine were electrophoresed and analyzed by Western blot with specific anti–Pgp antibodies. Chemiluminescent detection of the immune complexes was performed with ECL.

DISCUSSION Intestinal P-gp Distribution The bibliography on the distribution of P-gp along the intestinal tract of the rat and other laboratory animals is rather contradictory. In several reports,19–21 some authors indicate that P-gp concentrates toward the final fractions of the small intestine, whereas other authors find a preferential distribution in colon22 or proximal segment.23 Our in situ absorption experiments clearly indicate that from the highest to the lowest, the salbutamol absorption yield sequence is as follows: colon—proximal—mean—distal. Taking into consideration that the small intestine is the preferent site of the absorption process, we performed the comparative absorption experiments only in this tract (i.e., the colon segment was excluded). As the proximal and distal segments exhibit the most different absorption rate constants, the hypothesis of different expression of P-gp in these segments was made. To test this assumption, we performed inhibition assays with verapamil in the two segments. Addition of verapamil in salbutamol solutions when the experiments were performed in proximal and distal segments produced no differences in the ka values in the proximal segment and increased 2.24-fold in the distal segment. Taking into account that the metabolism can be considered constant, any increase in the absorption rate JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 6, JUNE 2004

constant should be attributed to a decrease in the excretion process mediated by P-gp. These results can be explained if the expression of the system is more concentrated in the distal segment. Therefore, if the P-gp–mediated secretion of salbutamol were clearly related to its absorption yield, the enzyme content of the above intestinal fractions should follow a completely inverse sequence. And, indeed, from our P-gp mRNA expression measurements, it can easily be deduced that this is the case, for the sequence found is: distal—mean—colon/proximal. According to the results shown above, it appears that the expression of the mRNA encoding the P-gp in the small intestine follows a gradient, increasing from the proximal to distal portion. A recent article24 shows the same mRNA tissue distribution in the rat using branched-DNA signal amplification technology. To confirm these results at the protein level, we performed Western blot with an antibody raised against the human P-gp, that reacts also with the corresponding rat and mouse proteins. The antibody recognizes a broad band of approximately 170 kD, that corresponds to the glycosylated protein. The reactivity of the antibody reproduces the results obtained for the mRNA levels, and a gradient of P-gp reactivity in the small intestine becomes evident. P-gp levels were low in the proximal portion, increased in the mean part, and were highest in the distal part of the small intestine. Expression of the P-gp is almost

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undetectable in stomach and oral mucosa, and clearly detectable in colon and lung. Therefore, a conclusion is that the absorption yield of salbutamol could, in fact, depend on its concomitant or parallel P-gp–mediated secretion to luminal fluid, at least to a large extent. This assumption is supported by the fact that the ka value of salbutamol is higher in the proximal segment of the rat small intestine (ka ¼ 0.47 h
1) than in its mean (ka ¼ 0.34 h
1) or distal (ka ¼ 0.33 h
1) segments, whereas the sulfation metabolism seems to be more important in the proximal small intestine (three holds) than in the distal one. However, the effect of the pH changes was negligible on the absorption process because the dissociation of the drug is very similar in the different segments of the small intestine. Biopharmaceutical Implications For many drugs, poor oral bioavailability could be due to intestinal metabolic enzymes and efflux transporters, working coordinately as a protective mechanism.25 Before administering drugs affected by P-gp– mediated secretion, it would be desirable to know as exactly as possible where the enzyme predominates, in order to intend that preferential cession will be achieved in a segment with a minimal P-gp content. Although the lung is rich in P-gp, as shown in Figure 2, treatment involving a massive dose of salbutamol administered by inhalation is usually effective, and it is considered a good alternative for acute or emergency cases. It is possible that the oral mucosa may offer a better alternative for salbutamol treatment. Its P-gp content (Fig. 1) is not high (in fact, it is similar to that found in the mean intestinal segment). Therefore, buccal or sublingual administration of bioadhesive forms could provide sustained, effective plasma levels of the drug. These results show that the absorption rate constant, ka, of salbutamol in a 0.29-mM solution is higher in the colon (ka ¼ 0.50 h
1) and in the proximal segment of the rat small intestine (ka ¼ 0.47 h
1) than in its mean (ka ¼ 0.34 h
1) or distal (ka ¼ 0.33 h
1) segments. From our perspective, salbutamol metabolism is not the main cause of the ka changes across the gastrointestinal tract. Recent articles1,2 showed that salbutamol metabolism is higher in the proximal intestinal segment, where the ka is also higher. In fact, the addition of verapamil to the

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salbutamol solution does not affect the ka in the proximal segment, whereas it is 2.24-fold higher in the distal segment. Because the P-gp content, as assessed by RTPCR analysis of the transcripts, is lower in the first two of these fractions than in the latter two, one can conclude, in principle, that the active P-gp– mediated secretion of the drug to luminal fluid can reduce its absorption yield, and Western blot analysis confirmed these previous results.

ACKNOWLEDGMENTS This work was supported by the CICYT of the Ministry of Education and Science of Spain (Projects SAF 96-1710 to A.M.-V. and PM 960074 to D.B.). The authors are indebted to the University of Valencia for a grant to B.V. (500th Centenary) and to the Conselleria de Cultura de Educacio´n y Ciencia de la GV for a grant to G.L.-C. The authors are grateful to Laboratories Aldo-Unio´n (Barcelona, Spain) for supplying salbutamol. The authors express gratitude to Prof. J.M. Pla´-Delfina for his constructive criticism and discussion and to Jane Heilker for the review of the English manuscript.

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