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Evaluation of colonic absorbability of drugs in dogs using a novel colon-targeted delivery capsule (CTDC)
Journal of Controlled Release 59 (1999) 361–376
Evaluation of colonic absorbability of drugs in dogs using a novel colon-targeted delivery capsule (CTDC) Takashi Ishibashi*, Kengo Ikegami, Hiroaki Kubo, Masao Kobayashi, Masakazu Mizobe, Hiroyuki Yoshino Pharmaceutics Research Laboratory, Tanabe Seiyaku Co., Ltd., 16 -89, Kashima 3 -chome, Yodogawa-ku, Osaka 532, Japan Received 23 July 1998; received in revised form 21 October 1998; accepted 8 December 1998
Abstract A series of dog studies were performed to examine the in vitro / in vivo relationship of drug release behavior of the newly developed colon-targeted delivery capsule (CTDC). The four kinds of CTDCs containing theophylline, each of which has a different in vitro dissolution lag time, were orally administered to four beagle dogs under fasted condition, and the onset times of drug absorption were compared. The CTDC with longer in vitro lag time had a later onset of drug absorption. It was also found that the time difference between the gastric emptying and the onset of drug absorption was almost equal to the in vitro dissolution lag time of the capsule, suggesting a similar performance of CTDC in the gastrointestinal tract. From the comparison to the absorption behavior of the colon arrival marker, i.e. sulfasalazine, it was proved that the CTDC with the lag time of 3 h can deliver the drug directly to the colon. This result implied that the CTDC can be used as a non-invasive means for assessing the regional absorbability of drugs in the gastrointestinal tract. To evaluate the absorbability of drugs in the colon, three model drugs, theophylline (THEO), acetaminophen (ACET), and phenylpropanolamine hydrochloride (PPA) were directly delivered to the colons of beagle dogs using the CTDC with the lag time of about 3 h. The obtained relative bioavailabilities to the solution form were as high as 94.2%, 71.0%, and 91.5% for THEO, ACET and PPA, respectively, suggesting that the colonic absorbability of those drugs is essentially good. 1999 Elsevier Science B.V. All rights reserved. Keywords: Colon-targeted delivery capsule; Site-specific drug delivery; Beagle dog; Gastric emptying; Colonic bioavailability
1. Introduction The colon-targeted delivery of drugs via the oral route has attracted much interest for the local treatment of some colonic diseases, such as ulcer*Corresponding author. Tel.: 181-6-300-2788; fax: 181-6-3002799. E-mail address: [email protected] (T. Ishibashi)
ative colitis, Crohn’s disease, colon-cancer, etc. Another advantage of colon-targeting involves the improved oral bioavailability of peptide or protein drugs susceptible to acidic or enzymatic degradation in the stomach or small intestine [1]. The colon is thought suitable as the absorption site for those drugs because of less activity of proteolysis [2]. In the last decade, there has been much research aiming toward the colon-targeted delivery of drugs.
0168-3659 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 99 )00005-X
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Various methodologies and technologies have been proposed, such as pH-triggered [3,4], time-controlled [5–8], and microbially-controlled deliveries [9–11]. Nevertheless, it seems that only a few systems are applicable for practical use in terms of the selectivity to the target site, the safety of the materials to be used, and the preparation techniques to be applied. We recently developed a new capsule type colontargeted delivery system (colon-targeted delivery capsule: CTDC) from a practical point of view, which can be manufactured using currently applicable pharmaceutical technologies and materials recognized as safe. The CTDC was designed by imparting a pH-sensing function and a time-release function to the conventionally used hard gelatin capsule, so as to release the drug rapidly at the predetermined time after gastric emptying [12]. The lag time of the CTDC can be controlled by the coating amount of Eudragit E, and the kind of organic acid as a pH adjusting agent [13]. Since the transit time of pharmaceuticals in the small intestine is known to be less variable in humans, i.e. about 361 h [14], the colontargeting could be achieved with the CTDC when the onset time of drug release is adjusted at 3 h or more. If the reliable site-specificity to the colon is assured, the CTDC could be used to evaluate the colonic absorbability of drugs. As a matter of fact, the drug absorption from the colon is believed to be generally poor because less aqueous fluid existed in the lower part of the large intestine [15]. Therefore, when sustained-release formulations are designed, it is important to understand the rate or extent of the colonic absorption of the drug. The simple and relevant methodology to evaluate properly the colonic absorbability of drugs under the intact physiological condition, however, has not been offered to formulators. The CTDC may be a useful means for achieving this purpose, because the system enables the direct delivery of any type of drugs to the colon without any invasive treatment. The first purpose of the present study is to check if the CTDC can release the drugs at the targeted position in the gastrointestinal tract. Various CTDCs with different in vitro lag times were orally administered to beagle dogs. The in vitro / in vivo relationship of drug release behavior is demonstrated, and the in vitro lag time required for the colon-targeted drug delivery is estimated in dogs. The second
purpose is to evaluate the colonic absorbability of some drugs with different solubilities using the CTDC system.
2. Materials and methods
2.1. Materials Theophylline (THEO; Shiratori Seiyaku, Japan) and phenylpropanolamine hydrochloride (PPA; Alps Chemical Co., Japan) were used as model drugs. Acetaminophen (ACET; Yamamoto Chemical Co., Japan) was used as both a model drug and an indicator determining the gastric emptying time of the capsule. Sulfasalazine (SAS; Sigma Chemical Co., Germany) was used as an indicator determining arrival in the colon. Hard gelatin capsules (size [2; 17.8 mm in length; 6.07 mm and 6.36 mm in width of body and cap) were purchased from Warner Lambert. Aminoalkylmethacrylate copolymer B ¨ (Eudragit E 100, Rhom Pharm, Darmstadt, Germany) was used as the acid-soluble polymer, which is soluble in low pH aqueous medium up to pH 5. Hydroxypropyl-methylcellulose (TC-5 , type EW, Shin-etsu Chemical, Japan) was used as the hydrophilic polymer. Hydroxypropyl-methylcellulose acetate succinate (HPMC -AS, type MIF, Shin-etsu) was used as the enteric polymer. Ethylcellulose (10 cp grade, Shin-etsu) was used as the sealing agent of the hard gelatin capsules. Succinic acid (Katayama Chemical, Japan) was used as the pH-adjusting agent. 7-(2-Hydroxyethyl)-theophylline (Tokyo Kasei Kogyo, Japan) and phenethylamine hydrochloride (Katayama) were used as the internal standards. Tetragastrin was purchased from Mecto (Japan). All other chemicals and solvents were of reagent grade.
2.2. Preparation of CTDC with different lag time The powder mixture consisting of 20 mg of THEO, 50 mg of SAS and 100 mg of succinic acid was filled into a hard gelatin capsule, and the joint of the capsule body and cap was sealed with a small amount of the 5% (w / w) ethylcellulose ethanolic solution. Four batches of the sealed capsules were spray-coated with different amounts of Eudragit E (0, 11, 21, and 33 mg / capsule) to provide a different
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onset time of drug release. Each batch of the Eudragit E-coated capsules was then coated with TC-5 (15 mg / capsule) containing ACET (10 mg / capsule) as an intermediate layer, and finally with HPMC -AS (250 mg / capsule) as the outmost enteric layer. The coating process was carried out using a coating machine (Hicoater , Type HCT-mini, Freund Ind., Japan), and the formulas of the polymeric coating solutions for each layer are given in Table 1 along with the standard operating conditions.
2.3. Preparation of CTDC for colonic absorbability study The powder mixture consisting of three model drugs (20 mg of THEO, 30 mg of PPA, and 60 mg of ACET), a colon arrival indicator (50 mg of SAS), and a pH-adjusting agent (100 mg of succinic acid) was filled into a hard gelatin capsule (size [2). The joint of the capsule body and cap was sealed with a small amount of the 5% (w / w) ethylcellulose ethanolic solution. The sealed capsules were spraycoated with Eudragit E (30 mg / capsule), then with TC-5 (15 mg / capsule), and finally with HPMC AS (250 mg / capsule) using a coating machine (Hicoater , Type HCT-mini). The formulas of the polymeric coating solutions, and the operating conditions of coating for each layer are described in Table 1.
