International Journal of Pharmaceutics 409 (2011) 169–177

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Comparison of in vitro and in vivo performance of a colonic delivery system

Iman S. Ahmed a,∗ , James W. Ayres b
a
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr-El-Eini St., Cairo, Egypt
b
College of Pharmacy, Oregon State University, Corvallis, OR 97333, United States

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this work is to compare in vitro to in vivo performance of a colonic drug delivery system,
Received 20 December 2010 made of small pectin-ethylcellulose coated drug beads. The delivery system was evaluated in vitro by
Received in revised form 24 February 2011 conducting drug release studies in different dissolution media to mimic transit times and pH conditions
Accepted 25 February 2011
in the stomach, small intestine and colon and in vivo by using gamma-scintigraphic studies in dogs and
Available online 4 March 2011
absorption studies in human volunteers under fed and fasted conditions. In vitro release studies indicated
that drug release rate depended on the ratio of the pectin to ethylcellulose in the coat and the thickness of
Keywords:
the coat. In vivo release studies obtained by deconvolution of biostudy data did not correlate with in vitro
In vitro/in vivo correlation
Colonic delivery
results obtained from most coat formulations. Beads showing ideal release profiles in vitro showed very
Enzymic degradation poor performance in vivo and only those beads showing colonic premature drug release in vitro might
Absorption studies be able to deliver the drug to the colon. Scintigraphic studies of a selected formulation showed that the
Deconvolution labeled beads had an estimated gastric emptying time of 3 h, an estimated small intestine transit time of
2 h and an estimated colonic transit time of 36 h. Average in vivo lag times of the selected formulation
from absorption studies in humans were found to be 6.1 h and 4.8 h under fed and fasted conditions,
respectively. The Cmax was also observed at 6.8 h and 5.5 h on average, under fed and fasted conditions,
respectively, which might indicate that release of drug from the beads, resulted from degradation of
pectin in the coat by enzymatic action in the colon rather than by simple diffusion. Deconvolution of
biostudy data showed that drug absorption continued on average for at least 12 h under both fed and
fasted conditions.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction segment where the drug system usually resides for a short time
which may result in too early or too slow drug release at the wrong
Design and evaluation of colonic delivery systems requires moment or at the wrong site in the intestinal tract (Schellekens
knowledge of the drug, the delivery system and the targeted organ. et al., 2007; Ibekwe et al., 2006a,b,c; Ibekwe et al., 2008). The uni-
Physicochemical properties of the drug molecule and other mate- versal polysaccharide systems, on the other hand, appear promising
rials in the delivery system in relation to known physiological because of their practicality and simplicity as they depend on
variables such as gastro-intestinal pH gradients and transit times the most distinctive property of the colon which is the abundant
must be well understood. A large number of colonic drug deliv- microflora (Basit, 2005). From the natural polysaccharides, pectin
ery systems have been developed by several different approaches; (Xu et al., 2005; Ashford et al., 1993; Ashford and Fell, 1994), amy-
however only few studies were able to correlate in vitro/ in vivo lose (Milojevic et al., 1996; Bloor et al., 2002; Siew et al., 2004),
performance of these systems (Schellekens et al., 2008; Ueda et al., guar gum (Tugcu-Demiro et al., 2004) and chitosan (Norihito et al.,
1994; Sangalli et al., 2001). Colonic delivery systems include the 2002) have been heavily investigated for their potential use as
temporal control of delivery (Steed et al., 1997), pH-based systems colonic delivery systems. Pectin has a pKa-value of 3.5 and has
(Ibekwe et al., 2006a,b,c; Klein et al., 2005), pressure-based systems been especially useful compared to other enzyme-based systems
(Jeong et al., 2001), enzyme-based systems (Xi et al., 2005) and oth- as it is unionized in acid media and it has been reported that even a
ers. Although pH based systems are the only proven technology short time exposure of pectin beads in simulated gastric fluid media
used already in commercial products yet it has been reported that drastically decreased drug release in higher pH simulated intestinal
these systems are not very reliable because of intestinal physiol- media such as no enteric coating would be needed and more drug
ogy where a pH larger than 7 is encountered in a short intestinal could reach the colon (Ahmed, 2005). Pectin is also reported to be
very useful when used with water-insoluble polymers as film coat-
ings (Macleod et al., 1999; Wakerly et al., 1996, 1997; Ahmed, 2005;
∗ Corresponding author. Tel.: +971 503794374; fax: +971 65585812. Marianne et al., 2003; Liu et al., 2003) or in the form of matrix tablets
E-mail address: [email protected] (I.S. Ahmed). (Ahrabi et al., 2000; Fernandez-Hervas and Fell, 1998). Although