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2.4. In vitro release study The release profiles of ACET, THEO, and PPA from the capsule were determined according to the procedure described in the Japanese Pharmacopoeia XIII (the paddle method). The capsules were placed in a vessel with 900 mL of the JP 1st-fluid (pH 1.2) or 2nd-fluid (pH 6.8) at 3760.58C rotating at 100 rpm. The aliquots were removed periodically and assayed for ACET, THEO, and PPA using the high performance liquid chromatography (HPLC) method, of which analytical conditions are given in Section 2.6. Because of the different assay method applied, the release profile of SAS was determined by a separate run of the dissolution test. The capsules were placed in a vessel with 900 mL of the JP 1st-fluid (pH 1.2) or 2nd-fluid (pH 6.8) at 3760.58C rotating at 100 rpm. The released amount was periodically determined by the spectrophotometric method.
2.5. In vivo release /absorption study The in vivo absorption studies were carried out using tetragastrin-controlled beagle dogs, in order to minimize the intra-subject variance in gastric acidity. The healthy male beagle dogs, 11–12 kg, were used in this study under fasted condition. Tetragastrin (10 mg / kg) was injected intramuscularly twice, 15 min before and 45 min after oral administration of
Table 1 Formulas of coating solution and standard operating conditions Coating layer
Acid-soluble layer
Composition of coating solution (w / w%)
Eudragit E Ethanol
Operating conditions: Blower temperature (8C) Exhaust temperature (8C) Spray pressure (kg / cm 2 ) Air flow rate (L / cm 2 ) Spray rate (g / min) Rotating speed of coating pan (rpm)
45 30 2 30 2.5 40
5.0 95.0
Hydrophilic layer
TC-5 ACET Ethanol Water
65 35 2 30 1.8 40
1.5 4.0 23.0 71.5
Enteric layer HPMC -AS Talc Ethanol Water
60 40 2 30 2.5 40
5.0 2.5 55.8 36.7
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CTDC. The fasted dogs received no food, but had free access to water, for 18 h before the administration. The CTDC was orally administered to the male beagle dogs along with 50 mL of water. Blood samples (4 mL) were collected at every 1 h for the period from 0–14 h and again after 24 h. The plasma was obtained by centrifuging the blood samples at 3000 rpm for 5 min immediately after sampling. All the plasma samples were frozen at 2208C until analysis. The oral–caecal transit time was regarded as the time required for the first appearance of sulfapyridine (SP), a metabolite of SAS, in the plasma after administration [16,17].
2.6. Assay methods 2.6.1. THEO, ACET and SP The concentrations of THEO, ACET, and SP in plasma were determined together by the HPLC method. A 0.1 mL sample of 7-(2-hydroxyethyl)theophylline containing aqueous solution (20 mg / mL) as the internal standard was added to 0.5 mL of plasma. Then the mixture was shaken with 5 mL of ethylacetate for 5 min prior to centrifugation at 2000 rpm for 5 min. Four ml of the organic layer were taken out and evaporated to dryness at 408C under N 2 gas flow, and the residue was resolved in 0.5 mL of the mobile phase to make the sample solution for analysis. Next, 200 mL of the sample solution were loaded on to a HPLC system to determine the plasma concentration level of the drugs. The HPLC conditions applied were as follows: pump, L-6200 (Hitachi, Japan); UV detection, SPD-10A (Shimadzu, Japan); column, KC-PAK SMA C18 (4.63250 mm; Chemco Scientific, Osaka, Japan); mobile phase, acetonitrile: 0.01 M sodium acetate (pH 4) (1:20); flow rate, 0.8 mL / min; detection, wavelength at 254 nm; and column temperature, 508C. 2.6.2. PPA The concentration of PPA in plasma was determined by the HPLC method. A 0.5 mL sample of phenethylamine hydrochloride aqueous solution (5 mg / mL) as the internal standard was added to 0.5 mL of plasma. The mixture was shaken with 5 mL of
dichloromethane:diethylether (30:70) mixture for 10 min and centrifuged at 2000 rpm for 5 min. Then, 4 mL of the organic layer was taken out and added to 0.5 mL of 0.5% phosphate aqueous. After shaking for 10 min, the mixture was centrifuged at 2000 rpm for 5 min. Five mL of cyclohexane were added to the aqueous layer, and the mixture was shaken and centrifuged again under the above conditions. The organic layer was removed, and 200 mL of the aqueous solution were analyzed by HPLC. The HPLC system consisted of the same equipment as used in the assay of THEO, but the column used was a Hypersil 5-ODS (4.6 mm3250 mm; GL Science, Tokyo, Japan). The mobile phase, acetonitrile:0.2% phosphate buffer containing 0.01 M sodium lauryl sulfate (35:60), was pumped at 1.0 mL / min and monitored at 270 nm; column temperature, 508C.
3. Results and discussion The fundamental structures of CTDC used in this study are depicted schematically in Fig. 1, and the necessary information of individual capsules, regarding coating layers and drugs incorporated, are summarized in Table 2. All the capsules commonly contain SAS as a colon-arrival indicator and succinic acid as a pHadjusting agent besides drugs. Except CTDC 0 , the capsules are coated with a three-layered polymeric film comprising the innermost acid-soluble layer (Eudragit E), the intermediate hydrophilic layer (TC-5 ), and the outermost enteric layer (HPMC AS). CTDC 0 does not have the Eudragit E layer, so this can be regarded as a sort of enteric-coated capsule. For the three capsules, CTDC 1 , CTDC 2 and CTDC 3 , the coating amounts of the acid-soluble layer is purposely different to vary the onset time of drug release after the gastric emptying. Except CTDC 4 , ACET is embedded in the hydrophilic layer as an indicator of the gastric emptying. The small intestinal transit time can be estimated by monitoring the absorption of ACET and SP (a metabolite of SAS). Only CTDC 4 contains three drugs, THEO, ACET, and PPA, together in the capsule, which is used to compare the colonic absorbability of those drugs.
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3.1. Drug delivery to the colon by the CTDC
Fig. 1. Schematic representation of the fundamental structure of the colon-targeted delivery capsule (CTDC). (a) Drug, (b) succinic acid, (c) hard gelatin capsule, (d) acid-soluble layer, (e) hydrophilic layer, (f) enteric layer.
3.1.1. In vitro release characteristics CTDC 0 , CTDC 1 , CTDC 2 , and CTDC 3 were used in this study. The release profiles of THEO, SAS, and ACET from these capsules in the JP 1st-fluid (pH 1.2) and the JP 2nd-fluid (pH 6.8) are shown in Fig. 2. All the capsules did not release any drugs for over 8 h in the 1st-fluid, suggesting the excellent acidresistibility given by the enteric layer. The CTDC 0 , which has no acid-soluble layer, quickly released three drugs in the 2nd-fluid, even though a small difference in release rate was observed between them (Fig. 2a), which may be due to the difference in solubility. Nevertheless, a relatively good coincident in the release behavior of three drugs suggests that either the capsule shell, the TC-5 layer, or the enteric layer does not function essentially as a dissolution barrier, because each of those easily dissolves in the aqueous medium at pH 6.8. The CTDC 1 released ACET quickly after the dissolution test started, and about 60 min after that, THEO and SAS started to be released (Fig. 2b). This is the typical dissolution characteristic of CTDC, and the observed delayed-release was brought about by the
Table 2 Composition of the CTDC used in this study Composition
CTDC 0
CTDC 1
CTDC 2
CTDC 3
CTDC 4
Capsule contents Drugs Colon arrival indicator pH-adjusting agent
THEO SAS Succinic acid
THEO SAS Succinic acid
THEO SAS Succinic acid
THEO SAS Succinic acid
THEO, ACET, PPA SAS Succinic acid
–
Eudragit E (11 mg)
Eudragit E (21 mg)
Eudragit E (33 mg)
Eudragit E (30 mg)
TC-5 (15 mg) ACET*
TC-5 (15 mg) ACET*
TC-5 (15 mg) ACET*
TC-5 (15 mg) ACET*
TC-5 (15 mg) –
HPMC -AS (250 mg)
HPMC -AS (250 mg)
HPMC -AS (250 mg)
HPMC -AS (250 mg)
HPMC -AS (250 mg)
Coating layer Acid-soluble layer Polymer (Coating amount / capsule) Hydrophilic layer Polymer (Coating amount / capsule) Gastric emptying indicator Enteric layer Polymer (Coating amount / capsule)
THEO, theophylline (20 mg / capsule); ACET, acetaminophen (60 mg / capsule); PPA, phenylpropanolamine hydrochloride (30 mg / capsule); SAS, sulfasarazine (50 mg / capsule). *Used as the gastric emptying indicator (10 mg / capsule).
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Fig. 2. Release profiles of drugs from CTDC in the JP 1st-fluid (pH 1.2) and in the JP 2nd-fluid (pH 6.8); (a) CTDC 0 , (b) CTDC 1 , (c) CTDC 2 , (d) CTDC 3 ; h, j, ACET; n, m, THEO; s, d, SAS. Open symbols represent the release profiles in the JP 1st-fluid. Closed symbols represent the release profiles in the JP 2nd-fluid. Each data represents the mean6S.D. for six runs.
timed-corruption of the acid-soluble layer, which is triggered by the micro-environmental pH-change in the capsule induced by the dissolution of succinic acid [13]. The CTDC 2 and CTDC 3 provide similar release profiles as CTDC 1 , but it was noted that the dissolution lag time of THEO and SAS were extended in accordance with increasing the coating amount of the acid-soluble layer (Fig. 2c and d). The small difference in the starting time of dissolution between THEO and SAS could be attributed to the extremely poor water-solubility or water-repellent property of SAS. This phenomenon can be improved by adding the water soluble excipients or the disintegrate agents. From the results of this series of dissolution studies, the in vitro lag time of each capsule can be regarded as 0.2 h, 1.3 h, 2.0 h, and 3.5 h for CTDC 0 , CTDC 1 , CTDC 2 , and CTDC 3 , respectively.
3.1.2. In vivo release study In order to examine the drug release behavior of CTDC in vivo, and to estimate the small intestinal transit time, the dog study was performed. CTDC 0 , CTDC 1 , CTDC 2 , and CTDC 3 were orally administered to four tetragastrin-treated beagle dogs under fasted condition. The plasma concentration profiles of three drugs, i.e. THEO, ACET, and SP, were found to reflect well the in vitro release behavior of CTDC. Individual plasma concentrations vs. time profiles of three drugs after oral administration of CTDC 0 , CTDC 1 , CTDC 2 , and CTDC 3 to four beagle dogs are shown in Figs. 3–6, respectively. When CTDC 0 was orally given to Dog 1, ACET, an indicator of gastric emptying, was found in plasma on the third-hour for the first time after the administration (Fig. 3). This suggests that the gastric
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Fig. 3. Individual plasma concentration vs. time profiles of ACET, THEO, and SP after oral administration of CTDC 0 to four beagle dogs. j, ACET (gastric emptying indicator); m, THEO; d, SP (colon arrival indicator).
emptying of the capsule occurred between 2 h and 3 h. The first appearance of THEO in plasma was also observed in the third-hour, suggesting that THEO was released at almost the same time as ACET, as expected. However, SP was detected in plasma at 6 h after the administration. SP is known as the major metabolite of SAS, most of which was converted from SAS by the bacterial degradation in the colon. Since SAS cannot be absorbed from the intestine, SP can be regarded as a marker indicating the colonarrival of SAS. The above result, therefore, indicates that the oral–caecal transit time of SAS was about 6 h, and the small intestinal transit time can be estimated as about 3 h by subtracting the gastric emptying time from the oral–caecal transit time. When the CTDC 1 was administered to the same
dog, the first appearance of ACET in plasma was observed at 4 h after administration. The first appearance times (FAT) of THEO and SP were 5 h and 7 h, respectively (Fig. 4). Attention was also paid to the change in the FAT of drugs in the CTDC 2 and CTDC 3 (Fig. 5 and Fig. 6). It was clearly found that, with increasing the in vitro lag time, the difference in the FAT became larger in the case between ACET, and THEO and became closer in the case between THEO and SP. In order to analyze the in vivo / in vitro relationship of CTDC in dogs more quantitatively, three parameters, FATACET , FAT THEO , and FAT SP , which are defined as the first appearance times of ACET, THEO, and SP in plasma, respectively, were calculated from the plasma concentration profiles of
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Fig. 4. Individual plasma concentration vs. time profiles of ACET, THEO, and SP after oral administration of CTDC 1 to four beagle dogs. j, ACET (gastric emptying indicator); m, THEO; d, SP (colon arrival indicator).
individual dogs. The mean values obtained from four dogs are summarized in Table 3. Since ACET is used as a marker of gastric emptying, the value of FATACET corresponds to the gastric emptying time in itself. Although the observed values of FATACET implies that a certain relation might exist between FATACET and the in vitro lag time of CTDC, there was no statistically significant difference (P,0.05). In fact, considering the facts that one subject administered CTDC 0 provided a considerably short FATACET of less than 1 h, and another subject administered CTDC 3 provided an abnormally long FATACET of 7 h, the apparent difference should be judged as essentially negligible. The value of FAT SP corresponds to the oral–caecal transit time in itself as mentioned before. The mean
values were found to vary from 5.3 h (CTDC 0 ) to 8.0 h (CTDC 3 ) in accordance with the values of FATACET . The time difference between FAT SP and FATACET corresponds to the colon arrival time from the gastric emptying. This calculation provided quite closer values, such as 2.861.0 h for CTDC 0 , 2.760.5 h for CTDC 1 , 3.560.5 h for CTDC 2 , and 3.060.6 h for CTDC 3 , suggesting the relatively constant small intestinal transit time in dogs. The average value for all CTDCs was 3.0 h, which is almost coincident with the value reported by Mizuta et al. [17]. The value of FAT THEO was found to be increased with increasing the in vitro lag time of capsules, suggesting that all the capsules could release the drug as designed even in the gastrointestinal tract in
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Fig. 5. Individual plasma concentration vs. time profiles of ACET, THEO, and SP after oral administration of CTDC 2 to four beagle dogs. j, ACET (gastric emptying indicator); m, THEO; d, SP (colon arrival indicator).
dogs. The time difference between FATACET and FAT THEO means the onset time of theophylline release after the gastric emptying. The calculation provided 0.060.0 h, 1.060.0 h, 2.360.5 h, and 2.760.6 h for CTDC 0 , CTDC 1 , CTDC 2 , and CTDC 3 , respectively. In Fig. 7, all these values are plotted against the in vitro lag time of CTDCs, which are obtained from Fig. 2. As was clearly shown, it was proven that there was a good correlation between in vitro and in vivo drug release of the CTDC. The time difference between FAT SP and FAT THEO should be a parameter indicating the release site in the small intestine even though it is still difficult to define the anatomical position exactly. When the time difference is almost zero, it indicates that the CTDC has released theophylline in the colon. The smaller time difference means a closer release site to
the colon. The time differences calculated for all CTDCs are compared in Fig. 8. It was clearly shown that the time difference decreased with increasing the in vitro lag time of CTDC. This means that the CTDC with a longer lag time can deliver the drug to a deeper position in the small intestine. This result also indicates that, when the in vitro lag time of at least 3 h is given, the colonic delivery of drugs can be achieved in dogs. This rather linear relation between the in vitro lag time and the drug release site implies the possibility to use CTDC as the device for assessing the regional drug absorption in the gastrointestinal tract. Various experimental methods have been used to identify the specific regions where the maximum absorption and bioavailability of the drug are obtained in the gastrointestinal tract of animals or man. Examples of
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Fig. 6. Individual plasma concentration vs. time profiles of ACET, THEO, and SP after oral administration of CTDC 3 to four beagle dogs. j, ACET (gastric emptying indicator); m, THEO; d, SP (colon arrival indicator).
in situ methods include semi-loop, open-loop, and closed-loop techniques [18]. Examples of in vivo methods include double and triple lumen catheter perfusion [19], site-specific indwelling catheter in dogs, a multi-channel tube with two occluding balloons for segmental perfusion [20,21], and intubaTable 3 The first appearance times of ACET, THEO, and SAS after oral administration of CTDC under fasted condition First appearance time
CTDC 0
CTDC 1
CTDC 2
CTDC 3
FATACET (h) FAT THEO (h) FAT SP (h)
2.561.0 2.561.0 5.360.9
3.360.6 4.360.6 6.060.8
3.560.5 5.860.5 7.060.4
5.061.0 7.760.8 8.060.5
Each data represents the mean6S.D. of four dogs.
tion through single luminal naso-intestinal catheters [22–24]. Since each of those techniques forces some invasive treatment to subjects, however, the result may not always reflect the normal condition because the invasive procedures have been demonstrated conclusively to profoundly affect the function of the gastrointestinal tract. The recently developed high frequency capsule (HF capsule) [25] and the InteliSite capsule [26] are considered more sophisticated technologies for the collection of absorption data by non-invasive, operator-controlled delivery of drugs to specific regions of the gastrointestinal tract. Nevertheless, considering some limitations for the use of those capsules in routine drug absorption studies, such as complicated procedures and cost, the CTDC may be a useful addition to
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Fig. 7. In vivo / in vitro correlation in the lag time of THEO release from CTDC. In vitro lag time was directly determined by the dissolution study in the JP 2nd-fluid (pH 6.8). In vivo lag time was estimated from FAT THEO 2FATACET . Each data of in vivo lag time and in vitro lag time represents the mean6S.D. for four runs and six runs, respectively.
currently available non-invasive methodologies because of its simple structure and ease of practical use.