0378-5173/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijpharm.2011.02.061
170 I.S. Ahmed, J.W. Ayres / International Journal of Pharmaceutics 409 (2011) 169–177

single unit dosage forms are easier to prepare, multi-particulate onto the drug beads to different film thickness. Coat formulation
systems have been useful especially with film-coatings. Multiple- one (F1) was prepared by mixing a 2% (w/v) solution of pectin USP in
unit systems have many advantages for colonic delivery when distilled water with Surelease and distilled water to result in a ratio
compared to single unit dosage forms such as decreased variabil- of pectin (P) to Surelease® (S) of 1:12 by weight. Coat formulation
ity in gastric emptying time, are less influenced by the presence of two (F2) was prepared in a similar way but by using a 3% (w/v)
food in the stomach (Hardy et al., 1985), reduced risk of high local solution of pectin USP in distilled water with Surelease and distilled
concentrations of released drugs, stagnation at the ileo-cecal junc- water to result in a ratio of pectin (P) to Surelease® (S) of 1:6 by
tion is less likely to occur than with larger single units (Feely et al., weight. Coat formulation three (F3) was prepared by mixing a 5%
1985), slower transit of small particles through the colon which (w/v) solution of pectin USP in distilled water with Surelease and
prolongs contact between the formulation and the absorptive sur- distilled water to result in a ratio of pectin (P) to Surelease® (S)
face resulting in a greater proportion of drug being absorbed (Adkin of 1:3 by weight. 25 g of acetaminophen beads were used for each
et al., 1993) and higher surface area of multiple-unit systems leads batch of coating and film coat thickness.
to higher area being exposed to bacterial attack in the colon with
subsequent rapid drug release (Fernandez-Hervas and Fell, 1998).
2.2.3. Coating process
In pH-based and temporal systems it is often reported that
Coating was performed using a laboratory Aeromatic Strea-
in vitro characteristics of these systems correlate with in vivo per-
1 fluidized bed coater (Niro-Aeromatic, Columbia, MD). Coating
formance (Schellekens et al., 2008; Ueda et al., 1994; Sangalli et al.,
was performed at 50 ◦ C inlet temperature and coating solution
2001). In this work in vitro and in vivo performances of selected
was applied through a 1.0 mm spray nozzle at a spray rate of
pectin-ethylcellulose coat formulations on small drug loaded beads
2 ml/min using a rabbit peristaltic pump (Rainin Instrument Co.
as enzyme-based colonic delivery systems have been evaluated
Inc., Woburn, MA) and 25 psi atomizing air pressure. Coating solu-
in vitro using release studies in different dissolution media to mimic
tions were coated onto drug beads to result in a different coat
transit times and pH conditions in the gastrointestinal tract and
thickness. The film thickness is expressed as the theoretical per-
in vivo by using gamma-scintigraphic studies in dogs and absorp-
centage of the weight gained (TWG) used relative to the weight
tion studies in human volunteers under fed and fasted conditions.
of the uncoated beads. Beads were coated with 9%, 14%, 16% and
The inter-relationship between in vitro release studies, gamma-
18% weight gains for coat formulation F1, 12%, 25%, 30% and 35%
scintigraphic studies in dogs and in vivo absorption studies in
weight gains for coat formulation F2, and 25%, 35%, 45% and 55%
human volunteers is explored and investigated. Acetaminophen
weight gains for coat formulation F3. Coated beads were cured for
was chosen as a model drug to incorporate into the colonic deliv-
30 min at 50 ◦ C. The coated beads met established requirements for
ery system because it is known to be absorbed from the colon
drug content.
(Forrest et al., 1982), and it can be easily assayed in saliva. Saliva
acetaminophen concentrations have been reported to be propor-
tional and virtually equivalent to serum concentrations (Adithan 2.2.4. Dissolution studies
and Thangam, 1982). Drug release from coated beads was determined in a dissolution
tester (VK 7000 Dissolution Testing Station, Vankel Industries, Inc.,
2. Materials and methods NJ), following the USP paddle method. All tests were conducted in
900 ml dissolution medium maintained at 37 ± 0.5 ◦ C with a pad-
2.1. Materials dle rotation speed at 50 rpm. Dissolution studies were carried out
under conditions simulating pH and times likely to be encountered
Acetominophen (Spectrum Chemical Mfg Corp., Gardena, CA) during transit in the GI tract. Testing was carried out using sim-
was used as the model drug, ethylcellulose in the form of Surelease® ulated gastric fluid (SGF) for 2 h at pH 1.4, followed by simulated
of 24.8% (w/w) solids content (Colorcon Ltd., West Point, PA), pectin small intestinal fluid (SSIF) for 4 h at pH 7.4, followed by simulated
USP from apple (Sigma Chemicals, St. Louis, MO), microcrystalline cecal fluid (SCF) at pH 6 for at least 6 h (or until 100% drug release)
cellulose (Avicel® PH101, FMC Corp., Newark, DE), polyethylene with or without the addition of 3 ml enzymes. The buffer pH 6 was
oxide, mol. wt. 200,000 (Polyox N-80, Union Carbide Corp., Dan- used to compromise between the mean pH of the cecum and the
burg, CT), pectinolytic enzymes (Pectinex Ultra SP-L, Novo Nordisk optimum pH for the activity of the enzymes. Acetaminophen con-
Biochem., North America, Inc., Franklinton, NC) were obtained from centrations were determined at 242–244 nm using a Beckman DU
the indicated sources. Samarium III oxide (Sigma Chemicals, St. 640 Spectrophotometer (Beckman Instruments, Inc., CA). For each
Louis, MO) was used as a radiotracer in scintigraphic studies. dissolution experiment, a duplicate was run at the same time under
Tylenol® Extra Strength 500 mg caplets (McNeil, Fort Washing- the same conditions. After the 4 h in SSIF, enzymes were added
ton, PA) were used as a control treatment. to one of the dissolution vessels but not to the other. Thus, one
is a release study with enzymes and the other one is a release
2.2. Methods study without enzymes. Each dissolution experiment was repeated
3 times (n = 3).
2.2.1. Bead preparation
Beads (1.5–2 mm in diameter) containing 80% acetaminophen,
15% Avicel PH101 and 5% polyox N-80, were prepared by extrusion 2.2.5. Preliminary in vivo absorption studies in humans
and spheronisation using a bench top laboratory extruder model Seven coat formulations were selected according to dissolution
20/25 and spheronizer model 120 (Caleva Process Ltd., England). results to be tested each in 2 human volunteers under fed con-
The beads were left to dry overnight in an oven at 50 ◦ C. 100 g ditions to see if in vitro dissolution results correlate with in vivo
of acetaminophen beads were prepared for each batch. The beads absorption results. These preliminary in vivo studies were per-
met established requirements for drug content. The mean % drug formed similar to the methodology described below under in vivo
content was found to be more than 95% from all batches. absorption studies. From the seven coat formulations, one coat
formulation (55% F3) was chosen to be tested in vivo in dogs by con-
2.2.2. Coat preparation ducting gamma scintigraphic studies and also tested in six human
Three coat formulations containing different amounts of pectin volunteers under fasted and fed conditions for determination of
and Surelease® were prepared and coating solutions were coated colonic in vivo performance.
 I.S. Ahmed, J.W. Ayres / International Journal of Pharmaceutics 409 (2011) 169–177 171

2.2.6. Gamma scintigraphic studies and the protocol complies with the declarations of Helsinki and
GI behavior of a single multiparticulate capsule containing Tokyo for humans. All subjects were regular in their bowel habits
acetaminophen beads coated with a selected coat formulation was and had no known history of intestinal disease. All subjects fasted
observed by gamma scintigraphy in two dogs under fed condi- for at least 10 h before the study day. The subjects were randomly
tions. The radioactive beads were prepared as described previously assigned to each of the two groups of equal size under the fasted
but such as to contain 13% Sm oxide. Beads containing Sm oxide conditions with a one-week washout period in a crossover design.
were irradiated in a TRIGA type reactor at Oregon State Univer- After a two-week washout period the study was repeated under the
sity with a neutron flux of 3.0 × 1012 n cm−2 s−1 for 10 min. Such fed conditions. The first group received the commercially available
irradiation transformed stable Sm-152 into radioactive Sm-153, immediate release caplets, Tylenol® with 200 ml of water (treat-
a radionuclide with a half-life of 46.7 h and gamma ray emitting ment A), the second group received the colonic capsule (size # 0
at 103 keV. This provided a radioactivity level of 10 ␮Ci per bead Capsugel filled with beads equivalent to a 500 mg acetaminophen
24 h after irradiation. Each capsule included five beads radiolabeled dose) with 200 ml of water (treatment B). No food was allowed
with neutron-activated Sm-153. The residence time of radiolabeled for 4 h after dosing. In the fed study all subjects received a high
beads in different regions of the GI tract was determined and image fat meal consisting of 2 eggs fried in butter, 2 beef sausages, 2
displays were observed throughout a 85-h period. The Radiation slices of toast with butter, 4 ounces of hash brown potatoes and
Safety Review Committee and the Institutional Animal Care and 8 ounces of whole milk, i.e. approximately 150 protein calories,
Use Committee (IACUC) at Oregon State University approved the 250 carbohydrate calories, and 500–600 fat calories half an hour
protocol of the study. before receiving the treatment (FDA, 2002). The subjects were then
divided into the two treatment groups in a similar way described
2.2.6.1. Dosage form preparation. A hard gelatin capsule (size under the fasted conditions and then crossed over the groups with
# 0, Capsugel) was filled with beads equivalent to a 100 mg a one week washout period.
acetaminophen dose. The capsule comprised approximately 50
coated beads. On the study day the capsule was opened and five 2.2.7.2. Collection of saliva samples. Subjects provided a zero time
of the beads were replaced with five Sm-153 radiolabeled beads saliva sample prior to dosing and then ingested a formulation.
with a total radioactivity dose of 50 ␮Ci. Saliva samples were collected by chewing squares (1 in. × 1 in.) of
Parafilm® (American National Can, Menasha, WI) for 2 min with
2.2.6.2. Gamma camera imaging. On the study day, one capsule continuous spitting into a 15-ml polypropylene centrifuge tube.
was administered to each dog with 60 ml water. The five radiola- Saliva samples were frozen until delivered. Subjects collected saliva
beled beads were visualized using the gamma camera. An external at 0.0, 15.0, 30.0, 45.0 min, and at 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0 12.0 h
anatomical marker containing 0.1 MBq Technetium-99m (Tc-99m) for Tylenol® caplets. Saliva was collected at 0.0, 1.0, 2.0, 3.0, 4.0, 5.0,
was taped to the shaved skin on the right-hand side of the body at 6.0, 7.0, 8.0, 9.0, 10.0, 12.0 and 24.0 h for the colonic capsules.
the level of thoracic number 12 to mark the stomach and the colon
and to allow correct alignment of dogs during imaging. Anterior
2.2.7.3. Chromatographic conditions. Concentrations of acetamino-