3.2. Drug absorbability in the colon Based on the findings obtained in the first dog study, the CTDC 4 , of which in vitro lag time was about 3 h, was used for the next dog study. This capsule contains three model drugs with different solubilities, ACET, THEO, and PPA, and also it contains SAS as the colon arrival indicator. Fig. 9 shows the in vitro release profiles of those drugs in
Fig. 8. Comparison of the time difference between FAT THEO and FAT SP for the CTDCs with different in vitro lag times. Each data represents the mean6S.D. for four runs.
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the JP 1st-fluid (pH 1.2) and the JP 2nd-fluid (pH 6.8). As was shown, all the drugs were not released for over 8 h in the 1st-fluid, whereas they were quickly released after the lag time of about 3 h in the 2nd-fluid. This release characteristic is quite suitable to evaluate the colonic absorbability of drugs, because the drugs can be released directly on the dog colon with this system. The capsule was orally administered to three beagle dogs under fasted condition. The aqueous solution containing three drugs was also administered under the identical condition as the reference. The individual plasma concentration vs. time profiles of THEO, ACET, and PPA are compared with those obtained from the aqueous form in Figs. 10–12, respectively. When the drugs were administered in the solution form, the blood concentration of each drug quickly rose and attained the maximum level (Cmax ) by 2 h in all cases. On the other hand, when the CTDC was given, the onset of drug absorption was found to be considerably delayed, up to 6 h or 7 h. It is also noted that in the individual dog, the absorption of three drugs occurred at the same time and the onset time was almost coincident with that of SP (indicated by an arrow in each profile). Necessary pharmacokinetic parameters for discussion, calculated from the plasma drug concentration vs. time profiles, are listed in Table 4. T max is the time required to reach Cmax , AUC 24h is the area under the plasma concentration vs. time curve calculated from 0–24 h, and the relative bioavailability was the percentage of the AUC 24h of CTDC to the AUC 24h of the aqueous solution form for each drug. The first appearance times of each drug in plasma were all 6.360.5 h without any difference among drug species, and this value was very close to that of SP (7.060.0 h) as mentioned above. These results suggest that the CTDC successfully delivered the drugs to the caecum or proximal colon. The Cmax of ACET was found to be 8236150 ng / mL when administered with CTDC, which was almost half of that obtained from the administration in the solution form. In the cases of THEO and PPA, however, the Cmax values were quite close to those of the solution form. The relative bioavailabilities of THEO and
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Fig. 9. Release profiles of drugs from CTDC 4 in the JP 1st-fluid (pH 1.2) and in the JP 2nd-fluid (pH 6.8). (a) ACET and THEO, (b) PPA and SAS. h, j, ACET; n, m, THEO; 앳, ♦, PPA; s, d, SAS. Open symbols represent the release profiles in the JP 1st-fluid. Closed symbols represent the release profiles in the JP 2nd-fluid. Each data represents the mean6S.D. for six runs.
Fig. 10. Individual plasma concentration vs. time profile of THEO after oral administration of solution and CTDC 4 to beagle dogs. s, solution; d, CTDC 4 . The arrow indicates the onset time of SP absorption.
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Fig. 11. Individual plasma concentration vs. time profile of ACET after oral administration of solution and CTDC 4 to beagle dogs. s, solution; d, CTDC 4 . The arrow indicates the onset time of SP absorption.
PPA were 94.2610.2% and 91.5622.6%, respectively, for the CTDC administration, which is almost the same level as those obtained from the solution form. However, the relative bioavailability of ACET was only 71.066.0%, considerably lower than that of solution. This implies that the absorption of ACET would decrease in the colon. This is also supported by the findings of Yamada et al., in which the absorption of ACET was relatively low from the colon, very poor from the stomach, and quite sufficient from the jejunum and ileum [27]. It has been often pointed out that when drugs were administered in the sustained release dosage form, the relative bioavailability would be inevitably decreased due to the poor absorption from the colon. In fact, it was reported that the relative bioavailability of THEO, ACET, and PPA in sustained release dosage form decreased to 52% [28], 31.1% [27], and 78.8% [29], respectively, in dogs. One of the most
likely reasons may be due to the poor absorbability of drugs from the colon. However, considering the fact that there is considerably less water in the colon, the interference in the drug release from the device or in the diffusion of released drug to the mucosal surface should be a possible reason. In addition, the bio-degradation by microflora in the colon could be a possible reason for some drugs [30,31]. The present study provided some fundamental information to understand the colonic behavior of the released drugs in dogs, that is, the mucosal absorption of THEO and PPA was sufficient, no specific degradation occurred in the colon, and no significant interference to the diffusion of the released drug happened in the colon. Therefore, the most possible reason for the commonly observed low bioavailability of those drugs in sustained release dosage forms may be attributed to the considerable decrease in the drug release rate from the dosage form due to
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374
Fig. 12. Individual plasma concentration vs. time profile of PPA after oral administration of solution and CTDC 4 to beagle dogs. s, solution; d, CTDC 4 . The arrow indicates the onset time of SP absorption.
Table 4 The first appearance time and pharmacokinetic parameters of three drugs after oral administration of the aqueous solution form and CTDC to beagle dogs under fasted condition Drug
Dosage form
FAT b (h)
Cmax (ng / mL)
T max (h)
AUC 24h (ng h / mL)
Relative bioavailability c (%)
THEO
Solution CTDC Solution CTDC Solution CTDC CTDC
060 6.360.5 060 6.360.5 060 6.360.5 7.060.0
26766123 26656117 456641 516681 1996626 8236150
1.360.6 9.760.5 1.360.6 9.760.5 0.560.0 8.760.5
25 59466067 23 66063890 37516504 33416510 37906430 27016436
100.0 94.2610.2 100.0 91.5622.6 100.0 71.066.0
PPA ACET SP a a
Colon arrival indicator. The first appearance time of drugs in plasma after oral administration. c The percent of (AUC of CTDC /AUC of solution). Each data represents the mean6S.D. for three beagle dogs. b
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a lower amount of fluids. The decreased bioavailability of ACET may be caused by the first-pass effect as was reported by Hirate et al. [32]. From these results, this device is useful for delivering the drugs to the colon and for estimating the regional drug absorption from the gastrointestinal tract. Moreover, this device might be useful for anacidity or achylia gastrica if the enteric polymers dissolving at high pH like Eudragit S are used.
[8] [9]
[10]
[11]
4. Conclusion Through the present in vivo dog studies, it was proved that the CTDC had the potential to deliver the drug to various positions in the gastrointestinal tract by adjusting the onset time of in vitro drug release in the JP 2nd-fluid (pH 6.8). It was also found that the colon-targeted delivery of drugs can be achieved in dogs, when the 3-h lag time is given to the CTDC. This device is useful not only for the colon-targeted delivery of drugs, but also for assessing the regional drug absorption from the gastrointestinal tract.
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[24] N. Vidon, A. Pfeiffer, J. Godbillon, M. Rongier, S. Gauron, J. Hirtz, J.J. Bernier, J.P. Doubois, Evaluation of the gastric absorption and emptying of drugs under various pH conditions using a simple intubation method: application to diclofenac, Br. J. Clin. Pharm. 28(1) (1989) 121–124. [25] K.H. Antonin, Other methods in studying colonic drug absorption, in: P.R. Bieck (Ed.), Colonic Drug Absorption and Metabolism, Marcel Dekker, New York, 1993, pp. 89– 107. [26] US Patent 5167626, 5170801. [27] K. Yamada, A. Furuya, M. Akimoto, T. Maki, T. Suwa, H. Ogata, Evaluation of gastrointestinal transit controlled-beagle dog as a suitable animal model for bioavailability testing of sustained-release acetaminophen dosage form, Int. J. Pharm. 119 (1995) 1–10. [28] M. Turkoglu, W.A. Ritschel, A. Sakr, In vivo evaluation of fluidized-bed coated pellets, Int. J. Pharm. 103 (1994) 115– 118.
[29] R.C. DiLuccio, M.A. Hussain, D. CoffinBeach, G. Torosian, E. Shefter, A.R. Hurwitz, Polyvinyl alcohol–methyl acrylate copolymers as a sustained-release oral delivery system, Pharm. Res. 6(10) (1989) 844–847. ¨ [30] P. Langguth, G. Breves, A. Stockli, H.P. Merkle, S. Wolffram, Colonic absorption and bioavailability of the pentapeptide metkephamid in the rat, Pharm. Res. 11(11) (1994) 1640–1645. [31] M. Kuroiwa, N. Inotsume, R. Iwaoku, M. Nakano, Degradation of drugs by gut microflora which may affect the bioavailability after oral administration, J. Pharm. Sci. 76(11) (1987) S79. [32] J. Hirate, C.Y. Zhu, I. Horikoshi, V.O. Bhargava, First-pass metabolism of acetaminophen in rats after low and high doses, Biopharm. Drug Dispos. 11 (1990) 245–252.
Evaluation of colonic absorbability of drugs in dogs using a novel colon-targeted delivery capsule (CTDC) Takashi Ishibashi*, Kengo Ikegami, Hiroaki Kubo, Masao Kobayashi, Masakazu Mizobe, Hiroyuki Yoshino Pharmaceutics Research Laboratory, Tanabe Seiyaku Co., Ltd., 16 -89, Kashima 3 -chome, Yodogawa-ku, Osaka 532, Japan Received 23 July 1998; received in revised form 21 October 1998; accepted 8 December 1998
Abstract A series of dog studies were performed to examine the in vitro / in vivo relationship of drug release behavior of the newly developed colon-targeted delivery capsule (CTDC). The four kinds of CTDCs containing theophylline, each of which has a different in vitro dissolution lag time, were orally administered to four beagle dogs under fasted condition, and the onset times of drug absorption were compared. The CTDC with longer in vitro lag time had a later onset of drug absorption. It was also found that the time difference between the gastric emptying and the onset of drug absorption was almost equal to the in vitro dissolution lag time of the capsule, suggesting a similar performance of CTDC in the gastrointestinal tract. From the comparison to the absorption behavior of the colon arrival marker, i.e. sulfasalazine, it was proved that the CTDC with the lag time of 3 h can deliver the drug directly to the colon. This result implied that the CTDC can be used as a non-invasive means for assessing the regional absorbability of drugs in the gastrointestinal tract. To evaluate the absorbability of drugs in the colon, three model drugs, theophylline (THEO), acetaminophen (ACET), and phenylpropanolamine hydrochloride (PPA) were directly delivered to the colons of beagle dogs using the CTDC with the lag time of about 3 h. The obtained relative bioavailabilities to the solution form were as high as 94.2%, 71.0%, and 91.5% for THEO, ACET and PPA, respectively, suggesting that the colonic absorbability of those drugs is essentially good. 1999 Elsevier Science B.V. All rights reserved. Keywords: Colon-targeted delivery capsule; Site-specific drug delivery; Beagle dog; Gastric emptying; Colonic bioavailability
1. Introduction The colon-targeted delivery of drugs via the oral route has attracted much interest for the local treatment of some colonic diseases, such as ulcer*Corresponding author. Tel.: 181-6-300-2788; fax: 181-6-3002799. E-mail address: [email protected] (T. Ishibashi)
ative colitis, Crohn’s disease, colon-cancer, etc. Another advantage of colon-targeting involves the improved oral bioavailability of peptide or protein drugs susceptible to acidic or enzymatic degradation in the stomach or small intestine [1]. The colon is thought suitable as the absorption site for those drugs because of less activity of proteolysis [2]. In the last decade, there has been much research aiming toward the colon-targeted delivery of drugs.
0168-3659 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 99 )00005-X
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Various methodologies and technologies have been proposed, such as pH-triggered [3,4], time-controlled [5–8], and microbially-controlled deliveries [9–11]. Nevertheless, it seems that only a few systems are applicable for practical use in terms of the selectivity to the target site, the safety of the materials to be used, and the preparation techniques to be applied. We recently developed a new capsule type colontargeted delivery system (colon-targeted delivery capsule: CTDC) from a practical point of view, which can be manufactured using currently applicable pharmaceutical technologies and materials recognized as safe. The CTDC was designed by imparting a pH-sensing function and a time-release function to the conventionally used hard gelatin capsule, so as to release the drug rapidly at the predetermined time after gastric emptying [12]. The lag time of the CTDC can be controlled by the coating amount of Eudragit E, and the kind of organic acid as a pH adjusting agent [13]. Since the transit time of pharmaceuticals in the small intestine is known to be less variable in humans, i.e. about 361 h [14], the colontargeting could be achieved with the CTDC when the onset time of drug release is adjusted at 3 h or more. If the reliable site-specificity to the colon is assured, the CTDC could be used to evaluate the colonic absorbability of drugs. As a matter of fact, the drug absorption from the colon is believed to be generally poor because less aqueous fluid existed in the lower part of the large intestine [15]. Therefore, when sustained-release formulations are designed, it is important to understand the rate or extent of the colonic absorption of the drug. The simple and relevant methodology to evaluate properly the colonic absorbability of drugs under the intact physiological condition, however, has not been offered to formulators. The CTDC may be a useful means for achieving this purpose, because the system enables the direct delivery of any type of drugs to the colon without any invasive treatment. The first purpose of the present study is to check if the CTDC can release the drugs at the targeted position in the gastrointestinal tract. Various CTDCs with different in vitro lag times were orally administered to beagle dogs. The in vitro / in vivo relationship of drug release behavior is demonstrated, and the in vitro lag time required for the colon-targeted drug delivery is estimated in dogs. The second
purpose is to evaluate the colonic absorbability of some drugs with different solubilities using the CTDC system.
2. Materials and methods
2.1. Materials Theophylline (THEO; Shiratori Seiyaku, Japan) and phenylpropanolamine hydrochloride (PPA; Alps Chemical Co., Japan) were used as model drugs. Acetaminophen (ACET; Yamamoto Chemical Co., Japan) was used as both a model drug and an indicator determining the gastric emptying time of the capsule. Sulfasalazine (SAS; Sigma Chemical Co., Germany) was used as an indicator determining arrival in the colon. Hard gelatin capsules (size [2; 17.8 mm in length; 6.07 mm and 6.36 mm in width of body and cap) were purchased from Warner Lambert. Aminoalkylmethacrylate copolymer B ¨ (Eudragit E 100, Rhom Pharm, Darmstadt, Germany) was used as the acid-soluble polymer, which is soluble in low pH aqueous medium up to pH 5. Hydroxypropyl-methylcellulose (TC-5 , type EW, Shin-etsu Chemical, Japan) was used as the hydrophilic polymer. Hydroxypropyl-methylcellulose acetate succinate (HPMC -AS, type MIF, Shin-etsu) was used as the enteric polymer. Ethylcellulose (10 cp grade, Shin-etsu) was used as the sealing agent of the hard gelatin capsules. Succinic acid (Katayama Chemical, Japan) was used as the pH-adjusting agent. 7-(2-Hydroxyethyl)-theophylline (Tokyo Kasei Kogyo, Japan) and phenethylamine hydrochloride (Katayama) were used as the internal standards. Tetragastrin was purchased from Mecto (Japan). All other chemicals and solvents were of reagent grade.
2.2. Preparation of CTDC with different lag time The powder mixture consisting of 20 mg of THEO, 50 mg of SAS and 100 mg of succinic acid was filled into a hard gelatin capsule, and the joint of the capsule body and cap was sealed with a small amount of the 5% (w / w) ethylcellulose ethanolic solution. Four batches of the sealed capsules were spray-coated with different amounts of Eudragit E (0, 11, 21, and 33 mg / capsule) to provide a different
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onset time of drug release. Each batch of the Eudragit E-coated capsules was then coated with TC-5 (15 mg / capsule) containing ACET (10 mg / capsule) as an intermediate layer, and finally with HPMC -AS (250 mg / capsule) as the outmost enteric layer. The coating process was carried out using a coating machine (Hicoater , Type HCT-mini, Freund Ind., Japan), and the formulas of the polymeric coating solutions for each layer are given in Table 1 along with the standard operating conditions.