scintigraphic images were recorded at frequent intervals for up to
phen in saliva were determined using a modification of an HPLC
72 h. Images of 60 s were recorded at approximately 15 min inter-
procedure used by Borin and Ayres (1989). The column used was
vals up to 1.5 h post dose and then at approximately 1 h intervals
a reverse-phase micro-particulate C18 (Microsorb MV C18 , parti-
until 12 h post dose and then at different time intervals until the
cle size 5 ␮m, 25 cm × 4.6 mm, pore size 100 Å, Varian Analytical
release of the dogs.
Instruments, Walnut Creek, CA). Mobile phase was water:methanol
(85:15) at a flow rate of 1 ml/min. The detector was a fixed-
2.2.6.3. Scintigraphic data analysis. Anterior and posterior images
wavelength UV detector set at 254 nm (Waters model 440, Waters
were recorded by computer for subsequent analysis. The anatom-
Associates, Milford, MA).
ical position of the tracer was established by viewing the full
sequence of images and by reference to the external marker taped
to the skin of the dog to the right of the stomach and to X-rays 2.2.7.4. Standard solutions. Standards were prepared by spiking
of the dog’s abdomen taken after taping the external marker. The 900 ␮l of blank saliva with 100 ␮l of stock solutions and 100 ␮l
transit behavior of the multiparticulate system has been expressed (50 ␮g/ml) of internal standard (␤-hydroxyethyl-theophylline) to
in terms of the time for two-thirds of the beads to leave the stom- contain 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 15.0, 18.0 and 20.0 ␮g/ml
ach, or to arrive to the colon. A small intestine transit time has of acetaminophen. Mixtures were vortexed for 10 s and frozen at
been calculated as the difference between these two figures. Once −20 ◦ C for 24 h. Retention times of acetaminophen and internal
the position of the beads was determined, representative figures standard were 10 and 22 min, respectively. A standard curve was
of the GI tract were drawn on transparent sheets. The images from constructed by plotting the peak-area ratios of acetaminophen to
the gamma camera were then overlaid to the transparent sheets to internal standard against acetaminophen concentrations in saliva.
place the beads in their predetermined position. The lower limit of quantification was 0.2 ␮g/ml and linear response
across the full range of concentrations from 0.5 to 20.0 ␮g/ml
2.2.7. In vivo absorption studies (r2 = 0.999) was obtained. During the assay of the study samples,
2.2.7.1. Study design. The study was carried to compare the phar- the intra-batch precision and accuracy of the analytical procedure
macokinetics of acetaminophen from a selected coat formulation were evaluated after replicate analysis (n = 6) of control saliva sam-
(55% F3) to a conventional commercially available immediate ples spiked at four concentration levels: 0.5, 2, 8 and 15 ␮g/ml. The
release tablet (Tylenol® Extra Strength 500 mg caplets, McNeil, analysis of quality control samples showed a precision below 3%
Fort Washington, PA) following administration of single doses of relative standard variation and accuracy below ±5% for intra-batch
500 mg each using a non-blind, two-treatment, two-period, ran- analysis. The coefficient of variation for inter-batch analysis was
domized crossover design under both the fasted and fed states less than 10%.
(2 × 2 crossover). Six healthy subjects (3 males and 3 females) par-
ticipated in the study after giving informed written consent. The 2.2.7.5. Saliva analysis. Approximately 2.5 ml of saliva samples
study protocol was approved by the Oregon State University Insti- were centrifuged at 7000 rpm for 5 min. One ml of the salivary
tutional Review Board (IRB) for the protection of human subjects supernatants was then transferred to 1.5-ml microcentrifuge tubes
172 I.S. Ahmed, J.W. Ayres / International Journal of Pharmaceutics 409 (2011) 169–177