2.3. Preparation of CTDC for colonic absorbability study The powder mixture consisting of three model drugs (20 mg of THEO, 30 mg of PPA, and 60 mg of ACET), a colon arrival indicator (50 mg of SAS), and a pH-adjusting agent (100 mg of succinic acid) was filled into a hard gelatin capsule (size [2). The joint of the capsule body and cap was sealed with a small amount of the 5% (w / w) ethylcellulose ethanolic solution. The sealed capsules were spraycoated with Eudragit E (30 mg / capsule), then with TC-5 (15 mg / capsule), and finally with HPMC AS (250 mg / capsule) using a coating machine (Hicoater , Type HCT-mini). The formulas of the polymeric coating solutions, and the operating conditions of coating for each layer are described in Table 1.
363
2.4. In vitro release study The release profiles of ACET, THEO, and PPA from the capsule were determined according to the procedure described in the Japanese Pharmacopoeia XIII (the paddle method). The capsules were placed in a vessel with 900 mL of the JP 1st-fluid (pH 1.2) or 2nd-fluid (pH 6.8) at 3760.58C rotating at 100 rpm. The aliquots were removed periodically and assayed for ACET, THEO, and PPA using the high performance liquid chromatography (HPLC) method, of which analytical conditions are given in Section 2.6. Because of the different assay method applied, the release profile of SAS was determined by a separate run of the dissolution test. The capsules were placed in a vessel with 900 mL of the JP 1st-fluid (pH 1.2) or 2nd-fluid (pH 6.8) at 3760.58C rotating at 100 rpm. The released amount was periodically determined by the spectrophotometric method.
2.5. In vivo release /absorption study The in vivo absorption studies were carried out using tetragastrin-controlled beagle dogs, in order to minimize the intra-subject variance in gastric acidity. The healthy male beagle dogs, 11–12 kg, were used in this study under fasted condition. Tetragastrin (10 mg / kg) was injected intramuscularly twice, 15 min before and 45 min after oral administration of
Table 1 Formulas of coating solution and standard operating conditions Coating layer
Acid-soluble layer
Composition of coating solution (w / w%)
Eudragit E Ethanol
Operating conditions: Blower temperature (8C) Exhaust temperature (8C) Spray pressure (kg / cm 2 ) Air flow rate (L / cm 2 ) Spray rate (g / min) Rotating speed of coating pan (rpm)
45 30 2 30 2.5 40
5.0 95.0
Hydrophilic layer
TC-5 ACET Ethanol Water
65 35 2 30 1.8 40
1.5 4.0 23.0 71.5
Enteric layer HPMC -AS Talc Ethanol Water
60 40 2 30 2.5 40
5.0 2.5 55.8 36.7
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CTDC. The fasted dogs received no food, but had free access to water, for 18 h before the administration. The CTDC was orally administered to the male beagle dogs along with 50 mL of water. Blood samples (4 mL) were collected at every 1 h for the period from 0–14 h and again after 24 h. The plasma was obtained by centrifuging the blood samples at 3000 rpm for 5 min immediately after sampling. All the plasma samples were frozen at 2208C until analysis. The oral–caecal transit time was regarded as the time required for the first appearance of sulfapyridine (SP), a metabolite of SAS, in the plasma after administration [16,17].
2.6. Assay methods 2.6.1. THEO, ACET and SP The concentrations of THEO, ACET, and SP in plasma were determined together by the HPLC method. A 0.1 mL sample of 7-(2-hydroxyethyl)theophylline containing aqueous solution (20 mg / mL) as the internal standard was added to 0.5 mL of plasma. Then the mixture was shaken with 5 mL of ethylacetate for 5 min prior to centrifugation at 2000 rpm for 5 min. Four ml of the organic layer were taken out and evaporated to dryness at 408C under N 2 gas flow, and the residue was resolved in 0.5 mL of the mobile phase to make the sample solution for analysis. Next, 200 mL of the sample solution were loaded on to a HPLC system to determine the plasma concentration level of the drugs. The HPLC conditions applied were as follows: pump, L-6200 (Hitachi, Japan); UV detection, SPD-10A (Shimadzu, Japan); column, KC-PAK SMA C18 (4.63250 mm; Chemco Scientific, Osaka, Japan); mobile phase, acetonitrile: 0.01 M sodium acetate (pH 4) (1:20); flow rate, 0.8 mL / min; detection, wavelength at 254 nm; and column temperature, 508C. 2.6.2. PPA The concentration of PPA in plasma was determined by the HPLC method. A 0.5 mL sample of phenethylamine hydrochloride aqueous solution (5 mg / mL) as the internal standard was added to 0.5 mL of plasma. The mixture was shaken with 5 mL of
dichloromethane:diethylether (30:70) mixture for 10 min and centrifuged at 2000 rpm for 5 min. Then, 4 mL of the organic layer was taken out and added to 0.5 mL of 0.5% phosphate aqueous. After shaking for 10 min, the mixture was centrifuged at 2000 rpm for 5 min. Five mL of cyclohexane were added to the aqueous layer, and the mixture was shaken and centrifuged again under the above conditions. The organic layer was removed, and 200 mL of the aqueous solution were analyzed by HPLC. The HPLC system consisted of the same equipment as used in the assay of THEO, but the column used was a Hypersil 5-ODS (4.6 mm3250 mm; GL Science, Tokyo, Japan). The mobile phase, acetonitrile:0.2% phosphate buffer containing 0.01 M sodium lauryl sulfate (35:60), was pumped at 1.0 mL / min and monitored at 270 nm; column temperature, 508C.
3. Results and discussion The fundamental structures of CTDC used in this study are depicted schematically in Fig. 1, and the necessary information of individual capsules, regarding coating layers and drugs incorporated, are summarized in Table 2. All the capsules commonly contain SAS as a colon-arrival indicator and succinic acid as a pHadjusting agent besides drugs. Except CTDC 0 , the capsules are coated with a three-layered polymeric film comprising the innermost acid-soluble layer (Eudragit E), the intermediate hydrophilic layer (TC-5 ), and the outermost enteric layer (HPMC AS). CTDC 0 does not have the Eudragit E layer, so this can be regarded as a sort of enteric-coated capsule. For the three capsules, CTDC 1 , CTDC 2 and CTDC 3 , the coating amounts of the acid-soluble layer is purposely different to vary the onset time of drug release after the gastric emptying. Except CTDC 4 , ACET is embedded in the hydrophilic layer as an indicator of the gastric emptying. The small intestinal transit time can be estimated by monitoring the absorption of ACET and SP (a metabolite of SAS). Only CTDC 4 contains three drugs, THEO, ACET, and PPA, together in the capsule, which is used to compare the colonic absorbability of those drugs.
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3.1. Drug delivery to the colon by the CTDC
Fig. 1. Schematic representation of the fundamental structure of the colon-targeted delivery capsule (CTDC). (a) Drug, (b) succinic acid, (c) hard gelatin capsule, (d) acid-soluble layer, (e) hydrophilic layer, (f) enteric layer.
3.1.1. In vitro release characteristics CTDC 0 , CTDC 1 , CTDC 2 , and CTDC 3 were used in this study. The release profiles of THEO, SAS, and ACET from these capsules in the JP 1st-fluid (pH 1.2) and the JP 2nd-fluid (pH 6.8) are shown in Fig. 2. All the capsules did not release any drugs for over 8 h in the 1st-fluid, suggesting the excellent acidresistibility given by the enteric layer. The CTDC 0 , which has no acid-soluble layer, quickly released three drugs in the 2nd-fluid, even though a small difference in release rate was observed between them (Fig. 2a), which may be due to the difference in solubility. Nevertheless, a relatively good coincident in the release behavior of three drugs suggests that either the capsule shell, the TC-5 layer, or the enteric layer does not function essentially as a dissolution barrier, because each of those easily dissolves in the aqueous medium at pH 6.8. The CTDC 1 released ACET quickly after the dissolution test started, and about 60 min after that, THEO and SAS started to be released (Fig. 2b). This is the typical dissolution characteristic of CTDC, and the observed delayed-release was brought about by the
Table 2 Composition of the CTDC used in this study Composition
CTDC 0
CTDC 1
CTDC 2
CTDC 3
CTDC 4
Capsule contents Drugs Colon arrival indicator pH-adjusting agent
THEO SAS Succinic acid
THEO SAS Succinic acid
THEO SAS Succinic acid
THEO SAS Succinic acid
THEO, ACET, PPA SAS Succinic acid
–
Eudragit E (11 mg)
Eudragit E (21 mg)
Eudragit E (33 mg)
Eudragit E (30 mg)
TC-5 (15 mg) ACET*
TC-5 (15 mg) ACET*
TC-5 (15 mg) ACET*
TC-5 (15 mg) ACET*
TC-5 (15 mg) –
HPMC -AS (250 mg)
HPMC -AS (250 mg)
HPMC -AS (250 mg)
HPMC -AS (250 mg)
HPMC -AS (250 mg)
Coating layer Acid-soluble layer Polymer (Coating amount / capsule) Hydrophilic layer Polymer (Coating amount / capsule) Gastric emptying indicator Enteric layer Polymer (Coating amount / capsule)
THEO, theophylline (20 mg / capsule); ACET, acetaminophen (60 mg / capsule); PPA, phenylpropanolamine hydrochloride (30 mg / capsule); SAS, sulfasarazine (50 mg / capsule). *Used as the gastric emptying indicator (10 mg / capsule).
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Fig. 2. Release profiles of drugs from CTDC in the JP 1st-fluid (pH 1.2) and in the JP 2nd-fluid (pH 6.8); (a) CTDC 0 , (b) CTDC 1 , (c) CTDC 2 , (d) CTDC 3 ; h, j, ACET; n, m, THEO; s, d, SAS. Open symbols represent the release profiles in the JP 1st-fluid. Closed symbols represent the release profiles in the JP 2nd-fluid. Each data represents the mean6S.D. for six runs.
timed-corruption of the acid-soluble layer, which is triggered by the micro-environmental pH-change in the capsule induced by the dissolution of succinic acid [13]. The CTDC 2 and CTDC 3 provide similar release profiles as CTDC 1 , but it was noted that the dissolution lag time of THEO and SAS were extended in accordance with increasing the coating amount of the acid-soluble layer (Fig. 2c and d). The small difference in the starting time of dissolution between THEO and SAS could be attributed to the extremely poor water-solubility or water-repellent property of SAS. This phenomenon can be improved by adding the water soluble excipients or the disintegrate agents. From the results of this series of dissolution studies, the in vitro lag time of each capsule can be regarded as 0.2 h, 1.3 h, 2.0 h, and 3.5 h for CTDC 0 , CTDC 1 , CTDC 2 , and CTDC 3 , respectively.
3.1.2. In vivo release study In order to examine the drug release behavior of CTDC in vivo, and to estimate the small intestinal transit time, the dog study was performed. CTDC 0 , CTDC 1 , CTDC 2 , and CTDC 3 were orally administered to four tetragastrin-treated beagle dogs under fasted condition. The plasma concentration profiles of three drugs, i.e. THEO, ACET, and SP, were found to reflect well the in vitro release behavior of CTDC. Individual plasma concentrations vs. time profiles of three drugs after oral administration of CTDC 0 , CTDC 1 , CTDC 2 , and CTDC 3 to four beagle dogs are shown in Figs. 3–6, respectively. When CTDC 0 was orally given to Dog 1, ACET, an indicator of gastric emptying, was found in plasma on the third-hour for the first time after the administration (Fig. 3). This suggests that the gastric
T. Ishibashi et al. / Journal of Controlled Release 59 (1999) 361 – 376
367
Fig. 3. Individual plasma concentration vs. time profiles of ACET, THEO, and SP after oral administration of CTDC 0 to four beagle dogs. j, ACET (gastric emptying indicator); m, THEO; d, SP (colon arrival indicator).
emptying of the capsule occurred between 2 h and 3 h. The first appearance of THEO in plasma was also observed in the third-hour, suggesting that THEO was released at almost the same time as ACET, as expected. However, SP was detected in plasma at 6 h after the administration. SP is known as the major metabolite of SAS, most of which was converted from SAS by the bacterial degradation in the colon. Since SAS cannot be absorbed from the intestine, SP can be regarded as a marker indicating the colonarrival of SAS. The above result, therefore, indicates that the oral–caecal transit time of SAS was about 6 h, and the small intestinal transit time can be estimated as about 3 h by subtracting the gastric emptying time from the oral–caecal transit time. When the CTDC 1 was administered to the same
dog, the first appearance of ACET in plasma was observed at 4 h after administration. The first appearance times (FAT) of THEO and SP were 5 h and 7 h, respectively (Fig. 4). Attention was also paid to the change in the FAT of drugs in the CTDC 2 and CTDC 3 (Fig. 5 and Fig. 6). It was clearly found that, with increasing the in vitro lag time, the difference in the FAT became larger in the case between ACET, and THEO and became closer in the case between THEO and SP. In order to analyze the in vivo / in vitro relationship of CTDC in dogs more quantitatively, three parameters, FATACET , FAT THEO , and FAT SP , which are defined as the first appearance times of ACET, THEO, and SP in plasma, respectively, were calculated from the plasma concentration profiles of
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Fig. 4. Individual plasma concentration vs. time profiles of ACET, THEO, and SP after oral administration of CTDC 1 to four beagle dogs. j, ACET (gastric emptying indicator); m, THEO; d, SP (colon arrival indicator).
individual dogs. The mean values obtained from four dogs are summarized in Table 3. Since ACET is used as a marker of gastric emptying, the value of FATACET corresponds to the gastric emptying time in itself. Although the observed values of FATACET implies that a certain relation might exist between FATACET and the in vitro lag time of CTDC, there was no statistically significant difference (P,0.05). In fact, considering the facts that one subject administered CTDC 0 provided a considerably short FATACET of less than 1 h, and another subject administered CTDC 3 provided an abnormally long FATACET of 7 h, the apparent difference should be judged as essentially negligible. The value of FAT SP corresponds to the oral–caecal transit time in itself as mentioned before. The mean
values were found to vary from 5.3 h (CTDC 0 ) to 8.0 h (CTDC 3 ) in accordance with the values of FATACET . The time difference between FAT SP and FATACET corresponds to the colon arrival time from the gastric emptying. This calculation provided quite closer values, such as 2.861.0 h for CTDC 0 , 2.760.5 h for CTDC 1 , 3.560.5 h for CTDC 2 , and 3.060.6 h for CTDC 3 , suggesting the relatively constant small intestinal transit time in dogs. The average value for all CTDCs was 3.0 h, which is almost coincident with the value reported by Mizuta et al. [17]. The value of FAT THEO was found to be increased with increasing the in vitro lag time of capsules, suggesting that all the capsules could release the drug as designed even in the gastrointestinal tract in
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369
Fig. 5. Individual plasma concentration vs. time profiles of ACET, THEO, and SP after oral administration of CTDC 2 to four beagle dogs. j, ACET (gastric emptying indicator); m, THEO; d, SP (colon arrival indicator).
dogs. The time difference between FATACET and FAT THEO means the onset time of theophylline release after the gastric emptying. The calculation provided 0.060.0 h, 1.060.0 h, 2.360.5 h, and 2.760.6 h for CTDC 0 , CTDC 1 , CTDC 2 , and CTDC 3 , respectively. In Fig. 7, all these values are plotted against the in vitro lag time of CTDCs, which are obtained from Fig. 2. As was clearly shown, it was proven that there was a good correlation between in vitro and in vivo drug release of the CTDC. The time difference between FAT SP and FAT THEO should be a parameter indicating the release site in the small intestine even though it is still difficult to define the anatomical position exactly. When the time difference is almost zero, it indicates that the CTDC has released theophylline in the colon. The smaller time difference means a closer release site to
the colon. The time differences calculated for all CTDCs are compared in Fig. 8. It was clearly shown that the time difference decreased with increasing the in vitro lag time of CTDC. This means that the CTDC with a longer lag time can deliver the drug to a deeper position in the small intestine. This result also indicates that, when the in vitro lag time of at least 3 h is given, the colonic delivery of drugs can be achieved in dogs. This rather linear relation between the in vitro lag time and the drug release site implies the possibility to use CTDC as the device for assessing the regional drug absorption in the gastrointestinal tract. Various experimental methods have been used to identify the specific regions where the maximum absorption and bioavailability of the drug are obtained in the gastrointestinal tract of animals or man. Examples of
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370
Fig. 6. Individual plasma concentration vs. time profiles of ACET, THEO, and SP after oral administration of CTDC 3 to four beagle dogs. j, ACET (gastric emptying indicator); m, THEO; d, SP (colon arrival indicator).