and 100 ␮l of internal standard was added. The mixtures were vor- 100
texed for 10 s and then frozen at −20 ◦ C for 24 h. The samples were 90 14% F1-E
thawed and centrifuged at 14,000 rpm for 5 min before analysis. 80 16% F1-E

% drug released
Portions of supernatant (100 ␮l) were transferred to HPLC tubes 70
and assayed as described above. 18% F1-E
60
9% F1-NE
50
2.2.7.6. Pharmacokinetic analysis. Pharmacokinetic characteristics
40 14% F1-NE
from saliva data following administration of all treatments
were estimated for each subject by using a computer program, 30 16% F1-NE
WinNonlin® (version 1.5, Scientific Consulting, Inc., Cary, NC, USA). 20
18% F1-NE
Non-compartmental analysis was used. Cmax (␮g/ml) and tmax 10
(h) were the observed maximal drug concentration and its time, 0
respectively. AUCs were calculated using trapezoidal rule to deter- 0 5 10 15 20 25 30 35 40
mine relative bioavailability. AUC 0–∞ is the area under the saliva Time (h)
concentration vs. time curve calculated from 0 to ∞ and the rel-
ative bioavailability (frel ) was the percentage of the AUC0–∞ of Fig. 1. Release of acetaminophen from beads coated with F1 coat formulation at
acetaminophen from the colonic capsule (OSU formulation) com- different percentage weight gains in the presence of enzymes (E) and absence of
enzymes (NE) (n = 3).
pared to the AUC 0–∞ of acetaminophen from Tylenol Extra Strength
500 mg caplet. In vivo lag times were observed before any appre-
ciable amount of acetaminophen could be detected. 100
12% F2-E
90
2.2.7.7. Statistical analysis. An analysis of variance (ANOVA) was

% drug released
80 25% F2-E
performed for untransformed data for the pharmacokinetic param- 70 30% F2-E
eters Cmax , AUC0–t and AUC0–∞ using the software SPSS 11.0 (SPSS 60 35% F2-E
Inc., Chicago, USA). The nonparametric Signed Rank Test was used 50 12% F2-NE
to compare the tmax for the colonic capsule under fasted and fed 40
25% F2-NE
conditions. The level of significance was ˛ = 0.05. A p-value of ≤0.05 30
was considered statistically significant. In vitro and in vivo lag times 30% F2-NE
20
were checked for statistical significance using the one-way analy- 10 35% F2-NE
sis of variance (ANOVA) F-test for testing the equality of several
0
means. A p-value > 0.05 was considered statistically insignificant. 0 5 10 15 20 25 30 35 40
Time (h)
2.2.7.8. Deconvolution of acetaminophen saliva concentration data.
In vivo release/absorption of acetaminophen from the delivery sys- Fig. 2. Release of acetaminophen from beads coated with F2 coat formulation at
tem under fed and fasted conditions were estimated by a model different percentage weight gains in the presence of enzymes (E) and absence of
independent numerical deconvolution of pharmacokinetic data. enzymes (NE) (n = 3).

Deconvolved input functions from biostudy data were determined

using computer software KineticaTM 2000 (Innaphase Corpora- 18% F1 coat (highest coat thickness used in F1 coats) compared
tion, PA). Because of the instability of numerical algorithms when to 18 h from 25% F2 coat and only to less than 5 h from 25% F3
there is noise in the supplied raw data (Langenbucher and Moller, coat. Also, 50% drug was released in 24 h from 35% F2 coat com-
1983), as was observed in the present study due to multiple peaks, pared to less than 5 h from 35% F3 coat. The difference in the rate
mean saliva concentrations obtained in fed and fasted conditions of drug release is related in this case to the amount of pectin in
were used as input response functions because mean values gave the coat. While for the same coat formulation the percentage drug
a smoother curve. Intravenous data for acetaminophen, required released depended on the thickness of the coat, for example, in 6 h
to perform model independent deconvolution, were obtained from 50% drug was released from 12% F2 coat compared to less than 7%
previous published data (Rawlins et al., 1977). In vitro/in vivo cor- drug released from 35% F2. Also, after 6 h 70% drug was released
relation is described using linear regression model and correlation from 25% F3 coat compared to only 37% drug released from 55%
coefficient for describing the degree of linear association between F3. The slow release observed with thicker coats is typical of rate
the two variables was calculated.

25% F3-E
3. Results and discussion 100
90 35% F3-E
% drug released

3.1. In vitro dissolution studies 80 45% F3-E

70 55% F3-E

Drug release from beads coated with F1, F2 and F3 coat formu- 60 25% F3-NE
lations, at all weight gain applied, after 2 h in SGF followed by 4 h 50
35% F3-NE
in SSIF followed by different hours in SCF (until almost 100% drug 40
45% F3-NE
release from all coats) with and without pectinolytic enzymes are 30
55% F3-NE
grouped in Figs. 1–3, respectively. 20
Release profiles from F1, F2 and F3 coats showed that the per- 10
centage of drug released depended on the ratio of pectin in the 0
0 3 6 9 12 15
coat and the thickness of the coat. The more pectin in the coat
(F3 > F2 > F1) and the thinner the film (smaller weight gains vs. Time (h)
higher weight gains), the faster the drug was released in SGF, SSIF
Fig. 3. Release of acetaminophen from beads coated with F3 coat formulation at
and SCF in presence and absence of enzymes. For example, in the different percentage weight gains in the presence of enzymes (E) and absence of
absence of enzymes, 50% drug was released in about 28 h from enzymes (NE) (n = 3).
 I.S. Ahmed, J.W. Ayres / International Journal of Pharmaceutics 409 (2011) 169–177 173

release coating membranes. Ethylcellulose, which is a hydropho- 120

bic sustained-release material, is known to form non porous films.

% Acetaminophen dissolved
However, a non porous film will not be formed in the current case 100
due to the leaching of the water-soluble pectin even after a com-
plete film is formed (Tang et al., 2000). It was found that for F3 coats, 80
it took a relatively high coat thickness to start to slow drug release in In vitro
both SGF and SSIF which could be due to the relatively small amount
60 In vivo
of ethylcellulose in these coats and/or incomplete film forming at
low coat thickness. However, some studies reported that in vitro
40
release experiments showed a significantly faster release of the
model drug from capsules coated with enzyme-dependent poly-
mer when compared with in vivo results (Van den Mooter et al., 20
1994). Thus, due to possible slower dissolution in vivo compared
to in vitro, these coats should also be tested in vivo for site specific 0
drug release. 0 4 8 12 16 20 24
The percentage of drug released in presence of pectinolytic Time (h)
enzymes was always higher from the different coats when com-
Fig. 4. In vitro and in vivo dissolution from 14% F1 coat formulation.
pared to no enzymes. As the ratio of pectin in the coat increased,
the effect of pectinolytic enzymes on drug release also increased.
It could be that pectinolytic enzymes accelerate pore formation by three coat formulations tested in this study and only coat formu-
attacking the pectin in the coat, but the coat remained intact and lations showing very fast release (9%F1 and 25%F3) or very slow
did not breakdown in most formulations and showed some cracks release (30%F2 and 35%F2) were excluded from being tested in vivo.
in formulations containing higher amounts of pectin (F3 coats). Dis- All formulations were tested under fed conditions because this is
solution studies also showed that coated beads resisted release in the condition where it is most difficult to provide protection against
SGF for 2 h. Thus it can be suggested that pectin in the coat did not pre-colonic drug release. Saliva drug concentration–time curves
dissolve during 2 h in SGF for most formulations. from the different formulations were obtained to determine the
Drug release from all formulations in the absence of enzymes lag time before any appreciable drug absorption could be detected
shows that the three pectin/ethylcellulose coat formulations give and to determine relative bioavailability compared to the reference
a nearly zero-order (linear) release over time. The release rate formulation (Tylenol). In vitro lag time was calculated as the time
depended on the amount of the pectin in the coat; the more pectin elapsed until a % drug release of more than 5% was detected in dis-
in the coat the faster the release is while maintaining linearity. For solution media while in vivo lag time was calculated as the time
example, release profile from the 55% F3 coat formulation showed elapsed until a concentration of >0.1 mg/l of acetaminophen was
linear 100% release over 14 h, while release from 25% F2 coat formu- detected in saliva. Table 1 shows a summary of in vitro and in vivo
lation showed 75% linear release over 24 h indicating the possibility findings for the tested formulations.
of obtaining different linear release profiles over any period of time Results show that in vitro behavior of all coat formulations did
that can be tailored to specific dosage form for individual drugs. not correlate with in vivo results except for 12%F2 and 35%F3 coats
In summary when the ratio of pectin to Surelease® was high (i.e. which showed good correlation with in vivo results (correlation
1:3, w/w), drug release was relatively rapid and low percentage coefficients were 0.84 and 0.92, respectively) and which could be
coat thicknesses were accompanied by cracking of the coat. How- due to the rapid dissolution of these coats. Comparison of in vitro
ever, there was a significant decrease in drug release when pectin and in vivo dissolution from one of the F1 coat formulations (14%F1)
was mixed with Surelease® in 1:6 and 1:12 (w/w) ratio which was is shown in Fig. 4 after deconvolution of corresponding in vivo
expected since Surelease® controls the swelling of pectin and some saliva concentration vs. time data from biostudy. Data presented in
of the coats thicknesses showed what could be considered as ideal this figure indicates that in vitro dissolution did not predict in vivo
drug release profiles for colonic delivery. dissolution, showing much faster in vitro dissolution compared to
in vivo dissolution from the same coat. The correlation coefficient
3.2. Preliminary in vivo absorption studies in humans calculated was only 0.41. All other coats showed correlation coef-
ficients of less than 0.5. It was also noticed that coat formulations
In vitro dissolution tests can provide essential information on showing very little drug release in SSIF in vitro did not show any
the mechanism of drug release but are not necessarily good pre- appreciable amount of drug absorbed in vivo up to 24 h indicat-
dictors of in vivo results. For this reason seven coat formulations ing very poor bioavailability from these coat formulations when
showing different dissolution profiles and containing different compared to Tylenol. In vitro lag times from most formulations
amounts of pectin and Surelease® and which were coated onto drug were also significantly shorter than in vivo lag times. Differences
beads to result in different percentage weight gains, were tested between in vitro performances when compared to in vivo per-
each in two subjects. The seven coat formulations represented all formances could be due to several factors such as differences in

Table 1
In vitro and in vivo average data relevant to the different formulations tested each in 2 subjects in vivo.