in situ methods include semi-loop, open-loop, and closed-loop techniques [18]. Examples of in vivo methods include double and triple lumen catheter perfusion [19], site-specific indwelling catheter in dogs, a multi-channel tube with two occluding balloons for segmental perfusion [20,21], and intubaTable 3 The first appearance times of ACET, THEO, and SAS after oral administration of CTDC under fasted condition First appearance time
CTDC 0
CTDC 1
CTDC 2
CTDC 3
FATACET (h) FAT THEO (h) FAT SP (h)
2.561.0 2.561.0 5.360.9
3.360.6 4.360.6 6.060.8
3.560.5 5.860.5 7.060.4
5.061.0 7.760.8 8.060.5
Each data represents the mean6S.D. of four dogs.
tion through single luminal naso-intestinal catheters [22–24]. Since each of those techniques forces some invasive treatment to subjects, however, the result may not always reflect the normal condition because the invasive procedures have been demonstrated conclusively to profoundly affect the function of the gastrointestinal tract. The recently developed high frequency capsule (HF capsule) [25] and the InteliSite capsule [26] are considered more sophisticated technologies for the collection of absorption data by non-invasive, operator-controlled delivery of drugs to specific regions of the gastrointestinal tract. Nevertheless, considering some limitations for the use of those capsules in routine drug absorption studies, such as complicated procedures and cost, the CTDC may be a useful addition to
T. Ishibashi et al. / Journal of Controlled Release 59 (1999) 361 – 376
Fig. 7. In vivo / in vitro correlation in the lag time of THEO release from CTDC. In vitro lag time was directly determined by the dissolution study in the JP 2nd-fluid (pH 6.8). In vivo lag time was estimated from FAT THEO 2FATACET . Each data of in vivo lag time and in vitro lag time represents the mean6S.D. for four runs and six runs, respectively.
currently available non-invasive methodologies because of its simple structure and ease of practical use.
3.2. Drug absorbability in the colon Based on the findings obtained in the first dog study, the CTDC 4 , of which in vitro lag time was about 3 h, was used for the next dog study. This capsule contains three model drugs with different solubilities, ACET, THEO, and PPA, and also it contains SAS as the colon arrival indicator. Fig. 9 shows the in vitro release profiles of those drugs in
Fig. 8. Comparison of the time difference between FAT THEO and FAT SP for the CTDCs with different in vitro lag times. Each data represents the mean6S.D. for four runs.
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the JP 1st-fluid (pH 1.2) and the JP 2nd-fluid (pH 6.8). As was shown, all the drugs were not released for over 8 h in the 1st-fluid, whereas they were quickly released after the lag time of about 3 h in the 2nd-fluid. This release characteristic is quite suitable to evaluate the colonic absorbability of drugs, because the drugs can be released directly on the dog colon with this system. The capsule was orally administered to three beagle dogs under fasted condition. The aqueous solution containing three drugs was also administered under the identical condition as the reference. The individual plasma concentration vs. time profiles of THEO, ACET, and PPA are compared with those obtained from the aqueous form in Figs. 10–12, respectively. When the drugs were administered in the solution form, the blood concentration of each drug quickly rose and attained the maximum level (Cmax ) by 2 h in all cases. On the other hand, when the CTDC was given, the onset of drug absorption was found to be considerably delayed, up to 6 h or 7 h. It is also noted that in the individual dog, the absorption of three drugs occurred at the same time and the onset time was almost coincident with that of SP (indicated by an arrow in each profile). Necessary pharmacokinetic parameters for discussion, calculated from the plasma drug concentration vs. time profiles, are listed in Table 4. T max is the time required to reach Cmax , AUC 24h is the area under the plasma concentration vs. time curve calculated from 0–24 h, and the relative bioavailability was the percentage of the AUC 24h of CTDC to the AUC 24h of the aqueous solution form for each drug. The first appearance times of each drug in plasma were all 6.360.5 h without any difference among drug species, and this value was very close to that of SP (7.060.0 h) as mentioned above. These results suggest that the CTDC successfully delivered the drugs to the caecum or proximal colon. The Cmax of ACET was found to be 8236150 ng / mL when administered with CTDC, which was almost half of that obtained from the administration in the solution form. In the cases of THEO and PPA, however, the Cmax values were quite close to those of the solution form. The relative bioavailabilities of THEO and
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Fig. 9. Release profiles of drugs from CTDC 4 in the JP 1st-fluid (pH 1.2) and in the JP 2nd-fluid (pH 6.8). (a) ACET and THEO, (b) PPA and SAS. h, j, ACET; n, m, THEO; 앳, ♦, PPA; s, d, SAS. Open symbols represent the release profiles in the JP 1st-fluid. Closed symbols represent the release profiles in the JP 2nd-fluid. Each data represents the mean6S.D. for six runs.
Fig. 10. Individual plasma concentration vs. time profile of THEO after oral administration of solution and CTDC 4 to beagle dogs. s, solution; d, CTDC 4 . The arrow indicates the onset time of SP absorption.
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373
Fig. 11. Individual plasma concentration vs. time profile of ACET after oral administration of solution and CTDC 4 to beagle dogs. s, solution; d, CTDC 4 . The arrow indicates the onset time of SP absorption.
PPA were 94.2610.2% and 91.5622.6%, respectively, for the CTDC administration, which is almost the same level as those obtained from the solution form. However, the relative bioavailability of ACET was only 71.066.0%, considerably lower than that of solution. This implies that the absorption of ACET would decrease in the colon. This is also supported by the findings of Yamada et al., in which the absorption of ACET was relatively low from the colon, very poor from the stomach, and quite sufficient from the jejunum and ileum [27]. It has been often pointed out that when drugs were administered in the sustained release dosage form, the relative bioavailability would be inevitably decreased due to the poor absorption from the colon. In fact, it was reported that the relative bioavailability of THEO, ACET, and PPA in sustained release dosage form decreased to 52% [28], 31.1% [27], and 78.8% [29], respectively, in dogs. One of the most
likely reasons may be due to the poor absorbability of drugs from the colon. However, considering the fact that there is considerably less water in the colon, the interference in the drug release from the device or in the diffusion of released drug to the mucosal surface should be a possible reason. In addition, the bio-degradation by microflora in the colon could be a possible reason for some drugs [30,31]. The present study provided some fundamental information to understand the colonic behavior of the released drugs in dogs, that is, the mucosal absorption of THEO and PPA was sufficient, no specific degradation occurred in the colon, and no significant interference to the diffusion of the released drug happened in the colon. Therefore, the most possible reason for the commonly observed low bioavailability of those drugs in sustained release dosage forms may be attributed to the considerable decrease in the drug release rate from the dosage form due to
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Fig. 12. Individual plasma concentration vs. time profile of PPA after oral administration of solution and CTDC 4 to beagle dogs. s, solution; d, CTDC 4 . The arrow indicates the onset time of SP absorption.
Table 4 The first appearance time and pharmacokinetic parameters of three drugs after oral administration of the aqueous solution form and CTDC to beagle dogs under fasted condition Drug
Dosage form
FAT b (h)
Cmax (ng / mL)
T max (h)
AUC 24h (ng h / mL)
Relative bioavailability c (%)
THEO
Solution CTDC Solution CTDC Solution CTDC CTDC
060 6.360.5 060 6.360.5 060 6.360.5 7.060.0
26766123 26656117 456641 516681 1996626 8236150
1.360.6 9.760.5 1.360.6 9.760.5 0.560.0 8.760.5
25 59466067 23 66063890 37516504 33416510 37906430 27016436
100.0 94.2610.2 100.0 91.5622.6 100.0 71.066.0
PPA ACET SP a a
Colon arrival indicator. The first appearance time of drugs in plasma after oral administration. c The percent of (AUC of CTDC /AUC of solution). Each data represents the mean6S.D. for three beagle dogs. b
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a lower amount of fluids. The decreased bioavailability of ACET may be caused by the first-pass effect as was reported by Hirate et al. [32]. From these results, this device is useful for delivering the drugs to the colon and for estimating the regional drug absorption from the gastrointestinal tract. Moreover, this device might be useful for anacidity or achylia gastrica if the enteric polymers dissolving at high pH like Eudragit S are used.
[8] [9]
[10]
[11]
4. Conclusion Through the present in vivo dog studies, it was proved that the CTDC had the potential to deliver the drug to various positions in the gastrointestinal tract by adjusting the onset time of in vitro drug release in the JP 2nd-fluid (pH 6.8). It was also found that the colon-targeted delivery of drugs can be achieved in dogs, when the 3-h lag time is given to the CTDC. This device is useful not only for the colon-targeted delivery of drugs, but also for assessing the regional drug absorption from the gastrointestinal tract.
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