Formulation In vitro lag time (h) In vitro release (%) In vivo lag time (h) Relative bioavailability

14% F1 1 ± 0.0 60 4.5 ± 0.7 Less than 5%

16% F1 4.3 ± 0.57 52 – No drug absorption
18% F1 6 ± 0.63 32 – No drug absorption
12% F2 1 ± 0.0 85 2± 0.0 91%
25% F2 4 ± 0.0 55 – No drug absorption
35% F3 1.16 ± 0.28 100 1± 0.0 84%
55% F3 2.2 ± 0.57 87 5.5 ± 0.7 61%

In vitro release is the % drug release in presence of enzymes within 8 h following lag time in vitro.
174 I.S. Ahmed, J.W. Ayres / International Journal of Pharmaceutics 409 (2011) 169–177

Fig. 5. Representative scintigraphs showing the GI transit of labeled beads in the stomach, small intestine and colon.

hydration rates, bacterial contents, enzymatic activities, viscosi- 3.3. Gamma scintigraphic studies
ties, fluid contents and different physiologic conditions. Although
the number of subjects used in this preliminary study is small, the Anterior and posterior images were taken at different time
results obtained were further investigated by selecting one of the intervals between 5 min and 85 h post dosing. Representative
coat formulations to be tested for GI behavior in two dogs and scintigraphs showing the GI transit of labeled beads in one of the
further in six healthy volunteers for its colonic delivery potential. dogs are shown in Fig. 5. At 5 min post dosing a cluster of beads
The selected coat formulation was the 55% F3 because preliminary was clearly seen in the stomach (A). At 20 min post dosing beads
results in two subjects of this coat showed a lag time of about 5.5 h could be distinguished and were spread in the different parts of the
before any appreciable amount of drug was detected in saliva. This stomach which indicate complete release from the capsule shell (B).
coat formulation consisted of a ratio of pectin (P) to Surelease® (S) At 1.5 h post dosing individual beads could be seen in the stomach
of 1:3 by weight and was coated onto drug beads to result in a and in the small intestine which indicate that beads were emptying
weight gain of 55%. from the fed stomach (C). At 4.5 h the beads were moving down and
In summary, results indicate that coats containing larger some of the beads reached the ascending colon (D). At 5.5 h beads
amounts of pectin relative to ethylcellulose but at high coat could be only seen in the ascending colon with probably some beads
thickness might be able to deliver drug specifically to the colon disintegration (E). At 7.5 h beads could be seen in the ascending
compared to thinner coats containing larger amounts of ethylcel- and transverse colon (F) and dispersion of radioactive material in
lulose. Thick coats containing a high ratio of pectin could be able to the ascending colon was very noticable which might indicate the
prevent premature release of drug from the beads before reaching disintegration of most of the beads in the colon. At 24 h radioactiv-
the colon while being hydrophilic enough to ensure a good frac- ity could only be seen in the descending colon and remained there
tion of drug being released under colonic conditions. Studies have until 42 h (G and H). The dog was released at 85 h post dosing after
shown that pectin must swell enough to be able to absorb enzyme- a last image showing no radioactivity in his body. A similar pat-
rich fluids in the colon. Therefore, hydration or swelling of pectin is tern of beads movement through the GIT was also observed with
a major requirement so that colonic enzymes obtain access to the the second dog (scintigraphs are not shown). Analysis of scinti-
glycosidic linkage in the polysaccharide. On the other hand, thinner graphic results of both dogs suggests that the labeled beads had an
coats containing high ratio of ethylcellulose slow down the release estimated gastric emptying time of 3 h, an estimated small intes-
in vivo to a large degree and control the swelling of pectin resulting tine transit time of 2 h and an estimated colonic transit time of
in a very small fraction of drug being released. 36 h.
 I.S. Ahmed, J.W. Ayres / International Journal of Pharmaceutics 409 (2011) 169–177 175

3.4. In vivo absorption studies Table 2

Mean pharmacokinetic parameters of acetaminophen after oral administration of
500 mg in the tested colonic capsule in 6 subjects under fed and fasted conditions.
Considering the aim of this work and the system under investi-
gation, the duration of the lag phase before appearance of drug in Parameter Fed Fast Statistical test (p)
biological fluid (saliva) and the relative bioavailability are the fun- In vivo lag time 6.16 ± 2.14 4.83 ± 2.23 p = 0.223
damental parameters to evaluate in vivo behavior of this delivery Cmax (mg/l) 2.14 ± 0.84 2.45 ± 0.76 p = 0.125
system. The IR Tylenol formulation is thought to be valuable in this tmax (h) 6.83 ± 2.31 5.51 ± 1.74 p = 0.345
AUC(0–t) (mg h/l) 12.32 ± 4.34 13.68 ± 3.10 p = 0.309
study because comparing pharmacokinetic parameters between
frel (%) 62.19 67.80 p = 0.203
the IR tablet and the colonic capsule (such as Cmax and tmax ) would
Data are mean values (n = 6) ± SD.
indicate that there was enough delay for the colonic beads to
reach the colon and release its drug content in the colon. Since
acetaminophen absorption in the colon is expected to be less com- Table 2 also summarizes some pharmacokinetic data relevant to
pared to upper GI tract, a reduced bioavailability is also to be the tested formulation under fed and fasted conditions.
expected. The multiple peaks observed in the biostudy data under fed con-
When the 55% F3 beads were administered to six healthy volun- ditions could be due to the gradual emptying of the beads from the
teers, acetaminophen appeared on average after 6.16 h (±2.14 h) in fed stomach which was also reported for small beads up to 3 mm in
saliva when administered after a standard breakfast and at 4.83 h diameter (Khosla and Davis, 1989). Such multiple peaks were not
(±2.23 h) when administered after an overnight fast, however, the observed under fasted conditions indicating bolus emptying of the
difference in time was not statistically significant (p = 0.223) which beads from the fasted stomach. These results are consistent with
is expected since small particles (less than 7 mm) were reported to scintigraphic studies in dogs. A constant saliva drug concentration
empty from the stomach even though it is in the digestive phase was also maintained for 7 h with minimal fluctuations under fasted
(Davis, 1989). These results suggest that beads coated with 55% F3 conditions (Fig. 7). These results suggest that the beads transit time
might successfully deliver drugs to the colon. These findings could in the ascending colon (where most of the pectinolytic enzymes are
not have been predicted from in vitro release studies which showed located) could be as long as 7 h which is also consistent with pub-
an in vitro lag time of 2.2 h and 35% drug release in SSIF. Similar lished work on the slow transit of small particles through the colon
findings were observed with in vivo published data in rats using (Adkin et al., 1993).
capsules coated with azo-polymers and containing ibuprofen as a The relative bioavailability of the colonic delivery system for-
model drug (Nubuchi et al., 1986). In this study the results of in vitro mulation to Tylenol under fed and fasted conditions was 62.19
release experiments with ibuprofen in sonicated rat cecal contents and 67.80%, respectively (Table 2). Relative bioavailability from the
(cell-free extract) showed a significantly faster release of the model delivery system under fasted conditions was slightly higher than
drug from capsules coated with azo-polymers that are susceptible under fed conditions but not statistically different (p-value = 0.203).
to bacterial azo reductase activity when compared to in vivo results. The reduced bioavailability, as assessed by AUC, compared to
It has to be mentioned that pharmacokinetic measurements Tylenol could be due to several factors such as incomplete drug
indicate solely drug absorption and not the mechanism responsible release from the beads and hence the drug was unavailable for
for drug release. Average Cmax estimated from the tested delivery absorption and would then be excreted along with the non-
system was found to be 2.14 and 2.45 mg/l under fed and fasted disintegrating beads. Assuming that the drug was released from
conditions, respectively compared to 5.96 and 6.35 mg/l under fed the beads, the reduced AUC may be due to erratic and incomplete
and fasted conditions, respectively from Tylenol caplet. Cmax was absorption in the colon as reported for many drugs (Koch-Weser
also observed at 6.8 h and 5.5 h on average, under fed and fasted and Schechter, 1981). Another contributing factor could be incom-
conditions, respectively from the colonic capsule, and since small plete drug dissolution due to less water in the colon especially in the
drug beads are known to show small variability in gastric emptying more distal regions. Although drugs that are well absorbed from the
time, are less influenced by the presence of food and are less likely intestines are expected to have lower bioavailability from the colon,
to stagnate at the ileo-cecal junction this might indicate that most of the colonic delivery system formulation developed in this study
the drug release from the beads resulted from degradation of pectin might be useful in improving bioavailability of drugs destroyed or
in the coat by enzymatic action in the colon rather than by simple metabolized in upper GI tract such as peptides drugs or deliver-
diffusion. Averages of acetaminophen saliva concentration–time
curves under fed and fasted conditions are shown in Figs. 6 and 7.
9

8 8

7 7
Saliva Conc (mg/L)

Colonic Capsule
Saliva Conc (mg/L)

6 Colonic Capsule 6
Tylenol
5 Tylenol 5

4 4

3 3

2 2

1 1

0 0
0 4 8 12 16 20 24 0 4 8 12 16 20 24
Time (h) Time (h)

Fig. 6. Mean saliva concentrations of acetaminophen following single 500-mg oral Fig. 7. Mean saliva concentrations of acetaminophen following single 500-mg oral
doses under fed conditions in six subjects. Error bars represent standard deviations doses under fasted conditions in six subjects. Error bars represent standard devia-
(n = 6). tions (n = 6).
176 I.S. Ahmed, J.W. Ayres / International Journal of Pharmaceutics 409 (2011) 169–177

120 in vitro studies. However, scintigraphic studies in human volun-

teers would be necessary to confirm these findings.
Cumulative input (% of dose)

100 Fed
Fast 4. Conclusions
80 Tylenol
In vitro performance of small pectin-ethylcellulose coated drug
beads for colonic delivery in simulated GI fluids did not predict
60
or correlate with in vivo performance. Beads showing ideal release
profiles in vitro resulted in no or little drug absorption in vivo while
40 beads showing premature drug release in vitro might successfully
deliver the drug to the colon as indicated by performing gamma
20 scintigraphic studies in dogs and deconvolution and absorption
studies in humans under fed and fasted conditions.
0
0 5 10 15 20 25 30 Conflict of interest
Time post dosing (h)
The authors report no declarations of interest.
Fig. 8. Mean in vivo acetaminophen release from the colonic delivery system under
fed and fasted conditions in six healthy volunteers.
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